Academic literature on the topic 'C-Cdh1'

Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

Abstract Hypermethylation of promoter contributes to the transcriptional repression of a number of cancer associated genes. In lymphoma, the promoter hypermethylation of many tumor suppressor genes (TSGs), such as p16, has been already known. Using methylation-specific PCR (MSP) and quantitative real-time PCR (qRT-PCR), we examined promoter methylation status and mRNA expression levels of E-cadherin (CDH1), H-cadherin (CDH13) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS18) which are putative TSGs located on chromosome 16q, and also examined the mRNA expression levels of DNA methyltransferases (DNMT1, 3A and 3B) and studied the correlation between these different tested parameters in 36 of lymphomas [included 29 diffuse large B cell (DLBCL) and seven mantle cell lymphoma (MCL)] and 16 non-malignant lymphoid tissues after obtaining informed consent. The expression of DNMTs mRNA were significantly higher in lymphomas compared to non-malignant tissues (p<0.05). Promoter hypermethylation of CDH1, CDH13, and ADAMTS18 was detected in 31/36 (86%), 33/36 (91.6%) and 28/36 (77.7%) of lymphoma, respectively. The expression of CDH1 and ADAMTS18 was significantly reduced in the patients with hypermethylated promoter when compared to unmethylated (p<0.01 and p<0.05, respectively), while no significant difference was found in CDH13. CDH1 and ADAMTS18 expressions were significantly reduced in lymphomas compared to non-malignant tissues (p<0.01), while CDH13 showed no significant difference. Notably, there was significant positive correlation between the expression levels of CDH1 and CDH13 (r = 0.735, p<0.01) (Fig. 1A). Moreover, ADAMTS18 expression showed significant positive correlation with both CDH1 and CDH13 expression levels (r = 0.625, p<0.01; r = 0.720, p<0.01, respectively) (Fig. 1B, C). Also there was significant negative correlation between the expression levels of DNMT3A and 3B with ADAMTS18 (r = -0.396, p<0.01; r = -0.364, p<0.01, respectively), but not with CDH1 and CDH13 (Fig. 2A and B). We could not find any correlation between the levels of DNMTs mRNA and the methylation status of CDH1, CDH13 and ADAMTS18. We examined the effect of 5-Aza-2-deoxycytidine (5-aza-dC) treatment on CDH1, CDH13 and ADAMTS18 expression levels and their methylated promoters in 3 of lymphoma cell lines (Raji, CTB-1 and SLVL) and one patient primary DLBCL cell line. We found that 5-aza-dC treatment of CDH1, CDH13 and ADAMTS18-methylated cell lines led to restoration of their expression levels (Fig. 3A, B, and C). Our results showed that CDH1, CDH13 and ADAMTS18, tumor suppressor genes adjacently located at chromosome 16q, are remarkably correlated and frequently methylated in human lymphoma and their methylation could not be explained solely by the expression level of DNMTs mRNA. Disclosures: No relevant conflicts of interest to declare.

The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/CCDC20 ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/CCDH1 has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/CCDC20, releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/CCDH1 substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/CCDC20 and APC/CCDH1 during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity.

In vivo analysis in budding yeast identifies APC/C-Cdh1–specific minimal degrons carrying either a D or a KEN box and a nuclear localization sequence. APC/C-Cdh1 activity is restricted to the nucleus, maximal in the nucleoplasm, and absent from the cytoplasm, allowing for spatiotemporal control of Cdh1 substrate proteolysis.

23 Background: CDH1 germline mutations are found to be associated with the development of hereditary diffuse gastric cancer (HDGC) and the early-onset diffuse gastric cancer (EODGC). But the impact of CDH1 gene mutations and large deletions on HDGC and EODGC has not been fully determined in Asians. Although the incidence of gastric cancer is relatively high in China, the detection rate of CDH1 germline mutations in Chinese patients with EODGC is rare compared to that in European patients. Methods: We investigated the mutation status of the CDH1 gene in 57 Chinese EODGC patients younger than 40 years old who met the clinical criteria for HDGC. Polymerase chain reaction-direct sequencing was performed, and multiplex ligation-dependent probe amplification (MLPA) was used to evaluate the patients with negative sequencing results. Associations between mutation, clinicopathologic, and overall survival data were analyzed by SPSS 19. Results: The germline mutations of CDH1gene were identified in 51 (89.5%) of the 57 EODGC patients. The nonsense mutation in exon 13 (c.2200T>C, p.Ala692*) occurred in fourty-six EODGC patients. The missense mutations were detected in twenty patients (Eighteen in exon 5: c.778G>C, p.Glu218Asp; Two in exon 12: c.2012C>G, p.Leu630Val). No deletion or duplication in any patient. Most of the patients carrying the CDH1 mutation in exon 13 had lymph node metastasis when compared with patients lacking CDH1 mutation (87.2% vs 60.0%) ( P < 0.05 ). EODGC patients, lacking germline CDH1 alterations, showed a longer median overall survival (mOS) than patients carrying CDH1 mutation in exon 13 ( P < 0.05 ). Moreover, the presence of CDH1 mutation in exon 13 was associated with the incidence of neural invasion ( P < 0.05 ). Conclusions: This study reveals novel CDH1 mutations in Chinese EODGC patients which had been poorly investigated. The presence of CDH1 mutation in EODGC patients may result in lymph node metastasis and poor prognosis. More research is needed to determine additional genetic targets that trigger EODGC.

ABSTRACT Cdh1 is a coactivator of the anaphase-promoting complex/cyclosome (APC/C) and contributes to mitotic exit and G1 maintenance by facilitating the polyubiquitination and subsequent proteolysis of specific substrates. Here, we report that budding yeast Cdh1 is a component of a cell cycle-regulated complex that includes the 14-3-3 homologs Bmh1 and Bmh2 and a previously uncharacterized protein, which we name Acm1 (APC/C Cdh1 modulator 1). Association of Cdh1 with Bmh1 and Bmh2 requires Acm1, and the Acm1 protein is cell cycle regulated, appearing late in G1 and disappearing in late M. In acm1Δ strains, Cdh1 localization to the bud neck and association with two substrates, Clb2 and Hsl1, were strongly enhanced. Several lines of evidence suggest that Acm1 can suppress APC/CCdh1-mediated proteolysis of mitotic cyclins. First, overexpression of Acm1 fully restored viability to cells expressing toxic levels of Cdh1 or a constitutively active Cdh1 mutant lacking inhibitory phosphorylation sites. Second, overexpression of Acm1 was toxic in sic1Δ cells. Third, ACM1 deletion exacerbated a low-penetrance elongated-bud phenotype caused by modest overexpression of Cdh1. This bud elongation was independent of the morphogenesis checkpoint, and the combination of acm1Δ and hsl1Δ resulted in a dramatic enhancement of bud elongation and G2/M delay. Effects on bud elongation were attenuated when Cdh1 was replaced with a mutant lacking the C-terminal IR dipeptide, suggesting that APC/C-dependent proteolysis is required for this phenotype. We propose that Acm1 and Bmh1/Bmh2 constitute a specialized inhibitor of APC/CCdh1.

Abstract Introduction We have previously proposed that Cdh1 is a tumor suppressor by maintaining genomic stability. We also found Cdh1 downregulated in several tumor cell lines including AML (Oncogene 2008; 27:907-17). Heterozygous Cdh1 knockout mice develop epithelial tumors, myelodysplasia and plasma cell dyscrasias (Nat. Cell Biol. 2008;10:802-11). By analyzing primary AML samples from bone marrow (BM) or peripheral blood (PB) we detected downregulation of Cdh1 in the vast majority of samples when compared to normal CD34+ HSCs. Progression through the cell cycle is tightly regulated by different cyclin-dependent kinases (Cdks) and their activating cyclin subunits. Stage-specific proteolysis of cyclins and other cell cycle regulators is important for transition to the next cell cycle phase. The anaphase-promoting complex/cyclosome (APC/C) is an E3-ubiquitin ligase that controls mitosis and G1 through degradation of these proteins. Through its activating subunits Cdh1 and Cdc20 the APC/C ensures substrate-specifity. While Cdc20 regulates progression through mitosis, Cdh1 is activated in late mitosis to coordinate accurate entry into S-phase. Thereby, the APC/C is crucial for maintaining genomic stability during the cell cycle. Suppression of APC/C-Cdh1 can lead to unscheduled cyclin expression and Cdk activity, which can cause cell cycle defects leading to the accumulation of DNA alterations and further to malignant transformations. However, the exact nature of the origin of genomic instability upon downregulation of Cdh1 is unclear. Methods To investigate stability of cyclins in Cdh1-knockdown (kd) cells, origin loading and start of replication, cells were released from a mitotic block and samples were taken every 2 h until S-phase entry for FACS and immunoblotting. For live-cell imaging cells were seeded 24 h before imaging in chambered coverslips, after which progression through the cell cycle was analyzed by automated microscopy. Results Characterization of a Cdh1-kd revealed strong stabilization of the substrates cyclin A/B leading to diminished loading of mini-chromosome maintenance (MCM) proteins on replication origins in G1. Stabilization of cyclin A/B and unscheduled Cdk1/2 activity may cause the observed premature entry into S-phase, while the reduced loading of MCMs in G1 could be responsible for the prolonged replication in S-phase seen in Cdh1-kd cells. Accordingly, treatment with the Cdk1 inhibitor RO-3306 restored reduced MCM loading. Polo-like kinase 1 (Plk1) was stabilized in Cdh1-kd cells, which may cause bypass of the Cdc14B-Cdh1-Plk1 dependent DNA damage checkpoint. Indeed, potential replication stress in Cdh1-kd cells did not lead to G2/M arrest, but was enforced by inhibition of the Cdh1 substrate Plk1. Underreplicated DNA and replication intermediates in mitosis may be the reason for increased genomic instability, namely lagging chromosomes, anaphase bridges and micronuclei in Cdh1-kd cells detected by live-cell imaging. In addition, aberrant cytokinesis and the development of polyploid cells generated by misseparation of chromosomes during mitosis were enhanced in Cdh1-kd cells. Finally, monitoring of 53BP1, a DNA-repair marker, in living cells showed amplified DNA-damage through increased double-strand breaks in Cdh1-kd cells. Conclusions Downregulation of the tumor suppressor APC/C-Cdh1 leads to deregulation of DNA-replication by stabilizing cyclin A and B in G1 and reduced loading of replication origins with MCM proteins resulting in the accumulation of enhanced genomic instability and DNA damage. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1563 Introduction: The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression. This is achieved by targeting various cell cycle regulators for proteasomal destruction. APC/C in conjunction with its adaptor protein Cdh1, both stabilizes G1-Phase (a pre-condition for an accurate cell cycle progression) and is involved in the induction of cell cycle arrest and differentiation. Further evidence suggests that Cdh1 is involved in the differentiation of a variety of cells such as neurons, myocytes, hepatocytes and lens epithelial cells. During differentiation Cdh1 interacts with the TGFb signaling pathway, targets Id2 for destruction and indirectly leads to accumulation of p27 by Skp2 degradation. We have been able to demonstrate that the expression of Cdh1 is decreased in both, AML cell lines and in primary blast samples carrying the translocation t(8;21), which consequently leads to an AML1/Eto fusion protein and is one of the most common chromosomal rearrangements in AML. Furthermore, we have data suggesting that APC/C-Cdh1 significantly influences the differentiation of malignant myeloid cells. Here, we have analyzed the expression of Cdh1, its target proteins and relevant cell cycle regulators during normal myeloid differentiation. Methods: A cytokine cocktail consisting of SCF (50ng/ml), IL-3 (5 ng/ml) and G-CSF (100 ng/ml) was used to induce differentiation of CD34+ cells into CD11b+ macrophage-like cells over seven days. Daily protein isolation, CD11b-FACS and FACS analysis of propidium iodide staining were performed to analyze Cdh1 status, differentiation kinetics and cell cycle distribution. In addition, we have established a Cdh1 knockdown in CD34+ cells by lentiviral vector mediated RNA interference. By means of GFP-cell-sorting the initially achieved transduction efficiency of 30% in CD34+ cells was increased to 70–80%. Results: The differentiation experiments carried out with normal CD34+ cells showed that after 7 days of stimulation the predominant majority of them had lost the CD34 marker and about 30% expressed CD11b on their surface confirming previous results. During the differentiation process an initial rise in Cdh1 levels, followed by a continuously high expression, was observed. Furthermore, we detected a downregulation of the Cdh1 target proteins Id2 and Skp2 and stable protein levels of p27. The cell cycle profile indicated an initial proliferation with an incremental G2/M-peak and at day 6 increasing apoptosis with a high Sub-G1-peak. In the transduced CD34+ cells we have been able to confirm by Western blotting analysis and RT-PCR that the CD34 positive cells harboring a Cdh1 shRNA had significantly decreased protein and RNA levels of Cdh1 compared to CD34 positive cells harboring a control shRNA against GFP. Analysis of the influence of Cdh1 knockdown on differentiation of CD34+ cells is ongoing and will be presented at the meeting. Conclusion: These results are consistent with the important role of Cdh1 in initiating differentiation and also show its sustained function in post mitotic myeloid cells. Studying the differentiation characteristics of CD34+ cells with a Cdh1 knockdown is likely to help to further determine its function. Disclosures: No relevant conflicts of interest to declare.

Orientador: Renée Laufer Amorim
Resumo: A próstata canina é um bom modelo para estudos comparados entre o cão e o homem, uma vez que essas duas espécies desenvolvem espontaneamente carcinoma de próstata (CP). Para melhor caracterização do CP canino, a presente pesquisa foi dividia em quatro capítulos que avaliam diferentes aspectos dos CPs em cães. A atrofia inflamatória proliferativa (PIA) é uma lesão pré-neoplásica descritas em humanos e pouco estuda em cães. Nós caracterizamos essa lesão em cães e identificamos uma forte relação entre a localização topográfica da PIA com os CPs. Além disso, foi identificada a perda de expressão gênica e proteica de PTEN e AR na PIA. Esses fatores associados corroboram com o potencial pré-neoplásico desta lesão em cães. Um achado interessante foi a alta expressão de P63 na PIA e em um grupo de CP caninos. Para melhor caracterizar este grupo, foi avaliada a expressão imuno-histoquímica de diferentes citoqueratinas e outras proteínas relacionadas ao desenvolvimento do CP em humanos. Os carcinomas que apresentam expressão de P63 apresentaram padrões morfológicos com escore de Gleason alto e um fenótipo mais agressivo quando comparado à tumores que não apresentação expressão de P63. Posteriormente, a expressão gênica e proteica de E-caderina, NKX3.1 e C-MYC foi avaliada em CP como marcadores nas diferentes lesões. Além disso, nós avaliamos a metilação como mecanismo regulatórios dos genes CDH1 e NKX3.1. Foi possível identificar a perda de E-caderina e NKX3.1 nos tumores, comparado à ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The canine prostate gland can be used as a model to human prostatic disease since dogs and men are the only species that spontaneously develop prostate carcinoma (PC). To better characterize the canine PC, this research was divided into four chapters that evaluated different aspects of the PC in dogs. The proliferative inflammatory atrophy (PIA) is a pre-neoplastic lesion described in humans and few studies in dogs describe it as a preneoplastic lesion. This study characterized PIA in dogs and identified a strong relationship between the PIA topography with PC. In addition, we identified the loss of PTEN and AR expression in PIA. These findings demonstrated the potential of PIA as a preneoplastic lesion in dogs. An interesting finding in this research was the high expression of P63 in PIA and a group of PC. This study found a group of PC showing P63 positive expression in neoplastic epithelial cells. Thus, these tumors were selected to better characterize them using immunohistochemistry. These tumors had an aggressive phenotype and presented high expression of AKT and C-MYC and loss of NKX3.1. Further, we selected a usual group of PC and evaluate the expression of E-cadherin, NKX3.1 and C-MYC. In addition, we evaluated the methylation as a regulatory mechanism of CDH1 and NKX3.1 genes. We have identified loss of E-cadherin and NKX3.1 in PC compared to normal prostate and C-MYC overexpression. The expression of E-cadherin was related to overall survival and Gleason score. The ... (Complete abstract click electronic access below)
Doutor

2010/2011
The calpains are a family of intracellular cysteine proteases, among which the best studied isoforms, micro- (CAPN1) and milli-calpain (CAPN2), are heterodimers consisting of a catalytic subunit and a common regulatory subunit, CAPNS1, required for function. Calpain is involved in many processes important for cancer biology, such as autophagy, indeed in calpain-depleted cells autophagy is impaired, with a subsequent increase in apoptosis sensitivity. Calpain is also important in all the stages of the stress response. A proteomic approach was employed for the identification of novel CAPNS1 interacting proteins. Proteins immunoprecipitating with endogenous CAPNS1 in HT1080 cell lysates were analyzed by Mass Spectrometry. We identified novel partners among which the deubiquitinating enzyme USP1, a key regulator of the DNA damage response and genome integrity maintenance via its specific action on FANCD2, involved in DNA repair and protection from chromosome instability, and PCNA, involved in the regulation of translesion DNA synthesis (TLS), that bypasses DNA lesions with low stringency basepairing requirements. We performed co-IP assays in lysates of 293T cells and confirmed that the interaction was specific. Furhermore, we observed that calpain is able to bind a USP1 C-terminal deleted mutant, suggesting that USP1 first 523 aminoacids were sufficient for the binding. To understand what is the effect exerted by calpain upon USP1, we depleted calpain activity in a series of cell lines, and followed the fate of endogenous USP1. We transfected CAPNS1 specific siRNAs, or treated cells with a specific inhibitor of calpain, and we observed a strong decrease in USP1 protein levels. This effect should be at a post-transcriptional level, since any significant change in USP1 mRNA levels is detected. We also obtained the same result by transfecting a siRNA specific for CAPN1, the gene encoding for the catalytic subunit micro-calpain. Moreover, we studied the role of calpain in the PCNA-mediated switch between high fidelity replication and TLS upon UV irradiation. In mouse embryonic fibroblasts knockout for CAPNS1, USP1 downregulation is coupled to an increase in PCNA monoubiquitination. Moreover, CAPNS1-depleted U2OS cells showed an increase in the percentage of nuclei containing PCNA-induced foci upon UV irradiation. Since we demonstrated that calpain can modulate an important regulator of DNA damage response such as USP1, we investigated if calpain could have a role in genome integrity maintenance. CAPNS1 depleted cells showed a reduced rescue in DNA repair compared to control cells, suggesting that increased levels in PCNA monoubiquitination could lead to an increased amount of errore-prone TLS. Calpain plays an important role in autophagy, so we asked if USP1 degradation in absence of calpain activity could involve autophagic pathways. We first blocked macroautophagy by silencing ATG5, and we observed that USP1 was downregulated, suggesting that the depletion of ATG5 could lead to an increased activity of other degradation pathways. To impaire chaperone-mediated autophagy (CMA), we silenced a protein important for autophagosome formation, LAMP-2A. Also in this case we observed a decrease in USP1 protein levels, thus suggesting that USP1 is alternatively degraded by different pathways. However, we observed that USP1 is stabilized upon inhibition of lysosomal enzymes, suggesting that USP1 may be degraded in the lysosome. To better understand the mechanism by which calpain affect USP1 stability we search for an effect of calpain upon USP1 co-factor and activator UAF1/WDR48. CAPNS1-depleted cells showed WDR48 downregulation, but WDR48 overexpression only partially rescue USP1 protein levels in this cells. Furthermore, we provided evidences that calpain regulation of p35/p25 activator of Cdk5 can affect Cdh1 phosphorylation and thus APC/Cdh1 activity, leading to a regulation of USP1 stabilization. In conclusion, we identified USP1 as a novel interactor of calpain, and we found that calpain is important for USP1 stability, since in its absence USP1 is downregulated. The importance of this novel regulation is strengthened by the recent findings that unveiled a role of USP1 in maintenance of a mesenchymal stem cell program in osteosarcoma, and thus placing calpain in a crucial regulatory position for cancer development.
XXIV Ciclo
1983

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A S?ndrome de c?ncer de mama e ov?rio heredit?rio corresponde a 10-15% de todos os casos diagnosticados de c?ncer de mama no mundo. A maioria das muta??es germinativas s?o identificadas nos genes BRCA1 e BRCA2, contudo, a aplica??o de pain?is multig?nicos tem aumentado o n?mero de variantes patog?nicas detectadas em outros genes supressores de tumor. De acordo com a vers?o atual do protocolo americano NCCN (National Comprehensive Cancer Network), as muta??es em BRCA1 e BRCA2, TP53 e PTEN conferem alto risco de desenvolver c?ncer de mama, e muta??es em CDH1, CHEK2, PALB2, ATM e BRIP podem aumentar em 20% o risco para o desenvolvimento desta doen?a. Neste estudo foram analisados 157 indiv?duos com hist?rico pessoal e/ou familiar de c?ncer de mama. O DNA gen?mico foi isolado a partir de sangue perif?rico por meio de extra??o ? base de solu??o salina e as amostras foram analisadas usando o sequenciamento de nova gera??o (NGS). Foram identificadas 15 variantes patog?nicas e 4 VUS (Variants of Uncertain Significance) em 27 indiv?duos (27/157; 17%), dos quais tr?s s?o assintom?ticos. Foram identificadas sete novas variantes em 4 genes: BRCA1_c.3409A>G; BRCA2_g.26826_30318del, BRCA2_c.5800C>T; BRCA2_c.5228G>A; BRCA2_c.5305delG; ATM_c.634delT e ATR_c.3043C>T. Sessenta e oito por cento (13/19; 68%) de variantes foi detectada nos genes BRCA1 e BRCA2, enquanto 32% (6/19) foram identificados nos genes de risco moderado ATM (2/19); ATR (1/19); CDH1 (1/19); MLH1 (1/19) e MSH6 (1/19). Os indiv?duos foram separados em dois grupos para a an?lise comparativa: portadores de muta??o nos genes de alto risco e nos genes de risco moderado. Entre os tr?s indiv?duos assintom?ticos, duas variantes est?o presentes nos genes de risco moderado ATM e MLH1. Entre os indiv?duos com c?ncer de mama, dezoito pacientes (18/24; 75%) apresentaram muta??es em genes de alto risco, enquanto seis (6/24; 25%) s?o portadores de muta??es em genes de risco moderado. Ambos os grupos apresentaram alta incid?ncia de c?ncer de mama precocemente (83% dos indiv?duos). O grupo de portadores de muta??o nos genes de alto risco apresentaram maior ocorr?ncia de tumores de alto grau (83% vs 67%, P = 0,0090). No grupo de indiv?duos com muta??es em genes de risco moderado, os tumores apresentaram um fen?tipo mais agressivo com c?ncer bilateral (33% versus 11%, P = 0,0002), ocorr?ncia de met?stases (33% vs 5,6%, P <0,0001) e ?bito (33% vs 5,6%, P <0,0001). Ao todo, 1/3 de variantes foram identificadas em genes de risco moderado em pacientes com c?ncer mais agressivo. Estes resultados refor?am a import?ncia da aplica??o de an?lise multig?nica em indiv?duos em situa??o de risco para c?ncer de mama, especialmente em uma popula??o heterog?nea como brasileira.
Hereditary breast and ovarian cancer (HBOC) corresponds to 10-15% of all diagnosed cases of breast cancer in the world. The majority germline mutations are identified in BRCA1 and BRCA2 genes, however the application of multigene panels has increased the number of pathogenic variations detected in DNA repair genes. According to the current version of NCCN (National Comprehensive Cancer Network) Guideline, mutations in BRCA1, BRCA2, TP53 and PTEN confers high risk to develop breast cancer, and mutations in CDH1, CHEK2, PALB2, ATM and BRIP can increases over than 20% this risk. We analyzed 157 individuals with personal and/or familial breast cancer history. Genomic DNA was isolated from peripheral blood through saline-based extraction and samples were analyzed using next-generation sequencing (NGS). We identified 15 pathogenic variants and 4 VUS (Variants of Uncertain Significance) in 27 individuals (27/157; 17%), in which three are asymptomatic. Seven novel variants in 4 genes were identified: BRCA1_c.3409A>G; BRCA2_g.26826_30318del, BRCA2_c.5800C>T; BRCA2_c.5228G>A; BRCA2_c.5305delG; ATM_c.634delT and ATR_c.3043C>T. Sixty-eight percent (13/19; 68%) of variants was detected in BRCA1 and BRCA2 genes, while 32% (6/19) were identified in moderate risk genes ATM (2/19); ATR (1/19); CDH1 (1/19); MLH1 (1/19) and MSH6 (1/19). The individuals were separated in two groups for comparative analysis: high-risk genes and moderate risk genes. Among three asymptomatic individuals, two present variants in moderate risk genes ATM and MLH1. Among breast cancer individuals, eighteen patients (18/24; 75%) presented mutations in high-risk genes, while six (6/24; 25%) harbored mutations in moderate risk genes. Both groups had a high incidence of early-onset breast cancer, 83%. The group of individuals harboring variants in high-risk genes presented a greater occurrence of high-grade tumors (83% vs. 67%, P= 0.0090). In the group of individuals harboring mutation in moderate risk genes, tumors presented a more aggressive phenotype with bilateral cancer (33% vs. 11%, P= 0.0002), occurrence of metastasis (33% vs. 5.6%, P<0.0001) and incidence of deaths (33% vs. 5.6%, P<0.0001). Altogether, 1/3 of variants were identified in moderate risk genes in patients presenting a more aggressive phenotype. These results reinforce the importance of applying multigene analysis in individuals at-risk for breast cancer, especially in a heterogeneous population as Brazilian.

Candida albicans is an important fungal pathogen of humans, and its ability to switch between different cell morphologies, including yeast, pseudohyphae and hyphae, is critical for virulence. The cell cycle plays an important, yet poorly understood, role in regulating cellular morphogenesis. Our previous work demonstrated that blocking the yeast cell cycle in mitosis, through depletion of the polo-like kinase Cdc5p, resulted in polarized growth of the yeast bud, producing filamentous cells that were distinct from pseudohyphae or true hyphae. Polarized growth was partially dependent on the spindle checkpoint factor Bub2p, suggesting that checkpoint-activated growth occurs in C. albicans . In contrast, similar checkpoint activation leads to cessation of cell proliferation in most other systems. In order to elucidate how mitotic progression and spindle checkpoints are linked to morphogenesis, a better understanding of the basic regulation of mitosis is required. To this end, we characterized homologues of Cdc20p and Cdh1p, which are targets of the spindle checkpoint and activators of the ubiquitin ligase anaphase-promoting complex (APC), a major regulator of mitotic progression in most systems. Cdc20p and Cdh1p were important for the metaphase-to-anaphase transition and mitotic exit, similar to their counterparts in S. cerevisiae , but strongly influenced morphogenesis in a different manner. Deletion of CDH1 resulted in a pleiotropic phenotype, including some enlarged yeast cells, while absence of the same factor in S. cerevisiae produced small cells. Absence of Cdc20p produced highly polarized yeast buds that resembled Cdc5p-depleted cells, in contrast to the large doublets resulting from loss of Cdc20p in S. cerevisiae . Overexpression of CDC20 in Cdc5p-depleted cells partially suppressed the cell cycle defect, suggesting that Cdc20p may be downstream of Cdc5p. However, polarized growth was not abolished. Deletion of the putative Cdc20p-binding and spindle checkpoint factor Mad2p partially compromised polarized growth in Cdc5p-depleted cells, implying that Mad2p may play a role in linking Cdc5p function with Cdc20p. Deletion of CDH1 had no effect on Cdc5p-dependent polarization, suggesting that Cdh1p is dispensible for the process. While Cdc20p was not required for serum-induced hyphal growth, some cells lacking Cdh1p were not able to form hyphae, yet invaded agar more readily than control cells, suggesting a complex role for this factor in regulating polarized growth. Thus, we provide genetic evidence that Cdc20p and Cdh1p play important roles in regulating mitosis and morphogenesis in C. albicans , but in a different manner than their homologues in S. cerevisiae . Our results extend our knowledge of the regulatory circuit governing mitosis in C. albicans and the potential pathway underlying checkpoint-activated polarized growth.

In this chapter, besides its biomedical applications, the synthesis and properties of brushite were investigated. Brushite consists of two types of crystals, platy and needle-like, and their formation depends on the pH of the medium during precipitation. Platy crystals are formed in a slightly acidic medium, pH = 5, and needle-like crystals at a higher pH = 6.5–7. In this study, the monoclinic brushite crystals were synthesized using dissolution-precipitation reactions. It is found that the brushite crystal growth occurs mainly along the (020) crystallographic plane. The thermogravimetric analysis confirms the presence of the two structural water molecules, which decompose at a temperature range between 80 and 220°C. Brushite was used in the preparation of tetracalcium phosphate mineral, which is the powder component for calcium phosphate cement (CPC). CPC was subsequently prepared from TTCP and phosphate-based hardening solution. In vitro evaluation of the resultant CPC using Hanks’ Balanced Salt Solution results in the growth of nanofibrous crystals of Calcium-deficient hydroxyapatite (CDHA) layers on the surfaces of the CPC. The cultured CPC exhibits new connective tissues and throughout the CaP matrix.