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PubMed2006

Cdc7 is an active kinase in human cancer cells undergoing replication stress.

Cdc7 kinase promotes and regulates DNA replication in eukaryotic organisms. Multiple mechanisms modulating kinase activity in response to DNA replication stress have been reported, supporting the opposing notions that Cdc7 either plays an active role under these conditions or, conversely, is a final target inactivated by a checkpoint response. We have developed new immnunological reagents to study the properties of human Cdc7 kinase in cells challenged with the ribonucleotide reductase inhibitor hydroxyurea or with the DNA topoisomerase II inhibitor etoposide. We show that Cdc7.Dbf4 and Cdc7.Drf1 complexes are stable and active in multiple cell lines upon drug treatment, with Cdc7.Dbf4 accumulating on chromatin-enriched fractions. Cdc7 depletion by small interfering RNA in hydroxyurea and etoposide impairs hyper-phosphorylation of Mcm2 at specific Cdc7-dependent phosphorylation sites and drug-induced hyper-phosphorylation of chromatin-bound Mcm4. Furthermore, sustained inhibition of Cdc7 in the presence of these drugs increases cell death supporting the notion that the Cdc7 kinase plays a role in maintaining cell viability during replication stress.

Apoptosis↗
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↗
PubMed2002

Early recurrence of benign meningioma correlates with expression of mini-chromosome maintenance-2 protein.

We have investigated the potential utility of monoclonal antibodies against mini-chromosome maintenance-2 protein (Mcm2) in predicting meningioma recurrence. MCM proteins are members of the DNA-binding prereplicative complex and are essential for eukaryotic DNA replication. They are present throughout the cell cycle, but are down-regulated in quiescence and cell differentiation, making them specific markers of proliferating cells. We analysed 10 benign meningiomas that subsequently recurred within a 5-year period, together with 20 matched non-recurrent benign meningiomas. There was no significant correlation between histological subtype, mitotic count or Ki-67 labelling index and tumour recurrence. We observed that whilst the average Mcm2 labelling index (LI) of the tumour section as a whole (LI(Ave)) is not significantly different between recurrent and nonrecurrent meningiomas, the Mcm2 labelling index in the area of highest proliferative activity within the tumour section (LI(Max)) is significantly higher in recurrent meningiomas (p < 0.0001). Seven out of the 10 recurrent meningiomas displayed a Mcm2 LI((Max) greater than 30%, compared to 0 out of 20 for non-recurrent tumours. In conclusion, these results suggest that analysis of Mcm2 expression may facilitate identification of patients with a high risk of meningioma recurrence, for whom adjuvant radiotherapy may be of benefit.

Adult↗
PubMed1995

BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication.

We have recently cloned and characterized a human member (BM28) of the MCM2-3-5 family of putative relication factors (Todorov, I.T., R. Pepperkok, R.N. Philipova, S. Kearsey, W. Ansorge, and D. Werner. 1994. J. Cell Sci. 107:253-265). While this protein is located in the nucleus throughout interphase, we report here a dramatic alteration in its nuclear binding during the cell cycle. BM28 is retained in the nucleus after Triton X-100 extraction in G1 and early S phase cells, but is progressively lost as S phase proceeds, and little BM28 is retained in detergent-extracted G2 nuclei. BM28 that is resistant to extraction in G1 nuclei is removed by DNase I digestion, suggesting that the protein is chromatin associated. In addition, we present evidence for variations in the electrophoretic mobility of BM28 that may reflect posttranslational modifications of BM28 during the cell cycle. During mitosis, BM28 is present as a fast-migrating form, but on entry into G1, the protein is converted into a slow-migrating form. With the onset of S phase, the slow-migrating form is progressively converted into the fast form. BM28 is phosphorylated at all stages of the cell cycle, but during interphase the fast form is hyperphosphorylated compared with the slow form. These apparent changes in modification may reflect or effect changes in the nuclear binding of BM28. The behavior of BM28 is not dissimilar to related proteins in Saccharomyces cerevisiae, such as Mcm2p, which are excluded from the nucleus after DNA replication. We speculate that BM28 may be involved in the control that limits eukaryotic DNA replication to one round per cell cycle.

3T3 Cells↗
PubMed1999

Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex.

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1-mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.

Animals↗
PubMed2006

Essential role of phosphorylation of MCM2 by Cdc7/Dbf4 in the initiation of DNA replication in mammalian cells.

We report the identification of Cdc7/Dbf4 phosphorylation sites in human MCM2 and the determination of the role of Cdc7/Dbf4 phosphorylation of MCM2 in the initiation of DNA replication. Using immunoblotting, immunofluorescence, and high-speed automated cell-imaging analyses with antibodies specific against MCM2 and Cdc7/Dbf4 phosphorylated MCM2, we show that the chromatin recruitment and phosphorylation of MCM2 are regulated during the cell cycle in HeLa cells. Chromatin-bound MCM2 is phosphorylated by Cdc7/Dbf4 during G1/S, which coincides with the initiation of DNA replication. Moreover, we show that baculovirus-expressed purified MCM2-7 complex and its phosphomimetic MCM2E-7 complex display higher ATPase activity when compared with the nonphosphorylatable MCM2A-7 complex in vitro. Furthermore, suppression of MCM2 expression in HeLa cells by siRNA results in the inhibition of DNA replication. The inhibition can be rescued by the coexpression of wild type MCM2 or MCM2E but not MCM2A. Taken together, these results indicate that Cdc7/Dbf4 phosphorylation of MCM2 is essential for the initiation of DNA replication in mammalian cells.

Adenosine Triphosphatases↗
PubMed2005

Cell proliferation and granule cell dispersion in human hippocampal sclerosis.

Granule cell dispersion (GCD) is observed in approximately 40% of cases of hippocampal sclerosis (HS) in patients with epilepsy. Studies in animal models suggest that GCD may be a consequence of enhanced proliferation of granule cell precursors as a result of seizures. We quantified the number of cells in cycle in subfields of the hippocampus with immunohistochemistry for Mcm2 in 14 HS cases with or without severe GCD compared to 6 epilepsy patients without classical HS or GCD as well as 5 postmortem controls. Higher numbers of Mcm2-positive cells were seen in the region of the granule cell layer in patients with severe GCD, and immunolabeling with Geminin and Ki-67 confirmed a proportion were progressing through cycle. Double labeling with Mcm2 and GFAP confirmed the majority of these cycling cells were GFAP-negative and occasional cells stained colocalized with stem cell marker nestin. These findings support the view that GCD may be a phenomenon related to increased progenitor cell proliferation in patients with hippocampal damage and chronic epilepsy.

Adult↗
PubMed2005

The expression of minichromosome maintenance protein-2 in normal and abnormal megakaryocytes and comparison with the proliferative marker Ki-67.

The minichromosome maintenance (Mcm) and Cdc6 proteins are important regulators of eucaryotic DNA replication. In most normal tissues, a similar proportion of cells express Mcm-2 and Ki-67. The present study showed that in both normal and abnormal states, the proportion of megakaryocytes expressing Mcm-2 is roughly seven times as many as those that express Ki-67. This is likely to be related to the process of endomitosis and endoreduplication. We also demonstrated that a significantly lower proportion of megakaryocytes in myelodysplastic syndrome express Mcm-2. These findings provide new insights into megakaryocyte biology.

Bone Marrow↗
PubMed2005

Mcm2 labelling of balloon cells in focal cortical dysplasia.

Balloon cells (BC) are the prominent and defining cellular component of type IIB Focal Cortical Dysplasia (FCD), a common cause of focal epilepsy in patients undergoing surgical treatment. BC are considered immature cells of uncommitted cellular differentiation having immunophenotypical characteristics of both neurones and glia. They are often located in the lower cortical layers and white matter underlying the dysplastic cortex, suggesting migratory arrest during development. We investigated the proliferative potential of BC in 15 cases of FCD from patients with a wide range of ages using immunohistochemistry for Mcm2 (mini chromosome maintenance protein) and Ki67. In the majority of cases, BC showed Mcm2 nuclear positivity. In addition, cells with intermediate neuronal-glial characteristics were labelled whilst the dysmorphic or hypertrophic pyramidal neuronal components of FCD were not. Ki67 labelled only occasional BC. These findings support the view that BC cells represent a pool of less differentiated glial cells with proliferative capacity which may have potential for delayed neuronal differentiation. Furthermore, as Mcm2 specifically identifies BC populations, this marker may be of diagnostic value in the subtyping of FCD lesions in patients with epilepsy.

Adolescent↗
PubMed1999

MCM proteins are associated with RNA polymerase II holoenzyme.

MCMs are a family of proteins related to ATP-dependent helicases that bind to origin recognition complexes and are required for initiation of DNA replication. We report that antibodies against MCM2(BM28) specifically inhibited transcription by RNA polymerase II (Pol II) in microinjected Xenopus oocytes. Consistent with this observation, MCM2 and other MCMs copurified with Pol II and general transcription factors (GTFs) in high-molecular-weight holoenzyme complexes isolated from Xenopus oocytes and HeLa cells. Pol II and GTFs also copurified with MCMs isolated by anti-MCM3 immunoaffinity chromatography. MCMs were specifically displaced from the holoenzyme complex by antibody against the C-terminal domain (CTD) of Pol II. In addition, MCMs bound to a CTD affinity column, suggesting that their association with holoenzyme depends in part on this domain of Pol II. These results suggest a new function for MCM proteins as components of the Pol II transcriptional apparatus.

Animals↗
PubMed2006

Cell cycle phase distribution analysis in chronic lymphocytic leukaemia: a significant number of cells reside in early G1-phase.

BACKGROUND AND AIMS: Chronic lymphocytic leukaemia (CLL) is a frequent non-Hodgkin lymphoma characterised by a heterogeneous clinical course. Assessment of cell cycle phase kinetics might be important for prediction of clinical behaviour and prognosis. METHODS: Distribution of neoplastic cells in CLL within the cell cycle was evaluated by determining the labelling indices (LI, i.e. percentage of positive cells) of markers specific for late G1-phase (cyclin E), S-phase (cyclin A), and G2/M-phase (cyclin B1), and Mcm2, a novel marker of proliferative potential, in a large cohort of patients (n = 79) using tissue microarray (TMA) technology. Utilising a combination of these markers, an algorithm was developed--subtracting the combined LIs of cyclin E, cyclin A and cyclin B1 from the LI of Mcm2--to determine the percentage of tumour cells residing in early G1-phase, which is probably a critical state for the malignant potential of CLL. RESULTS: 27.11% of cells had acquired proliferative potential as indicated by expression of Mcm2. Only a small number of cells were found to be in late G1-phase (7.16%), S-phase (3.31%) or G2/M-phase (0.98%), while 15.66% of cells were considered to be in early G1-phase. CONCLUSION: Cell cycle phase distribution can easily be assessed by immunohistochemistry in routinely processed paraffin-embedded specimens. A large number of neoplastic cells in CLL have proliferative potential, with a significant sub-population residing in early G1-phase. Estimates of these cells may identify cases likely to exhibit a more aggressive biological behaviour and adverse clinical course.

Adult↗
PubMed2003

Association between histology grade, expression of HsMCM2, and cyclin A in human invasive breast carcinomas.

AIM: Increased proliferation of tumour cells has prognostic value in human invasive breast carcinomas (IBCs), and high histology grade and cyclin A expression, which may reflect high proliferation rate, are associated with poor prognosis. Expression of HsMCM2 is related to cell proliferation. This study evaluates the correlation between the expression of cyclins A, D1, D3, and E, Ki-67, proliferating cell nuclear antigen (PCNA), histology grade, and HsMCM2 expression, in addition to the independent prognostic value of HsMCM2 expression in human IBCs. METHODS: Immunohistochemistry to evaluate HsMCM2, Ki-67, and PCNA expression in tumours from 147 patients with IBC. RESULTS: Nuclear staining for HsMCM2 was seen in 10-30% of the tumour cells in 30 samples, in 30-70% in 40 samples, in > 70% in 44 samples, and in < 10% in 33 samples. One way ANOVA showed a significant association between expression of HsMCM2 and cyclin A, D3, E, histology grade, and Ki-67. A borderline correlation was seen between HsMCM2 and PCNA. In multivariate analysis, the only association was with cyclin A, in addition to a borderline association with histology grade. In a Cox regression hazards model, expression of HsMCM2 was associated with poor patient survival, although it lost its independent prognostic value when cyclin A expression was included. Ki-67 and PCNA expression were not associated with patient survival. CONCLUSION: Cyclin A expression is independently associated with HsMCM2 expression, histology grade, and Ki-67. HsMCM2 expression is associated with poor patient survival, although it loses prognostic value when adjusted for cyclin A.

Biomarkers, Tumor↗
PubMed1994

The human gene for nuclear protein BM28 (CDCL1), a new member of the early S-phase family of proteins, maps to chromosome band 3q21.

BM28, a newly recognized human nuclear protein, possibly plays an important role in two crucial steps of the cell cycle e.g. the onset of DNA replication and cell division. It shows significant similarity to members of a recently defined family of early S-phase proteins. Using total plasmid DNA containing the complete coding sequence of the BM28 gene (CDCL1, for cdc-like 1) as a probe for fluorescence in situ hybridization, we have mapped the gene to chromosome band 3q21. This region is involved in specific structural chromosome aberrations found in acute myeloid leukemia (AML). Based on the function of BM28 and the chromosomal location of its gene, CDCL1 might prove to be a candidate for an oncogene affected by the chromosomal breaks and playing a pathogenetic role in AML.

Cell Cycle Proteins↗
PubMed1995

Isolation and mapping of a human gene (MCM2) encoding a product homologous to yeast proteins involved in DNA replication.

From a human fetal-lung cDNA library, we isolated a gene, human MCM2, whose nucleotide sequence predicts a protein product homologous to the yeast nuclear proteins MCM2, MCM3, CDC21, and CDC46. Determination of cDNA sequences covering the entire coding region demonstrated an open reading frame of 1,629 nucleotides encoding 543 amino acids. Sequence comparison revealed 30-40% amino acid identity with these four yeast proteins, all of which are considered to play important roles in DNA replication. Analysis by Northern blotting revealed that the human MCM2 gene is expressed ubiquitously in normal tissues. Furthermore, we localized this gene to chromosomal bands 7q21.3-->q22.1 by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed2005

Comparison of the proliferative capacity of human corneal endothelial cells from the central and peripheral areas.

PURPOSE: To compare the relative proliferative capacity between human corneal endothelial cells (HCECs) cultured from the central and peripheral areas of the cornea. METHODS: Human corneas were divided into two groups based on donor age (younger group, < or =30 years of age; older group, > or =50 years of age). Corneas were trephined, and Descemet's membrane with HCECs was stripped from the central (0-6.75 mm) and peripheral (6.75-9.5 mm) areas. HCECs were then isolated from Descemet's membrane and cultivated. An equal number of passage-1 endothelial cells from each area were seeded, and the number of cells was determined at various times after seeding. Doubling times of cells from each area were compared. The antibody against minichromosome maintenance-2 (MCM2) protein was tested for replication competence. RESULTS: Morphologically, HCECs from the central area were similar to cells from the peripheral area. The doubling time (in hours) of HCECs from the central area was 35.20 in the younger group (n = 4) and 54.54 in the older group (n = 4) and from the peripheral area, 29.37 in the younger group and 46.23 in the older group. There was no significant difference (younger: P = 0.515; older: P = 0.222) between the central and peripheral area in each age group. MCM2-positive cells were consistently observed in cultures from the central, as well as peripheral, area. There was no significant difference (younger: P = 0.929; older: P = 0.613) in the percentage of MCM2-positive cells between these two areas in either age group. Even though there was no significant difference, there was a tendency toward increased doubling time and decreased percentage of MCM2 in the central area of the older group. CONCLUSIONS: These results indicate that corneal endothelial cells from both the central and peripheral areas retain potential proliferative capacity.

Adolescent↗
PubMed2006

Prognostic significance of MCM2, Ki-67 and gelsolin in non-small cell lung cancer.

BACKGROUND: Uncontrolled proliferation and increased motility are hallmarks of neoplastic cells, therefore markers of proliferation and motility may be valuable in assessing tumor progression and prognosis. MCM2 is a member of the minichromosome maintenance (MCM) protein family. It plays critical roles in the initiation of DNA replication and in replication fork movement, and is intimately related to cell proliferation. Ki-67 is a proliferation antigen that is expressed during all but G0 phases of the cell cycle. Gelsolin is an actin-binding protein that regulates the integrity of the actin cytoskeletal structure and facilitates cell motility. In this study, we assessed the prognostic significance of MCM2 and Ki-67, two markers of proliferation, and gelsolin, a marker of motility, in non-small cell lung cancer (NSCLC). METHODS: 128 patients with pathologically confirmed, resectable NSCLC (stage I-IIIA) were included. Immunohistochemistry was utilized to measure the expressions of these markers in formalin-fixed, paraffin-embedded tumor tissues. Staining and scoring of MCM2, Ki-67 and gelsolin was independently performed. Analyses were performed to evaluate the prognostic significance of single expression of each marker, as well as the prognostic significance of composite expressions of MCM2 and gelsolin. Cox regression and Kaplan-Meier survival analysis were used for statistical analysis. RESULTS: Of the three markers, higher levels of gelsolin were significantly associated with an increased risk of death (adjusted RR = 1.89, 95% CI = 1.17-3.05, p = 0.01), and higher levels of MCM2 were associated with a non-significant increased risk of death (adjusted RR = 1.36, 95% CI = 0.84-2.20, p = 0.22). Combined, adjusted analyses revealed a significantly poor prognostic effect for higher expression of MCM2 and gelsolin compared to low expression of both biomarkers (RR = 2.32, 95% CI = 1.21-4.45, p = 0.01). Ki-67 did not display apparent prognostic effect in this study sample. CONCLUSION: The results suggest that higher tumor proliferation and motility may be important in the prognosis of NSCLC, and composite application of biomarkers might be of greater value than single marker application in assessing tumor prognosis.

Aged↗
PubMed2006

Alteration of proliferation and apoptotic markers in normal and premalignant tissue associated with prostate cancer.

BACKGROUND: Molecular markers identifying alterations in proliferation and apoptotic pathways could be particularly important in characterizing high-risk normal or pre-neoplastic tissue. We evaluated the following markers: Ki67, Minichromosome Maintenance Protein-2 (Mcm-2), activated caspase-3 (a-casp3) and Bcl-2 to determine if they showed differential expression across progressive degrees of intraepithelial neoplasia and cancer in the prostate. To identify field effects, we also evaluated whether high-risk expression patterns in normal tissue were more common in prostates containing cancer compared to those without cancer (supernormal), and in histologically normal glands adjacent to a cancer focus as opposed to equivalent glands that were more distant. METHODS: The aforementioned markers were studied in 13 radical prostatectomy (RP) and 6 cystoprostatectomy (CP) specimens. Tissue compartments representing normal, low grade prostatic intraepithelial neoplasia (LGPIN), high grade prostatic intraepithelial neoplasia (HGPIN), as well as different grades of cancer were mapped on H&E slides and adjacent sections were analyzed using immunohistochemistry. Normal glands within 1 mm distance of a tumor focus and glands beyond 5 mm were considered "near" and "far", respectively. Randomly selected nuclei and 40 x fields were scored by a single observer; basal and luminal epithelial layers were scored separately. RESULTS: Both Ki-67 and Mcm-2 showed an upward trend from normal tissue through HGPIN and cancer with a shift in proliferation from basal to luminal compartment. Activated caspase-3 showed a significant decrease in HGPIN and cancer compartments. Supernormal glands had significantly lower proliferation indices and higher a-casp3 expression compared to normal glands. "Near" normal glands had higher Mcm-2 indices compared to "far" glands; however, they also had higher a-casp3 expression. Bcl-2, which varied minimally in normal tissue, did not show any trend across compartments or evidence for field effects. CONCLUSION: These results demonstrate that proliferation and apoptosis are altered not only in preneoplastic lesions but also in apparently normal looking epithelium associated with cancer. Luminal cell expression of Mcm-2 appears to be particularly promising as a marker of high-risk normal epithelium. The role of apoptotic markers such as activated caspase-3 is more complex, and might depend on the proliferation status of the tissue in question.

Adenocarcinoma↗