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Results for “Minichromosome Maintenance Complex Component 3”

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PubMed2003

Thymine-rich single-stranded DNA activates Mcm4/6/7 helicase on Y-fork and bubble-like substrates.

The presence of multiple clusters of runs of asymmetric adenine or thymine is a feature commonly found in eukaryotic replication origins. Here we report that the helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine stretches. The Mcm helicase is specifically activated by a synthetic bubble structure which mimics an activated replication origin, as well as by a Y-fork structure, provided that a single-stranded DNA region of sufficient length is present in the unwound segment or 3' tail, respectively, and that it carries clusters of thymines. Sequences derived from the human lamin B2 origin can serve as a potent activator for the Mcm helicase, and substitution of its thymine clusters with guanine leads to loss of this activation. At the fork, Mcm displays marked processivity, expected for a replicative helicase. These findings lead us to propose that selective activation by stretches of thymine sequences of a fraction of Mcm helicases loaded onto chromatin may be the determinant for selection of initiation sites on mammalian genomes.

Adenosine Triphosphatases↗

PubMedDate not supplied

Phyllodes tumor of the breast: EGFR family expression and relation to clinicopathological features.

The expression of EGFR family members was examined by immunohistochemistry in 22 phyllodes tumors, and the results were evaluated together with immunohistochemical findings for proliferation markers Ki67 and BM28, and the tumor suppressor gene product p53. Light and electron microscopy were performed in all cases. Clinical information was obtained from the medial records. We did find that expression of EG FR, c-erbB-3 and c-erbB-4 proteins could be detected in the neoplastic mesenchymal cells, and that the expression increased with increasing malignancy. Increased expressions of Ki67, BM28, p53 and EGFR family members in neoplastic cells were associated with malignancy and unfavorable clinical course. Furthermore, the expression of ER-alpha and PR in the epithelial cells of phyllodes tumors was increased compared to that in normal breast epithelium. Finally, the application of electron microscopy helped to identify a group of malignant tumors, revealing neoplastic cells with characteristic nuclear indentations, as well as an increasing number of myofibroblasts.

Adolescent↗

PubMed2006

A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice.

Mcm4 (minichromosome maintenance-deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2-MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4(Chaos3) encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4(-)) caused preimplantation lethality, Mcm(Chaos3/-) embryos died late in gestation, indicating that Mcm4(Chaos3) is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4(Chaos3/Chaos3) females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.

Adenocarcinoma↗

PubMed2003

Mammalian Mcm2/4/6/7 complex forms a toroidal structure.

BACKGROUND: The Mcm proteins are a family of six homologous proteins (Mcm2-7) that play an important role in DNA replication. They form Mcm4/6/7 and Mcm2/4/6/7 complexes, but their structures are not known. RESULTS: We found that the human Mcm2/4/6/7 tetramer forms a toroidal structure, with a central cavity about 3-4 nm in diameter. Observations were made using electron microscopy, employing the image analysis of single particles. The most predominant averaged image displayed a toroid harbouring four bulges forming corners, one of which was larger than the others. This structure was very similar to the mouse Mcm2/4/6/7 tetramer that was independently prepared and analysed by electron microscopy. These toroidal structures are distinct from that of the Mcm4/6/7 hexamer, which was also examined by electron microscopy. GST(glutathione S-transferase)-pull down and two hybrid experiments suggest that a putative Mcm6-Mcm6 hinge contributes to the formation of the Mcm7/4/6/6/4/7 heterohexamer. CONCLUSIONS: The Mcm2/4/6/7 tetramer forms a toroidal structure that is distinct from that of the Mcm4/6/7 hexamer in size and shape.

Animals↗

PubMed2002

Aberrant expression of minichromosome maintenance proteins 2 and 5, and Ki-67 in dysplastic squamous oesophageal epithelium and Barrett's mucosa.

BACKGROUND: Minichromosome maintenance (Mcm) proteins are essential for eukaryotic DNA replication, and their expression implies potential for cell proliferation. Expression is dysregulated in dysplastic states but data for oesophageal squamous mucosa and Barrett's mucosa have not been published. AIM: To test the hypothesis that Mcm proteins are downregulated together with the proliferation marker Ki-67 in differentiating epithelial compartments of non-dysplastic squamous and Barrett's epithelium, and that this process does not occur in dysplastic mucosae. METHODS AND CASES: Forty five patients with Barrett's oesophagus included 20 with glandular dysplasia (10 low grade, eight high grade, two both, and four with invasive adenocarcinoma). Twenty five other patients included 12 with oesophageal squamous dysplasia (three low grade, six high grade, three both, and four with invasive squamous carcinoma). Formalin fixed paraffin embedded tissue sections from biopsy series and resections were immunostained using antibodies to Mcm2, Mcm5, and Ki-67. Percentage of nuclei positive for Mcm2, Mcm5, and Ki-67 was estimated and scored from 0 to 6 as: 0, none +; 1, <10%+; 2, 10-30%+; 3, 30-70%+; 4, 70-90%+; 5, >90%+; 6, all+. Four separate epithelial strata were scored: in squamous epithelium the basal layer and thirds to the surface, in Barrett's mucosa the luminal surface, upper and lower crypt, and deep glands. RESULTS: In non-dysplastic squamous epithelium and Barrett's mucosa, high level expression of Mcm2, Mcm5, and Ki-67 proteins was largely confined to the proliferative compartments and downregulated in differentiated compartments. Expression persisted up to the mucosal surface in dysplastic squamous epithelium and Barrett's mucosa. CONCLUSIONS: Persistent expression of Mcm2, Mcm5, and Ki-67 proteins in luminal compartments of dysplastic oesophageal squamous epithelium and dysplastic Barrett's mucosa may be diagnostic markers and imply disruption of cell cycle control and differentiation in these dysplastic epithelia.

Barrett Esophagus↗

PubMed2006

Cell proliferation marker MCM2, but not Ki67, is helpful for distinguishing between minimally invasive follicular carcinoma and follicular adenoma of the thyroid.

AIMS: To compare cell proliferation markers, minichromosome maintenance protein 2 (MCM2) and Ki67, in minimally invasive follicular carcinoma (MIFC) and follicular adenoma (FA) of the thyroid and among MIFCs with different diagnostic criteria. METHODS AND RESULTS: Twenty-two MIFCs and 20 FAs were immunohistochemically stained for MCM2 and Ki67. The MIFCs were subdivided into six Group 1 tumours with both capsular and vascular invasions, seven Group 2 tumours with vascular invasion only and nine Group 3 tumours with capsular invasion only. The MCM2 and Ki67 indices were calculated, counting more than 1000 tumour cells in the most frequently positive areas. In total and Groups 1-3 MIFCs and in FAs, the average MCM2 index was 26.7 +/- 11.0, 28.4 +/- 8.6, 26.3 +/- 14.8, 25.9 +/- 8.4 and 10.7 +/- 4.5, respectively, whereas the average Ki67 index was 2.07 +/- 1.65, 1.93 +/- 2.02, 2.49 +/-1.38, 1.84 +/- 1.5 and 1.78 +/- 0.92, respectively. There was a significant difference in the MCM2 index, but not in the Ki67 index, between each category of MIFCs and FA (P < 0.01). However, neither the MCM2 index nor the Ki67 index showed a statistically significant difference among the subgroups of MIFC. CONCLUSIONS: MCM2, but not Ki67, is a helpful marker for differentiating MIFC from FA. The tumour cell proliferative activity supports the histological criteria based on diagnosing MIFC by either capsular or vascular invasion only.

Adenocarcinoma, Follicular↗

PubMed2000

Identification of functional PDZ domain binding sites in several human proteins.

TIP-15 was previously identified as a cellular protein that can bind to the C-terminal end of the HTLV-1 Tax protein via its two PDZ domains. The sequence of the N-terminal part of TIP-15 is identical to that of the synaptic protein PSD-95. Both proteins are likely to be produced from the same gene by alternative splicing. Whereas expression of the PSD-95 mRNA was detected only with brain RNAs, that of TIP-15 was detected with RNAs from thymus, brain, skeletal muscle and Jurkat cells. The TIP-15 protein exhibits an apparent molecular weight of 40 kD and is weakly expressed in T cell lines. A two-hybrid screen performed with TIP-15 as bait revealed the presence of a PDZ binding site (PDZ-BS) in the following proteins: Lysyl tRNA synthetase, 6-phosphogluconolactonase (6-GPL), Stress-activated protein kinase 3 (SAPK3), NET-1, Diacylglycerol kinase zeta, MTMR1, MCM7, and hSec8. The sequence at the C-terminal ends of these proteins matches the X-S/T-X-V-COOH consensus previously defined for PDZ-BSs, with the exception of 6-GPL and SAPK3 which include a leucine as the C-terminal residue. For Lysyl tRNA synthetase, NET1, MTMR1 and hSec8, binding to TIP-15 was confirmed by co-immunoprecipitation experiments performed with the extracts of transfected COS7 cells. These results show the existence of functional PDZ-BSs in these proteins, but future studies will be necessary to establish whether or not TIP-15 represents a physiological partner. The significance of the presence of a PDZ-BS in these various proteins is discussed with respect to their function.

Amino Acid Sequence↗

PubMed1995

A ste12 allele having a differential effect on a versus alpha cells.

The transcriptional activator Ste12p is a key component of the yeast pheromone response pathway: phosphorylated as a consequence of signal transduction, it activates transcription of genes that promote mating and the subsequent fusion of the two cell types a and alpha. Activation by Ste12p requires three types of protein-protein interaction between DNA-binding activator proteins: (1) Ste12p by itself can induce non-cell-type-specific genes involved in mating; (2) cooperation of the transactivator Mcm1p with Ste12p induces a-specific genes; and (3) formation of a complex of the activator proteins Mcm1p and alpha 1 (a transcriptional activator of alpha-specific genes) with Ste12p is believed to induce alpha-specific genes. We isolated and characterized a partially functional ste12 allele (ste12-T50), that is defective only in the activation of alpha-specific genes. ste12-T50 was isolated as a second-site mutation conferring the a mating phenotype on mat alpha 2 mutant cells. In mat alpha 2 cells, where due to the lack of repressor, alpha 2, both sets of cell-type-specific genes are expressed, ste12-T50 apparently tips the balance in favor of a-specific gene expression. Thus, mat alpha 2 ste12-T50 cells mate like a cells. Additional ste12 mutants that confer the a mating phenotype on mat alpha 2 cells have also been isolated.

Alleles↗

PubMed2004

Correlation of MYCN amplification with MCM7 protein expression in neuroblastomas: a chromogenic in situ hybridization study in paraffin sections.

Amplification of the MYCN oncogene in neuroblastomas is generally associated with a more aggressive clinical course. Recently, 1 of the minichromosome maintenance proteins, MCM7, was found to be a direct target of the MYCN transcription factor in neuroblastoma. To confirm this correlation, chromogenic in situ hybridization (CISH) to detect MYCN amplification and immunohistochemical staining for MCM7 protein expression were performed on paraffin tissue sections of 26 neuroblastomas cases and of 4 recurrences of these tumors. Seven of the primary tumors showed MYCN amplification, and all were stage 3 or 4 tumors. Only 4 of these showed MCM7 overexpression. However, 11 primary tumors overexpressed MCM7. The 4 patients with MCM7 expression associated with MYCN amplification all died from the tumor. In contrast, the 7 patients with MCM7 overexpression but no MYCN amplification were all younger than 1 year of age and have shown good survival. This suggests that MCM7 overexpression by itself is not related to a poorer prognosis as is MYCN amplification. In addition, the 4 pairs of primary and recurrent tumors all showed changes in MCM7 expression from negative to positive, whereas none of them had MYCN amplification. This study showed that MCM7 overexpression is not necessarily correlated with MYCN amplification or an aggressive clinical course. Interpretation of the results of CISH was quite easy and straightforward because the preparations were viewed with an ordinary light microscope with good preservation of the tissue morphology.

Adolescent↗

PubMed2006

Stem/progenitor cell-specific enhanced green fluorescent protein expression driven by the endogenous Mcm2 promoter.

Previous studies have demonstrated expression of the minichromosome maintenance protein Mcm2 in cells that remain competent to divide, including stem/progenitor cells of the subventricular zone (SVZ) within the brain. Here, a transgenic mouse line in which the Mcm2 gene drives expression of enhanced green fluorescent protein (EGFP) was constructed by insertion of an internal ribosomal entry site (IRES)-EGFP cassette into the last exon of the gene, 3' to the stop codon. In these mice, expression of EGFP is observed in the SVZ and several other tissues with high proliferative activity, including the spleen, intestine, hair follicles, and bone marrow. These observations suggest that EGFP fluorescence in this mouse line provides an index of the proliferative capacity of different tissues. Immunohistological analysis demonstrates a direct concordance between expression of EGFP and Mcm2, consistent with a transcriptional level downregulation of Mcm2 expression in postmitotic cells. To test the utility of EGFP expression for recovery of live cells retaining the capacity to divide, EGFP-expressing and -nonexpressing cells from bone marrow and brain were isolated from an adult Mcm2(IRES-EGFP) mouse by fluorescence-activated cell sorting and assayed for clonal growth. The EGFP-positive fraction contained the entire clonogenic population of the bone marrow and greater than 90% of neurosphere-forming cells from the brain. Brain-derived clonogenic cells were shown to remain competent to differentiate towards all three neural lineages. These studies demonstrate that the Mcm2(IRES-EGFP) transgenic line constructed here can be used for recovery of proliferation competent cells from different tissue types.

Animals↗

PubMedDate not supplied

Follicular epithelial cell hypertrophy induced by chronic oral administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Harlan Sprague-Dawley rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects the thyroid morphologically and/or functionally in adult animals. Recently, the National Toxicology Program conducted a 2-year gavage study of TCDD in female Harlan Sprague-Dawley rats. The only treatment-related alterations found in thyroid follicles were decreased luminal size and increased height of the follicular epithelial cells, without prominent protrusion into the lumen. The present study elucidated the nature of these follicular lesions. Thyroid glands of 10 rats each from the control, high (100 ng/kg/day)-dose, and stop-study (100 ng/kg/day, 30 weeks; vehicle to study termination) groups in the 2-year study were evaluated microscopically. Twenty randomly selected follicles were measured morphometrically in each animal. TCDD treatment significantly decreased the mean ratio of luminal/epithelial areas and increased the mean sectional epithelial height of the high-dose group compared to controls. Thyroid sections were immunostained with antibody against minichromosome maintenance (MCM) proteins, a novel cell-cycle biomarker. The MCM labeling index of the high-dose group was significantly higher than that of the control; however, the TUNEL labeling index was also higher in the high-dose group than the control. All data from the stop group were comparable to those from controls. These results indicate that the follicular cell response was hypertrophic and reversible. This information should contribute to diagnosis of nonneoplastic thyroid follicular lesions in rats.

Administration, Oral↗

PubMed2003

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia.

Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium (n=6), undifferentiated HPV positive VIN 1 (n=3), VIN 2 (n=8) and VIN 3 (n=20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of 'snapshots' of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.

Biomarkers, Tumor↗