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Background Trisomy 18 syndrome (Edwards syndrome, ES) is a type of aneuploidy caused by the presence of an extra chromosome 18. Aneuploidy is the leading cause of early pregnancy loss, intellectual disability, and multiple congenital anomalies. The research of trisomy 18 is progressing slowly, and the molecular characteristics of the disease mechanism and phenotype are still largely unclear. Results In this study, we used the commercial Chromium platform (10× Genomics) to perform sc-ATAC-seq to measure chromatin accessibility in 11,611 single umbilical cord blood cells derived from one trisomy 18 syndrome patient and one healthy donor. We obtained 13 distinct major clusters of cells and identified them as 6 human umbilical cord blood mononuclear cell types using analysis tool. Compared with the NC group, the ES group had a lower ratio of T cells to NK cells, the ratio of monocytes/DC cell population did not change significantly, and the ratio of B cell nuclear progenitor and megakaryocyte erythroid cells was higher. The differential genes of ME-0 are enriched in Human T cell leukemia virus 1 infection pathway, and the differential peak genes of ME-1 are enriched in apopotosis pathway. We found that CCNB2 and MCM3 may be vital to the development of trisomy 18. CCNB2 and MCM3, which have been reported to be essential components of the cell cycle and chromatin. Conclusions We have identified 6 cell populations in cord blood. Disorder in megakaryocyte erythroid cells implicates trisomy 18 in perturbing fetal hematopoiesis. We identified a pathway in which the master differential regulatory pathway in the ME-0 cell population involves human T cell leukemia virus 1 infection, a pathway that is dysregulated in patients with trisomy 18 and which may increase the risk of leukemia in patients with trisomy 18. CCNB2 and MCM3 in progenitor may be vital to the development of trisomy 18. CCNB2 and MCM3, which have been reported to be essential components of the cell cycle and chromatin, may be related to chromosomal abnormalities in trisomy 18.

Despite diagnostic and therapeutic advances, hepatocellular carcinoma (HCC) remains a leading cause of morbidity/mortality worldwide. This retrospective study investigates the isolated and combined mini-chromosome maintenance complex component 3 (MCM3) and glypican-3 (GPC3) immunohistochemical (IHC) expression in HCC. A novel HCC immunosubtyping model based on combined MCM3/GPC3 expression is introduced and tested in comparison with prognostic variables and survival outcomes. Seventy-six HCC patients who underwent hepatectomy were enrolled. After the collection of clinicopathological, laboratory, and 3-year-survival data, IHC was applied to HCC tissue microarray-prepared sections using anti-MCM3 and GPC3. IHC scoring divided HCCs as: MCM3-high and MCM3-low expression, GPC3-positive and GPC3-negative expression, and combined scoring model immunosubtypes: MCM3-high/GPC3-positive; MCM3-low/GPC3-positive; MCM3-high/GPC3-negative, and MCM3-low/GPC3-negative. Statistical and Kaplan-Meier survival analyses were performed using SPSS version 23. MCM3 was expressed in 84.2% of HCCs. MCM3-high HCCs (60.5%) were significantly associated with lack of tumor capsulation, portal vein thrombosis, high grades, advanced stages, and Child-Pugh Scores B and C (all P≤0.05), and had a tendency for multiplicity, metastasis, solid growth pattern, shorter overall survival (OS) and disease-free survival (DFS). GPC3-positve HCCs (56.6%) were significantly associated with multiplicity and higher alfa-fetoprotein (all P≤0.05) with a tendency for shorter OS and DFS. Among all isolated and combined-expression immunosubtypes, MCM3-high/GPC3-positive HCCs had the worst prognosis and the shortest OS and DFS whereas MCM3-low/GPC3-negative immunosubtype showed the best prognosis and had the longest OS and DFS. MCM3 is defined as diagnostic, prognostic marker, and potential therapeutic target in HCC. The novel MCM3/GPC3 immunosubtyping model provides prognostic indications and stratification criteria for patients with HCC.

Background: Salivary gland tumors bear uncanny characteristics of being different based on their morphological aspects rather than the presence of clear demarcation. This ambiguity in the spectrum from benign to malignant salivary gland neoplasms while categorizing the neoplasm is having inherent pitfalls. The present study was, therefore, designed to characterize benign and malignant salivary gland tumors based on their proliferative indices. Materials and Method: Study samples comprised of 97 cases of histopathologically confirmed benign and malignant salivary gland tumors. The cases were immunohistochemically assessed for MCM3 and Ki-67 expressions and the molecular characterization was performed based on the findings. Results: The majority of benign and malignant salivary gland tumors were from the parotid gland, (51.2%) and (42.4%), respectively. Overall mean labeling index of MCM3 was higher i.e., (5.60 ± 3.99) in comparison to Ki-67 i.e., (2.82 ± 3.14) with P = 0.05 using paired t-test. Besides, malignant salivary gland neoplasms represented a higher mean score of MCM3 and Ki-67 than benign neoplasms. Conclusion: The requirement of a novel marker has led to the use of MCM3 which has a characteristic role in the entire spectrum of the cell cycle. The present study highlighted the extrapolation of MCM3 over Ki-67 for diagnosis and for true characterization of biologic behavior of salivary gland pathologies which may, in turn, influence the treatment modality employed for such lesions.

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan–Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81–0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36–0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

The minichromosome maintenance complex 3 (MCM3) is essential for the regulation of DNA replication and cell cycle progression. However, the expression and prognostic values of MCM3 in cervical cancer (CC) have not been well-studied. Herein, we investigated the expression patterns and survival data of MCM3 in cervical cancer patients from the ONCOMINE, GEPIA, Human Protein Atlas, UALCAN, Kaplan-Meier Plotter, and LinkedOmics databases. The expression level of MCM3 is negatively correlated with advanced tumor stage and metastatic status. Specifically, MCM3 is significantly differentially expressed between patients in stage 1 and stage 3 cervical cancer with p value 0.0138. Similarly, the p values between stage 1 and stage 4 cervical cancer, between stage 2 and stage 3, and between stage 2 and stage 4 are 0.00089, 0.0244, and 0.00197, respectively. Not only that, cervical cancer patients with high mRNA expression of MCM3 may indicate longer overall survival but indicate shorter relapse-free survival. PRIM2 and MCM6 are positively correlated genes of MCM3. Bioinformatics analysis revealed that MCM3 might be considered a biological indicator for prognostic evaluation of cervical cancer. However, it is currently limited to bioinformatics analysis, and more clinical tissue specimens and cell experiments are needed to further explore the role of MCM3 in the occurrence and progression of cervical cancer.

Uterine fibroids are the most common mesenchymal uterine neoplasms, their prevalence is estimated in 40%–60% of women under 35 and in 70%–80% of women over 50 years of age. The current research aims to focus on the etiopathogenesis of uterine fibroids, the factors that affect their growth, and markers with diagnostic and prognostic properties. The MCM (minichromosome maintenance) protein family consists of peptides whose primary function is participation in the molecular mechanism of creating replication forks while regulating DNA synthesis. The aim of this work was to determine the proliferative potential of uterine fibroid cells based on the expression of the Ki-67 antigen and the MCMs—i.e., MCM-3, MCM-5, and MCM-7. In addition, the expression of estrogen (ER) and progesterone (PgR) receptors was evaluated and correlated with the expression of the abovementioned observations. Ultimately, received results were analyzed in terms of clinical and pathological data. Materials and methods: In forty-four cases of uterine fibroids, immunohistochemical reactions were performed. A tissue microarray (TMA) technique was utilized and analyzed cases were assessed in triplicate. Immunohistochemistry was performed using antibodies against Ki-67 antigen, ER, PgR, MCM-3, MCM-5, and MCM-8 on an automated staining platform. Reactions were digitalized by a histologic scanner and quantified utilizing dedicated software for nuclear analysis. Assessment was based on quantification expression of the three histiospots, each representing one case in TMA. Results: In the study group (uterine fibroids), statistically significant stronger expression of all the investigated MCMs was observed, as compared to the control group. In addition, moderate and strong positive correlations were found between all tested proliferative markers. The expression of the MCM-7 protein also correlated positively with ER and PgR. With regard to clinical and pathological data, there was a negative correlation between the expression of MCMs and the number of both pregnancies and births. Significant reductions in MCM-5 and MCM-7 expression were observed in the group of women receiving oral hormonal contraceptives, while smoking women showed an increase in MCM-7, ER, and PgR. Conclusions: Uterine fibroid cells have greater proliferative potential, as evaluated by expression of the Ki-67 antigen and MCMs, than unaltered myometrial cells of the uterine corpus. The expression of MCM-7 was found to have strong or moderate correlations in all assessed relations. In the context of the clinical data, as well evident proliferative potential of MCMs, further studies are strongly recommended.

Basic transcription factor 3 (BTF3) is associated with the development of several cancers. The aim of our study was to elucidate the role of BTF3 in colorectal cancer (CRC) tissues. CRC tissues or their paired adjacent noncancerous (ANCT) tissues were obtained from 90 patients who underwent operations in our hospital from November 2011 to December 2016, and then we implemented a gene microarray assay for detecting significant changes in gene expression and confirmed expression in tissues using immunohistochemistry and real-time PCR. We transfected or injected the silencing BTF3 (BTF3-siRNA) plasmid into cells and nude mice, and measured the tumorigenicity of CRC cells with flow cytometry and studied the expression level of BTF3 downstream genes (MAD2L2, MCM3 and PLK1) in CRC cells. BTF3 expression level was not only significantly higher in CRC tissue than in ANCT tissue (2.61 ± 0.07 vs 1.90 ± 0.03, P

Background: The adult mammalian cardiomyocytes lose their proliferative capacity, which is responsible for cardiac dysfunction and heart failure following injury. The molecular mechanisms underlying the attenuation of adult cardiomyocyte proliferation remain largely unknown. Because long noncoding RNAs (lncRNAs) have a critical role in the development of cardiovascular problems, we investigated whether lncRNAs have any role in the regulation of cardiomyocyte proliferation and cardiac repair. Methods: Using bioinformatics and initial analysis, we identified an lncRNA, named CPR (cardiomyocyte proliferation regulator), that has a potential regulatory role in cardiomyocyte proliferation. For in vivo experiments, we generated CPR knockout and cardiac-specific CPR-overexpressing mice. In isolated cardiomyocytes, we used adenovirus for silencing (CPR–small interfering RNA) or overexpressing CPR. To investigate the mechanisms of CPR function in cardiomyocyte proliferation, we performed various analyses including quantitative reverse transcription–polymerase chain reaction, Western blot, histology, cardiac function (by echocardiography), transcriptome analyses (microarray assay), RNA pull-down assay, and chromatin immunoprecipitation assay. Results: CPR level is comparatively higher in the adult heart than in the fetal stage. The silencing of CPR significantly increased cardiomyocyte proliferation in postnatal and adult hearts. Moreover, CPR deletion restored the heart function after myocardial injury, which was evident from increased cardiomyocyte proliferation, improvement of myocardial function, and reduced scar formation. In contrast, the neonatal cardiomyocyte proliferation and cardiac regeneration were remarkably suppressed in CPR-overexpressing mice or adeno-associated virus serotype 9–CPR—overexpressing heart. These results indicate that CPR acts as a negative regulator of cardiomyocyte proliferation and regeneration. Next, we found that CPR targets minichromosome maintenance 3, an initiator of DNA replication and cell cycle progression, to suppress cardiomyocyte proliferation. CPR silenced minichromosome maintenance 3 expression through directly interacting and recruiting DNMT3A to its promoter cysteine-phosphate-guanine sites, as evident from decreased minichromosome maintenance 3 promoter methylation and increased minichromosome maintenance 3 expression in CPR knocked-down cardiomyocytes and CPR knockout mouse heart. These results were confirmed in CPR-overexpressing cardiomyocytes and CPR-overexpressing mouse heart. Conclusions: Together, our findings identified that CPR is a suppressor of cardiomyocyte proliferation and indicated that lncRNAs take part in the regulation of cardiomyocyte proliferation and cardiac repair. Our study provides an lncRNA-based therapeutic strategy for effective cardiac repair and regeneration.

Background/aim Morphological features, combined with Ki-67 proliferative index, remain the standard for discriminating benign and malignant adrenocortical tumors. The aim of this study was to evaluate the role of minichromosome maintenance proteins MCM-3, MCM-5, MCM-7, and Ki-67 as proliferative markers in adrenocortical tumors. Materials and methods Specimens of 81 adrenocortical adenomas and 3 adrenocortical carcinomas were stained with antibodies against MCM-3, 5, 7 and Ki-67. Results Malignant tumors were characterized by a greater size (p=0.017), volume (p=0.017), and higher levels of Ki-67 (p=0.005), MCM-3 (p=0.005), MCM-7 (p=0.008), but not MCM-5 (p=0.069). The markers' levels were independent from the tumors' size and volume, the patient's age and hormonal status. ROC curves showed Ki-67 (AUC 0.984), MCM-3 (AUC 0.984), and MCM-7 (AUC 0.950), but not MCM-5 (AUC 0.820) to be reliable markers. Conclusion Ki-67, MCM-3, and MCM-7, but not MCM-5 are reliable proliferative and diagnostic markers in discerning benign and malignant adrenocortical tumors.

DNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is replicated only once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both the initiation and elongation of DNA replication. Although only two DNA helicase activities are necessary to establish a bidirectional replication fork from each replication origin, a large excess of MCM complexes is amassed and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most are displaced from the DNA during the S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of a reduction in MCM complex. The alteration of MCM proteins induced a change in the activation of key factors of the DDR in response to Etoposide treatment. Etoposide-induced DNA damage affected the phosphorylation of γ-H2AX, CHK1 and CHK2 without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we identified a decrease in both HR and NHEJ associated with a decrease in MCM complex.

Human Pin1 is a peptidyl prolyl cis/trans isomerase with a unique preference for phosphorylated Ser/Thr-Pro substrate motifs. Here we report that MCM3 (minichromosome maintenance complex component 3) is a novel target of Pin1. MCM3 interacts directly with the WW domain of Pin1. Proline-directed phosphorylation of MCM3 at S112 and T722 are crucial for the interaction with Pin1. MCM3 as a subunit of the minichromosome maintenance heterocomplex MCM2-7 is part of the pre-replication complex responsible for replication licensing and is implicated in the formation of the replicative helicase during progression of replication. Our data suggest that Pin1 coordinates phosphorylation-dependently MCM3 loading onto chromatin and its unloading from chromatin, thereby mediating S phase control.

Sleeve gastrectomy (SG) is the most widely used bariatric procedures globally, which could improve glucose and lipid metabolism dramatically. Circular RNAs (circRNAs) are being increasingly implicated in numerous pathophysiological processes. However, for diabetes mellitus (DM), the expression and function of circRNAs remain largely undetermined, in particular, whether circRNAs mediate the amelioration of DM observed after SG. Using a diabetic rat model, we subjected liver tissue from SG and sham-operated rats to RNA sequencing. Amongst the 103 differentially regulated circRNAs identified in diabetic rats after SG, we focused on circDOCK7, a highly expressed circRNA derived from the back-splicing of the DOCK7 gene. Silencing of circDOCK7 significantly inhibited cellular proliferation and induction of apoptosis in insulin-resistant rat hepatocytes. Further analysis indicated circDOCK7 harbored binding sites for miR-139-3p and regulated the expression of minichromosome maintenance 3 (MCM3) through sequestration of miR-139-3p. Our findings therefore demonstrate a novel regulatory pathway involving circDOCK7 that regulates cellular proliferation and apoptosis through increasing the expression of MCM3. Overall, our study establishes a list of specific circRNAs expressed in diabetic rat liver after SG including circDOCK7 which serve as potential biomarkers and treatment targets for DM patients.

Hepatocellular carcinoma (HCC) is a common malignant tumor with insidious onset and rapid progression. Its treatment is often difficult owing to tumor resistance. In this study, we aimed to understand the different biological characteristics, gene expression profiles, and drug resistance mechanisms of HCC cells cultured under different conditions. A conventional adherence method and a liquid overlay technique were used to prepare two‐ and three‐dimensional cultures of Bel‐7402 and 5‐fluorouracil (5‐Fu)‐resistant Bel‐7402 (Bel‐7402/5‐Fu) cells. Morphological characteristics were assessed via microscopy, and cell cycle distribution and apoptotic rate were obtained using flow cytometry. Cell sensitivity to different concentrations of drugs was detected with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays. Gene expression profiles and signal transduction pathways of Bel‐7402 and Bel‐7402/5‐Fu cells under different culture conditions were determined using gene chips. Cells in three‐dimensional culture were suspended and they grew into dense multicellular spheroid (MCS) structures, aggregating with each other. In contrast to cells in the two‐dimensional culture, cell cycle arrest was observed in MCSs. The sensitivity of Bel‐7402 cells in the two‐dimensional culture to drugs at high concentrations was significantly higher than that of cells in the three‐dimensional culture (p, Biological features, gene expression profile, and mechanisms of drug resistance of two‐ and three‐dimensional hepatocellular carcinoma cell cultures.

Contains fulltext : 237838.pdf (Publisher’s version ) (Closed access) The MCM2-7 helicase is a heterohexameric complex with essential roles as part of both the pre-replication and pre-initiation complexes in the early stages of DNA replication. Meier-Gorlin syndrome, a rare primordial dwarfism, is strongly associated with disruption to the pre-replication complex, including a single case described with variants in MCM5. Conversely, a biallelic pathogenic variant in MCM4 underlies immune deficiency with growth retardation, features also seen in individuals with pathogenic variants in other pre-initiation complex encoding genes such as GINS1, MCM10, and POLE. Through exome and chromium genome sequencing, supported by functional studies, we identify biallelic pathogenic variants in MCM7 and a strong candidate biallelic pathogenic variant in MCM3. We confirm variants in MCM7 are deleterious and through interfering with MCM complex formation, impact efficiency of S phase progression. The associated phenotypes are striking; one patient has typical Meier-Gorlin syndrome, whereas the second case has a multi-system disorder with neonatal progeroid appearance, lipodystrophy and adrenal insufficiency. We provide further insight into the developmental complexity of disrupted MCM function, highlighted by two patients with a similar variant profile in MCM7 but disparate clinical features. Our results build on other genetic findings linked to disruption of the pre-replication and pre-initiation complexes, and the replisome, and expand the complex clinical genetics landscape emerging due to disruption of DNA replication.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)

Background: The minichromosome maintenance (MCM) family, a core component of DNA replication, is involved in cell cycle process. Abnormal proliferation has been identified as a crucial process in the evolution of colorectal cancer (CRC). However, the roles of the MCM family in CRC remain largely unknown. Methods: Here, the expression, prognostic significance and functions of the MCM family in CRC were systematically analyzed through a series of online databases including CCLE, Oncomine, HPA, cBioPortal and cancerSEA. Results: We found all MCM family members were highly expressed in CRC, but only elevation of MCM3 expression was associated with poor prognosis of patients with CRC. Further in vitro and in vivo experiments were performed to examine the role of MCM3 in CRC. Analysis of CCLE database and qRT-PCR assay confirmed that MCM3 was overexpressed in CRC cell lines. Moreover, knockdown of MCM3 significantly suppressed transition of G1 to S phase in CRC cells. Furthermore, down-regulation of MCM3 inhibited CRC cell proliferation, migration, invasion and promoted apoptosis. Conclusion: These findings reveal that MCM3 may function as an oncogene and a potential prognosis biomarker. Thus, the association between abnormal expression of MCM3 and the initiation of CRC deserves further exploration.

Accurate regulation of activity and level of the MCM complex is critical for precise DNA replication and genome transmission. Cyclin-dependent kinase (CDK) negatively regulates nuclear localization of the MCM complex via phosphorylation of the Mcm3 subunit. More recently, we found that Mcm3 is degraded via the Skp1–Cullin–F-box (SCF)–proteasome axis in budding yeast. However, how Mcm3 degradation is regulated is largely unknown. Here, we show that CDK represses Mcm3 degradation. Phosphorylated Mcm3 was excluded from the nucleus, where SCF is predominantly located, although CDK-mediated phosphorylation itself generated a phosphodegron of Mcm3, stimulating the degradation of Mcm3 resident in the nucleus. Thus, CDK negatively regulated nuclear MCM levels by exclusion from the nucleus and degradation in the nucleus via Mcm3 phosphorylation. We will discuss the physiological importance of Mcm3 degradation.

Coordination of DNA replication and cellular redox homeostasis mechanisms is essential for the sustained genome stability due to the sensitivity of replicating DNA to oxidation. However, substantial gaps remain in our knowledge of underlying molecular pathways. In this study, we characterise the interaction of Keap1, a central antioxidant response regulator in Metazoa, with the replicative helicase subunit protein MCM3. Our analysis suggests that structural determinants of the interaction of Keap1 with its critical downstream target - Nrf2 master transactivator of oxidative stress response genes – may have evolved in evolution to mimic the conserved helix-2-insert motif of MCM3. We show that this has led to a competition between MCM3 and Nrf2 proteins for Keap1 binding, and likely recruited MCM3 for the competitive binding dependent modulation of Keap1 controlled Nrf2 activities. We hypothesise that such mechanism could help to adjust the Keap1-Nrf2 antioxidant response pathway according to the proliferative and replicative status of the cell, with possible reciprocal implications also for the regulation of cellular functions of MCM3. Altogether this suggests about important role of Keap1-MCM3 interaction in the cross-talk between replisome and redox homeostasis machineries in metazoan cells.

Background: Current studies indicate irisin role in carcinogenesis. The aim of the study was to investigate the expression of irisin in LSCCs and to determine its association with clinicopathological factors, as well as recognized markers of proliferation, i.e., Ki-67 and MCM3,5,7 and MT-I/II proteins. Material and methods: The research material consisted of 140 cases of LSCCs, 57 cases of laryngeal papillomas (BLs) and 14 controls (benign hypertrophic changes). Tissue microarrays were used to perform IHC. Western blot and immunofluorescence were performed in laryngeal cancer cell lines and normal keratinocytes. Results: Irisin expression levels were significantly increased in LSCC compared to BLs (p < 0.0001) and controls (p = 0.001). We noted a positive moderate and weak correlation between irisin and Ki-67, MCM3 and MT-I/II. We observed an elevated level of irisin expression with increasing tumor size (T1–2 vs. T3–4; p = 0.0348). The levels of irisin were higher in N0 than in N1 and N2–3 (p = 0.0031 and p = 0.0457, respectively). Our in vitro study revealed a higher level of irisin in Larynx Epidermoid Carcinoma 2 (HEp-2) cells compared to the control Normal Human Keratinocyte (HaCat) cell line. Conclusions: Increased irisin expression levels in LSCC and its correlation with clinicopathological and proliferation factors may indicate the potential role of irisin as a biomarker in the diagnostic process of LSCC.

The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells.

// Paul A. Stewart 1, * , Zahraa I. Khamis 1, 4, * , Haiyen E. Zhau 2, * , Peng Duan 2 , Quanlin Li 2 , Leland W.K. Chung 2 and Qing-Xiang Amy Sang 1, 3 1 Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL, United States of America 2 Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, United States of America 3 Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, United States of America 4 Laboratory of Cancer Biology and Molecular Immunology, Department of Biochemistry, Faculty of Sciences, Lebanese University, Beirut, Lebanon * These authors have contributed equally to this work Correspondence to: Qing-Xiang Amy Sang, email: qxsang@chem.fsu.edu Haiyen E. Zhau, email: haiyen.zhau@cshs.org Keywords: epithelial-to-mesenchymal transition, minichromosome maintenance complex proteins, metastasis, androgen receptor, proteomics Received: September 06, 2016 Accepted: March 08, 2017 Published: April 05, 2017 ABSTRACT Metastasis is often associated with epithelial-to-mesenchymal transition (EMT). To understand the molecular mechanisms of this process, we conducted proteomic analysis of androgen-repressed cancer of the prostate (ARCaP), an experimental model of metastatic human prostate cancer. The protein signatures of epithelial (ARCaP E ) and mesenchymal (ARCaP M ) cells were consistent with their phenotypes. Importantly, the expression of mini-chromosome maintenance 3 (MCM3) protein, a crucial subunit of DNA helicase, was significantly higher in ARCaP M cells than that of ARCaP E cells. This increased MCM3 protein expression level was verified using Western blot analysis of the ARCaP cell lineages. Furthermore, immunohistochemical analysis of MCM3 protein levels in human prostate tissue specimens showed elevated expression in bone metastasis and advanced human prostate cancer tissue samples. Subcutaneous injection experiments using ARCaP E and ARCaP M cells in a mouse model also revealed increased MCM3 protein levels in mesenchymal-derived tumors. This study identifies MCM3 as an upregulated molecule in mesenchymal phenotype of human prostate cancer cells and advanced human prostate cancer specimens, suggesting MCM3 may be a new potential drug target for prostate cancer treatment.

// Dong-dong Cheng 1, * , Hui-zhen Zhang 2, * , Jun-qing Yuan 2, * , Shi-jie Li 1 , Qing-cheng Yang 1 , Cun-yi Fan 1 1 Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 200233, China 2 Department of Pathology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, 200233, China * Authors share co-first authorship Correspondence to: Cun-yi Fan, email: fancunyi888@hotmail.com Qing-cheng Yang, email: tjyqc@163.com Keywords: MCM2, MCM3, DHX9, proliferation, osteosarcoma Received: November 14, 2016 Accepted: February 07, 2017 Published: February 18, 2017 ABSTRACT A label free quantitative proteomic approach (SWATH™ experiment) was performed to identify tumor-associated nuclear proteins that are differentially expressed between osteosarcoma cells and osteoblast cells. By functional screening, minichromosome maintenance protein 2 (MCM2) and minichromosome maintenance protein 3 (MCM3) were found to be related to osteosarcoma cell growth. Here, we show that knockdown of MCM2 or MCM3 inhibits osteosarcoma growth in vitro and in vivo . In co-immunoprecipitation and co-localization experiments, MCM2 and MCM3 were found to interact with DExH-box helicase 9 (DHX9) in osteosarcoma cells. A rescue study showed that the decreased growth of osteosarcoma cells by MCM2 or MCM3 knockdown was reversed by DHX9 overexpression, indicating that MCM2 and MCM3 activity was DHX9-dependent. In addition, the depletion of DHX9 hindered osteosarcoma cell proliferation. Notably, MCM2 and MCM3 expression levels were positively correlated with the DHX9 expression level in tumor samples and were associated with a poor prognosis in patients with osteosarcoma. Taken together, these results suggest that the MCM2/MCM3–DHX9 axis has an important role in osteosarcoma progression.

Actinic prurigo (AP) is an idiopathic photodermatosis; the initial manifestations usually occur during the first decades of life but can appear at any age. Cases are usually diagnosed late once the lesions have exacerbated; due to the extensive involvement of the vermilion border and the etiology, it has been confused with and related to a potentially malignant process. Syndecan-1 and E-cadherin were positive in the epidermis, with moderate-to-intense staining in 100% of samples. Ki67 and MCM3 were expressed in the lower third of the epidermis and showed greater immunolabeling in samples that contained lymphoid follicles (Ki 67: epidermis [17.7% ± 6.79%] and dermis [7.73% ± 6.69%]; MCM3: epidermis [22.92% ± 10.12%] and dermis [6.13% ± 6.27%]). In conclusion AP is a disease in which there is no evidence that the lesions are potentially cancerous. AP cheilitis should not be confused with actinic cheilitis because they are separate entities.

Melanomas represent the malignant transformation of melanocytes, cells found primarily in the skin to protect epithelium and underlying connective tissues from harmful effects of ultraviolet light. Melanomas vary greatly in morphology and may continue to produce melanin markedly, mildly, or not at all. Performing and evaluating hematoxylin and eosin stains as well as immunohistochemical stains on pigmented melanomas has been a long-standing challenge due to the obscuring pigment. Protocols for removing melanin to reveal cellular morphology have been used successfully for years, but coupling these protocols with stains for immunohistochemistry represents an added challenge. In this study, the investigators evaluated results of various melanin bleaching protocols on tissue morphology, completeness of melanin removal, and immunohistochemistry staining quality. It was found that 1% formamide in 3% H2O2 under bright light without heating outperformed other tested protocols.

High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a “dam-and-diversion tunnel” mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation., The DNA duplex is known to be split apart in a steric exclusion manner during replication, but the specific mechanism has remained unclear. Here the authors present a cryo-EM structure of a eukaryotic replicative CMG helicase on forked DNA, revealing the mechanism of DNA unwinding.

Invasive ductal carcinoma (IDC) is the most common breast cancer. Our study used gene microarray data to select differentially expressed genes between normal and IDC mammary tissues. From these, we selected genes related to the proliferation of tumor cells and compared their prognostic value with known biomarker Ki67 for IDC. Analysis of publicly available Gene Expression Omnibus (GEO) data revealed 24 differentially expressed genes (DEGs) in normal and 31 DEGS in IDC tissues that were used for further analyses. Gene chip analysis software was used to identify DEGs. DEG profiles were confirmed using quantitative PCR (qPCR). DEG functions where shown to be related to cell proliferation. We confirmed MCM3 expression using immunohistochemical staining in 45 IDC patients. The relationship between MCM3 expression and survival was investigated using Kaplan-Meier survival curves and Cox proportional hazard regression models. A total of 1307 differentially expressed genes were identified between IDC and normal tissues, which were enriched in 32 Gene Ontology (GO) terms and 9 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. qPCR demonstrated that both COL1A1 and MCM3 were significantly up-regulated in IDC tissues, of which only MCM3 was related to cell proliferation. Ki67 is closely associated with the tumor grade, ER status, PR status and HER2 status, while MCM3 was shown to relate to tumor size, lymph node, and PR status. There was significant association between survival and MCM3, but not for Ki67. High MCM3 expression demonstrated statistically significant associations with poor prognosis in IDC patients. Findings from the gene microarray data analysis confirmed that MCM3 is associated with the response to cell proliferation. MCM3 represents a better proliferation marker than Ki67 making it a valuable prognostic tool that is independent of ER and HER2 status.

Cellular aging is characterized by the loss of DNA replication capability and is mainly brought about by various changes in chromatin structure. Here, we examined changes in MCM2-7 proteins, which act as a replicative DNA helicase, during aging of human WI38 fibroblasts at the single-cell level. We used nuclear accumulation of p21 as a marker of senescent cells, and examined changes in MCM2-7 by western blot analysis. First, we found that senescent cells are enriched for cells with a DNA content higher than 4N. Second, the levels of MCM2, MCM3, MCM4 and MCM6 proteins decreased in senescent cells. Third, cytoplasmic localization of MCM2 and MCM7 was observed in senescent cells, from an analysis of MCM2-7 except for MCM5. Consistent with this finding, fragmented MCM2 was predominant in these cells. These age-dependent changes in MCM2-7, a protein complex that directly affects cellular DNA replication, may play a critical role in cellular senescence.

Minichromosome maintenance 3 (MCM3) protein has been widely studied due to its essential role in DNA replication. In addition, it is overexpressed in several human tumor types. However, the role of this protein in renal cell carcinoma (RCC) is not widely known. In this study, we demonstrated that polo-like kinase 1 (PLK1)-mediated MCM3 phosphorylation regulates proliferation and apoptosis in RCC. Our results confirm that PLK1 and phospho-MCM3 (p-MCM3) are highly expressed in renal cell carcinoma. The expression of PLK1 is closely related to the clinical characteristics of renal cell carcinoma. They play important roles in the proliferation and apoptosis of RCC. In vitro, after overexpression of PLK1 or MCM3, the proliferation of RCC cells was significantly enhanced and cell apoptosis was inhibited, while after knockout, the proliferation of RCC cells was weakened and cell apoptosis was promoted. In addition, Mn2+-Phos-tag SDS-PAGE, western blotting, and immunofluorescence were utilized to determine that MCM3 is a physiological substrate of PLK1, which is phosphorylated on serine 112 (Ser112) in a PLK1-dependent manner. PLK1-mediated MCM3 phosphorylation promotes RCC cell cycle proliferation and suppresses apoptosis in vitro. Moreover, we found that PLK1-mediated MCM3 phosphorylation induced cellular proliferation and decreased apoptosis, as well as tumor growth in mice. Overall, we conclude that PLK1-mediated MCM3 phosphorylation is a novel mechanism to regulate RCC proliferation and apoptosis.

KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro. Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2–7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2–7 complex. KEAP1 is thus poised to affect MCM2–7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.

Pleomorphic adenoma, the most common benign nonvascular tumor of the parotid gland in juveniles, should be differentiated from other extremely rare tumors, including schwannoma. In this article, we present a rare case of an intraparotid schwannoma in a juvenile, along with the patient history, a description of pathological features, and the results of ultrastructural and immunohistochemical examination. The respective labeling indexes of Ki-67 and MCM-3, i.e., the mean proportions of positive tumor cells out of 1000 tumoral cells counted in 10 microscopic fields at ×400 magnification, given as a percentage, were found to be 0.82% and 0.4%, respectively.

Aberrant DNA replication is one of the driving forces behind oncogenesis. Furthermore, minichromosome maintenance complex component 3 (MCM3) serves an essential role in DNA replication. Therefore, in the present study, the diagnostic and prognostic value of MCM3 and its interacting proteins in hepatocellular carcinoma (HCC) were investigated. By utilizing The Cancer Genome Atlas (TCGA) database, global MCM3 mRNA levels were assessed in HCC and normal liver tissues. Its effects were further analyzed by reverse transcription-quantitative PCR (RT-qPCR), western blotting and immunohistochemistry in 78 paired HCC and adjacent tissues. Functional and pathway enrichment analyses were performed using the Search Tool for the Retrieval of Interacting Genes database. The expression levels of proteins that interact with MCM3 were also analyzed using the TCGA database and RT-qPCR. Finally, algorithms combining receiver operating characteristic (ROC) curves were constructed using binary logistic regression using the TCGA results. Increased MCM3 mRNA expression with high α-fetoprotein levels and advanced Edmondson-Steiner grade were found to be characteristic of HCC. Survival analysis revealed that high MCM3 expression was associated with poor outcomes in patients with HCC. In addition, MCM3 protein expression was associated with increased tumor invasion in HCC tissues. MCM3 and its interacting proteins were found to be primarily involved in DNA replication, cell cycle and a number of binding processes. Algorithms combining ROCs of MCM3 and its interacting proteins were found to have improved HCC diagnosis ability compared with MCM3 and other individual diagnostic markers. In conclusion, MCM3 appears to be a promising diagnostic biomarker for HCC. Additionally, the present study provides a basis for the multi-gene diagnosis of HCC using MCM3.

The ATR-interacting protein (ATRIP) is responsible for the recognition of DNA damage-induced structure and regulation of cellular responses to DNA damage and replication stress. The purpose of our study was to identify the underlying mechanism with respect to chromatin loading and phosphorylation of ATRIP in mantle cell lymphoma (MCL).JeKo cells were used in our study. Differently tagged ATRIP (Myc-, hemaglutinin (HA) or Flag) and minichromosome maintenance (MCM) complex (MCM2, MCM3, MCM5, and MCM6) were transfected into 293T cells. After 48 h, ATRIP-interacting protein was identified by mass spectrometry (MS). Cell fractionation was done to localize proteins inside the cells. Immunoprecipitation (IP) and immunoblot (IB) analysis were used to identify immunoreactive species, and Glutathione S-transferase (GST) pull-down assays were performed to detect protein-protein interaction between ATRIP and MCM complex. After silencing the expression of MCM2 and MCM6 by short hairpin RNA (shRNA), chromatin fraction were analyzed. The expression of ATRIP phosphorylation (pS224-ATRIP) was determined after application of different doses of MCM2 shRNA (0.5 μg, 1 μg, and 2.5 μg).ATRIP directly interacts with MCM2, MCM3, MCM6, and MCM7 in JeKo cells. Downregulation of MCM2 and MCM6 significantly reduced ATRIP chromatin fraction. Downregulation of MCM2 statistically decreased the expression of ATRIP phosphorylation. The expression levels of pS224-ATRIP were regulated by MCM2 shRNA in a dose-dependent manner.Our results suggest that interaction between ATRIP and MCM complex is required for ATRIP chromatin loading and ATRIP phosphorylation.

Objective. To evaluate the association between upregulated differentially expressed genes (DEGs) and the outcomes of patients with hepatocellular carcinoma (HCC). Methods. Using Gene Expression Omnibus (GEO) datasets including GSE45436, GSE55092, GSE60502, GSE84402, and GSE17548, we detected upregulated DEGs in tumors. KEGG, GO, and Reactome enrichment analysis of the DEGs was conducted to clarify their function. The impact of the upregulated DEGs on patients’ survival was analyzed based on TCGA profile. Results. 161 shared upregulated DEGs were identified among GSE45436, GSE55092, GSE60502, and GSE84402 profiles. Cell cycle was the shared pathway/biological process in the gene sets investigation among databases of KEGG, GO, and Reactome. After being validated in GSE17548, 13 genes including BUB1B, CCNA2, CCNB1, CCNE2, CDC20, CDC6, CDC7, CDK1, CDK4, CDKN2A, CHEK1, MAD2L1, and MCM3 in cell cycle pathway were shared in the three databases for enrichment. The expression of BUB1B, CCNB1, CDC7, CDC20, and MCM3 was upregulated in HCC tissues when compared with adjacent normal tissues in 6.67%, 7.5%, 8.06%, 5.56%, and 9.72% of HCC patients, respectively. Overexpression of BUB1B, CCNB1, CDC7, CDC20, and MCM3 in HCC tissues accounted for poorer overall survival (OS) and disease-free survival (DFS) in HCC patients (all log rank P < 0.05). BUB1B, CCNB1, CDC7, CDC20, and MCM3 were all overexpressed in HCC patients with neoplasm histologic grade G3-4 compared to those with G1-2 (all P < 0.05). BUB1B, CCNB1, and CDC20 were significantly upregulated in HCC patients with vascular invasion (all P < 0.05). Additionally, levels of BUB1B, CCNB1, CDC7, and CDC20 were significantly higher in HCC patients deceased, recurred, or progressed (all P < 0.05). Conclusion. Correlated with advanced histologic grade and/or vascular invasion, upregulation of BUB1B, CCNB1, CDC7, CDC20, and MCM3 in HCC tissues predicted worse OS and DFS in HCC patients. These genes could be novel therapeutic targets for HCC treatment.

Small cell lung cancer (SCLC) is one of the highly malignant tumors and a serious threat to human health. The aim of the present study was to explore the underlying molecular mechanisms of SCLC. mRNA microarray datasets GSE6044 and GSE11969 were downloaded from Gene Expression Omnibus database, and the differentially expressed genes (DEGs) between normal lung and SCLC samples were screened using GEO2R tool. Functional and pathway enrichment analyses were performed for common DEGs using the DAVID database, and the protein-protein interaction (PPI) network of common DEGs was constructed by the STRING database and visualized with Cytoscape software. In addition, the hub genes in the network and module analysis of the PPI network were performed using CentiScaPe and plugin Molecular Complex Detection. Finally, the mRNA expression levels of hub genes were validated in the Oncomine database. A total of 150 common DEGs with absolute fold-change >0.5, including 66 significantly downregulated DEGs and 84 upregulated DEGs were obtained. The Gene Ontology term enrichment analysis suggested that common upregulated DEGs were primarily enriched in biological processes (BPs), including ‘cell cycle’, ‘cell cycle phase’, ‘M phase’, ‘cell cycle process’ and ‘DNA metabolic process’. The common downregulated genes were significantly enriched in BPs, including ‘response to wounding’, ‘positive regulation of immune system process’, ‘immune response’, ‘acute inflammatory response’ and ‘inflammatory response’. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified that the common downregulated DEGs were primarily enriched in the ‘complement and coagulation cascades’ signaling pathway; the common upregulated DEGs were mainly enriched in ‘cell cycle’, ‘DNA replication’, ‘oocyte meiosis’ and the ‘mismatch repair’ signaling pathways. From the PPI network, the top 10 hub genes in SCLC were selected, including topoisomerase IIα, proliferating cell nuclear antigen, replication factor C subunit 4, checkpoint kinase 1, thymidylate synthase, minichromosome maintenance protein (MCM) 2, cell division cycle (CDC) 20, cyclin dependent kinase inhibitor 3, MCM3 and CDC6, the mRNA levels of which are upregulated in Oncomine SCLC datasets with the exception of MCM2. Furthermore, the genes in the significant module were enriched in ‘cell cycle’, ‘DNA replication’ and ‘oocyte meiosis’ signaling pathways. Therefore, the present study can shed new light on the understanding of molecular mechanisms of SCLC and may provide molecular targets and diagnostic biomarkers for the treatment and early diagnosis of SCLC.

OBJECTIVE: To observe the effects of deguelin on the proliferation of breast cancer MCF-7 cells and lung cancer H1299 cells in vitro and the expression of minichromosome maintenance protein 3 (MCM3) and CDC45 in the cells. METHODS: MTT assay was used to evaluate the proliferation of MCF-7 and H1299 cells exposed to different concentrations of deguelin for 48, 72 or 96 h. The growth of the cells was observed microscopically and the changes of MCM3 and CDC45 expressions in MCF-7 and H1299 cells following deguelin treatment were detected with fluorescence quantitative PCR. RESULTS: The proliferation of MCF-7 cells was significantly inhibited by exposure to 0.25, 0.5, 1, 5, 10, 30, and 50 μmol/L deguelin for 48, 72, and 96 h in a concentration-and time-dependent manner. In MCF-7 cells, the IC(50) of deguelin at 48, 72, and 96 h was 9, 3, and 2 μmol/L, respectively. Deguelin treatments of H1299 cells at 0.5, 1, 5, 10, 30, 50, and 100 μmol/L also resulted in a concentration-and time-dependent inhibition of the cell growth with an IC(50) at 96 h of 2 μmol/L. Optical microscopy of the cells revealed a decreased number of viable cells with obvious cell shrinkage following deguelin treatments. The expression of MCM3 and CDC45 were significantly reduced in the cells after deguelin treatments. CONCLUSION: Deguelin can inhibit the proliferation of MCF-7 and H1299 cells in vitro and down-regulate the expression of MCM3 and CDC45 in the cells.

The aim of this study was to identify the expression of Ki-67 and MCM3 in oral squamous cell carcinoma (OSCC) as well as to address the correlation with patient survival and clinical features. Samples were collected from 51 patients with OSCC who presented for follow-up. Immunohistochemical expression of Ki-67 and MCM3 in all groups was performed. The scoring system was previous published by Tsurutani in 2005. We used Kappa index to evaluate observers agreement degree. The associations between protein expression and clinical variables were examined for statistical significance using the chi-squared test. The overall survival rates were estimated by the Kaplan-Meier method and the relationship between protein expression and survival was compared using the log-rank test (p < 0.05). The overall survival time for a patient with positive immunostaining for Ki-67 is shorter than for a patient with negative immunostaining, (log-rank test, p = 0.00882). Patients with tumor size T3 and T4 showed a statistically significant relationship with Ki-67 immunoexpression (log-rank test, p = 0.0174). The relationship between Ki-67 expression and the relation between age, gender, smoking, tumor site, lymph node metastasis and disease stage was not significant. The examiners agreement degree by Kappa presented p value < 0.05. There was not a significant correlation when we evaluated MCM3 expression regarding clinical characteristics and survival rate. From these results, the present study suggests that positive Ki-67 expression found in OSCC patients may contribute to predict the survival in OSCC samples, as well as the relation between the protein and the tumor size.

The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2-7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2-7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2-7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.

The expression of p53 has been studied not only in primary human ovarian carcinomas, but also in borderline ovarian tumors, however, the results were discordant. Expression patterns of proteins involved in cell proliferation and apoptosis have been investigated in various human neoplasms, including female genital tract neoplasms.The aim of this investigation was to assess the staining pattern and immunolocalization of p53 and selected proliferative markers (Ki-67, MCM3, PCNA, and topoisomerase IIα) in borderline ovarian tumors (BOTs).The study group consisted of 42 women who underwent pelvic surgery between 2006-2015. The median patients' age was 46 years. The immunoperoxidase technique was employed using antibodies against p53, Ki-67, MCM3, PCNA, and topoisomerase IIα.For p53, nuclear expression was observed in BOTs, however, cytoplasmatic immunoreactivity was also detected. Altogether, 25 (60%) tumors demonstrated positive p53 immunostaining, including overexpression found in 6 (14%). There were no significant differences in p53 expression between subgroups of clinicopathological variables. Immunoexpression of Ki-67, MCM3, PCNA, and topoisomerase IIα was nuclear. Ki-67 expression was positive in 12 (29%) cases and there was a trend towards a relationship between patients' age and Ki-67 staining (P=0.08). Interestingly, a significantly higher Ki-67 expression was found in tumors of ≥10 cm in diameter compared to smaller tumors (P=0.008). MCM3 expression was detected in 38 (90%) tumors, and PCNA expression in 28 (67%), yet none of clinicopathological factors was related to them. Topoisomerase IIα expression was present in 14 (33%) cases and, interestingly, its significantly higher expression was observed in BOTs of ≥10 cm in diameter compared to smaller tumors (P=0.008). Moreover, Spearman's correlation revealed highly significant positive associations between Ki-67 and topoisomerase IIα (R=0.403, P=0.008) and Ki-67 and MCM3 (R=0.469, P=0.001).We report a high positive immunostaining rate for p53, suggesting a role of TP53 alterations in the development of BOTs in humans. The new finding of higher topoisomerase IIα immunostaining positivity in BOTs of ≥10 cm may be clinically relevant and requires further studies on larger patient groups.

Cyclin G-associated kinase (GAK) harbors a consensus phosphorylation motif (Y412) for c-Src; however, its physiological significance remains elusive. Here, we show that GAK is phosphorylated by c-Src not only at Y412 but also at Y1149. An anti-GAK-pY412 antibody recognized the shifted band of GAK during M phase. Immunofluorescence (IF) showed that GAK-pY412/pY1149 signals were present in the nucleus during interphase, translocated to chromosomes at prophase and prometaphase, moved to centrosomes at metaphase, and finally translocated to chromosomes at the end of telophase, when nuclear membrane formation was almost complete. These subcellular movements of GAK resemble those of DNA licensing factors. Indeed, mass spectrometry identified mini-chromosome maintenance (MCM) 3, an essential component of the DNA licensing system, as one of the association partners of GAK; immunoprecipitation-mediated Western blotting confirmed their association in vivo. These results suggest that the c-Src_GAK_MCM axis plays an important role in cell cycle progression through control of the DNA replication licensing system.

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.

Embryonic cell cycles of amphibians are rapid and lack zygotic transcription and checkpoint control. At the mid-blastula transition, zygotic transcription is initiated and cell divisions become asynchronous. Several cell cycle-related amphibian genes retain 2 distinct forms, maternal and zygotic, but little is known about the functional differences between these 2 forms of proteins. The minichromosome maintenance (MCM) 2-7 complex, consisting of 6 MCM proteins, plays a central role in the regulation of eukaryotic DNA replication. Almost all eukaryotes retain just a single MCM gene for each subunit. Here we report that Xenopus and zebrafish have 2 copies of MCM3 genes, one of which shows a maternal and the other a zygotic expression pattern. Phylogenetic analysis shows that the Xenopus and zebrafish zygotic MCM3 genes are more similar to their mammalian MCM3 ortholog, suggesting that maternal MCM3 was lost during evolution in most vertebrate lineages. Maternal MCM3 proteins in these 2 species are functionally different from zygotic MCM3 proteins because zygotic, but not maternal, MCM3 possesses an active nuclear localization signal in its C-terminal region, such as mammalian MCM3 orthologs do. mRNA injection experiments in zebrafish embryos show that overexpression of maternal MCM3 impairs proliferation and causes developmental defects, whereas zygotic MCM3 has a much weaker effect. This difference is brought about by the difference in their C-terminal regions, which contain putative nuclear localization signals; swapping the C-terminal region between maternal and zygotic genes diminishes the developmental defects. This study suggests that evolutionary diversification has occurred in MCM3 genes, leading to distinct functions, possibly as an adaption to the rapid DNA replication required for early development of Xenopus and zebrafish.

// Jayabal Panneerselvam 1 , Anna Pickering 1 , Bing Han 1 , Liantao Li 1,2 , Junnian Zheng 2 , Jun Zhang 3 , Yanbin Zhang 4 , and Peiwen Fei 1 , 1 University of Hawaii Cancer Center, University of Hawaii, Honolulu, HI 2 Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, China 3 Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 4 Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL Correspondence: Peiwen Fei, email: // Keywords : Replication, FANCD2 Received : February 11, 2014 Accepted : March 11, 2014 Published : March 13, 2014 Abstract Normal DNA replication starts following the stepwise recruitment of replication initiators to assemble Mini-chromosome Maintenance (MCM) 2-7 protein complexes at an adequate amount of DNA replication origins. Under normal conditions, the monoubiquitination of Fanconi Anemia (FA) group D2 protein (FANCD2) occurs in each S-phase of cell cycle, which is the basal level of FANCD2 monoubiquitination. However, little is known regarding the roles of this basal level of monoubiquitinated FANCD2. Here we show that monoubiquitinated FANCD2 in each S-phase of normal cell cycle is essential for replication origins to fire at a normal rate. We found that the basal level of the monoubiquitinated FANCD2 can interact with replication origins as well as mini-chromosome maintenance protein 3 (MCM3) in an S-phase specific manner to secure an enough number of the licensed-origins to fire. Non-monoubiquitinated FANCD2 or mutant MCM3 lacking AA 477-480 responsible for interacting with FANCD2 can lead to an insufficient amount of licensed origins to fire and, thereby, enlarged intervals between the fired origins. Our results demonstrate that the monoubiquitinated FANCD2 in each S-phase of normal cell cycle is required to maintain an enough number of licensed origins to initiate the normal DNA replication. This finding is the first to provide insights into how FANCD2 functions under normal condition of cell cycle to maintain genome stability, as well as resulting implications in the strategic improvement for the fight against human cancer.

Background The aim of this study was to identify the expression of MCM3, Ki-67 and p27 in normal mucosa, leucoplakia and oral squamous cell carcinoma (OSCC) and determine whether altered expression could serve as a prognostic marker of a malignant progression of dysplastic lesions. Methods The samples were collected from 37 patients with oral leucoplakia (13 with mild dysplasia – MLD, 12 with moderate dysplasia – MD and 12 with severe dysplasia – SD). Eleven samples of mouth floor mucocele (M) and 50 floor mouth and tongue samples OSCC of untreated patients were included in this study. Immunohistochemical expression of MCM3, Ki-67 and p27 of all the groups was analysed. Kruskal–Wallis and Dunn's test were used to determine differences among groups, and a Pearson's correlation test was used to evaluate the correlation between the proteins. Results Ki-67 expression was higher in OSCC than M (P

Hepatocellular carcinoma (HCC) is the most common tumors in the worldwide, it develops resistance to radiotherapy during treatment, understanding the regulatory mechanisms of radioresistance generation is the urgent need for HCC therapy.qRT-PCR, western blot and immunohistochemistry were used to examine MCM3 expression. MTT assay, colony formation assay, terminal deoxynucleotidyl transferase nick end labeling assay and In vivo xenograft assay were used to determine the effect of MCM3 on radioresistance. Gene set enrichment analysis, luciferase reporter assay, western blot and qRT-PCR were used to examine the effect of MCM3 on NF-κB pathway.We found DNA replication initiation protein Minichromosome Maintenance 3 (MCM3) was upregulated in HCC tissues and cells, patients with high MCM3 expression had poor outcome, it was an independent prognostic factor for HCC. Cells with high MCM3 expression or MCM3 overexpression increased the radioresistance determined by MTT assay, colony formation assay, TUNEL assay and orthotopic transplantation mouse model, while cells with low MCM3 expression or MCM3 knockdown reduced the radioresistance. Mechanism analysis showed MCM3 activated NF-κB pathway, characterized by increasing the nuclear translocation of p65, the expression of the downstream genes NF-κB pathway and the phosphorylation of IKK-β and IκBα. Inhibition of NF-κB in MCM3 overexpressing cells using small molecular inhibitor reduced the radioresistance, suggesting MCM3 increased radioresistance through activating NF-κB pathway. Moreover, we found MCM3 expression positively correlated with NF-κB pathway in clinic.Our findings revealed that MCM3 promoted radioresistance through activating NF-κB pathway, strengthening the role of MCM subunits in the tumor progression and providing a new target for HCC therapy.