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PubMed2001

Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle.

We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase, approximately 30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins.

Anaphase↗
PubMed2003

Localization of proteins bound to a replication origin of human DNA along the cell cycle.

The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site.

Base Sequence↗
PubMed1992

Properties of the nuclear P1 protein, a mammalian homologue of the yeast Mcm3 replication protein.

Polyclonal antibodies were raised against a multiprotein 'holoenzyme' form of calf thymus DNA polymerase alpha-primase and used to probe a human cDNA-protein expression library constructed in the lambda gt11 vector. The probe identified a series of cDNA clones derived from a 3.2 kb mRNA which encodes a novel 105 kDa polypeptide, the P1 protein. In intact cells, the P1 protein was specifically associated with the nucleus, and in cell extracts, it was associated with complex forms of DNA polymerase alpha-primase. The synthesis of human P1-specific mRNA was stimulated upon addition of fresh serum to growth-arrested cells, and RNA blot analyses with the human P1-cDNA probe indicated that P1 is encoded by a strictly conserved mammalian gene. The amino acid sequence deduced from a 240-codon open reading frame resident in the largest human P1-cDNA (0.84 kb) displayed greater than 96% identity with that deduced from the equivalent segment of a 795-codon open reading frame of a larger mouse P1-cDNA (2.8 kb). Throughout its length, the primary structure of mammalian P1 displayed strong homology with that of Mcm3, a 125 kDa yeast protein thought to be involved in the initiation of DNA replication (Gibson et al. 1990. Mol. Cell. Biol. 10: 5707-5720). The P1-Mcm3 homology, the strong conservation of P1 among mammals, its nuclear localization, and its association with the replication-specific DNA polymerase alpha strongly suggest an important role of the P1 protein in the replication of mammalian DNA.

Amino Acid Sequence↗
PubMed2003

Expression of replication-licensing factors MCM2 and MCM3 in normal, hyperplastic, and carcinomatous endometrium: correlation with expression of Ki-67 and estrogen and progesterone receptors.

Minichromosome maintenance (MCM) proteins are essential for cell cycling due to their function as replication-licensing factors. The aim of the present study was to investigate the clinicopathologic implications of the MCM2 and MCM3 in endometrial carcinogenesis. The authors investigated the immunohistochemical expression of MCM2 and MCM3, Ki-67, estrogen receptor, and progesterone receptor in 23 normal endometria, 9 endometrial hyperplasias, and 60 endometrial carcinomas. In the normal endometrial glands, the expression of MCM2 and MCM3 was significantly higher in the proliferative phase than in the secretory phase and was strongly correlated with Ki-67 expression. Similar correlation between the expression of MCMs and Ki-67 was also found in endometrial hyperplasia. In endometrial carcinomas, however, the expression of MCM2 and MCM3 was significantly lower than that in the normal proliferative endometrium. There was only a weak correlation between MCM2 and Ki-67, and no significant correlation between MCM3 and Ki-67 expression. These findings suggest that the expression of MCM2 and MCM3 directly reflects cell proliferation in normal and hyperplastic endometria. In endometrial carcinomas, however, there is a discrepancy between the expression of MCMs and cell proliferation, suggesting that the replication-licensing system may be aberrant in endometrial carcinomas.

Adult↗
PubMed1998

E2F3 activity is regulated during the cell cycle and is required for the induction of S phase.

Previous work has demonstrated the important role of E2F transcription activity in the induction of S phase during the transition from quiescence to proliferation. In addition to the E2F-dependent activation of a number of genes encoding DNA replication activities such as DNA Pol alpha, we now show that the majority of genes encoding initiation proteins, including Cdc6 and the Mcm proteins, are activated following the stimulation of cell growth and are regulated by E2F. The transcription of a subset of these genes, which includes Cdc6, cyclin E, and cdk2, is also regulated during the cell cycle. Moreover, whereas overall E2F DNA-binding activity accumulates during the initial G1 following a growth stimulus, only E2F3-binding activity reaccumulates at subsequent G1/S transitions, coincident with the expression of the cell-cycle-regulated subset of E2F-target genes. Finally, we show that immunodepletion of E2F3 activity inhibits the induction of S phase in proliferating cells. We propose that E2F3 activity plays an important role during the cell cycle of proliferating cells, controlling the expression of genes whose products are rate limiting for initiation of DNA replication, thereby imparting a more dramatic control of entry into S phase than would otherwise be achieved by post-transcriptional control alone.

CDC2-CDC28 Kinases↗
PubMed2004

Ciz1 promotes mammalian DNA replication.

Using a cell-free system that reconstitutes initiation of mammalian DNA replication, we identified a cyclin A-responsive protein, p21(Cip1)-interacting zinc finger protein 1 (Ciz1). In cell-free experiments, Ciz1 protein increases the number of nuclei that initiate DNA replication, and in intact cells GFP-tagged Ciz1 stimulates DNA synthesis, in both a wild-type and a p21(Cip1) null background. Furthermore, mutation of a putative cyclin-dependent kinase phosphorylation site at threonines 191/2 alters Ciz1 activity in vitro, indicating that this site plays a role in regulating Ciz1. Consistent with a role in DNA replication, endogenous Ciz1 is present in nuclear foci that co-localize with PCNA during S phase, and targeted depletion of Ciz1 transcripts restrains cell proliferation by inhibiting entry to S phase. Ciz1-depleted cells accumulate with chromatin bound Mcm3 and PCNA but fail to synthesize DNA efficiently. These cell-based and cell-free experiments suggest that Ciz1 functions to promote DNA replication after replication complex formation. Finally, alternatively spliced forms of Ciz1 occur in embryonic cells from mouse and man, raising the possibility that Ciz1 splicing contributes to the regulation of DNA replication during development.

3T3 Cells↗
PubMed1995

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication.

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

3T3 Cells↗
PubMed2000

A novel nuclear phosphoprotein, GANP, is up-regulated in centrocytes of the germinal center and associated with MCM3, a protein essential for DNA replication.

Antigen (Ag) immunization induces formation of the germinal center (GC), with large, rapidly proliferating centroblasts in the dark zone, and small, nondividing centrocytes in the light zone. We identified a novel nuclear protein, GANP, that is up-regulated in centrocytes. We found that GANP was up-regulated in GC B cells of Peyer's patches in normal mice and in spleens from Ag-immunized mice. GANP-positive cells appeared in the light zone of the GC, with coexpression of the peanut agglutinin (PNA) (PNA)-positive B220-positive phenotype. The expression of GANP was strikingly correlated with GC formation because Bcl6-deficient mice did not show the up-regulation of GANP. GANP-positive cells were mostly surrounded by follicular dendritic cells. Stimulation with anti-micro and anti-CD40 induced up-regulation of ganp messenger RNA as well as GANP protein in B220-positive B cells in vitro. GANP is a 210-kd protein localized in both the cytoplasm and nuclei, with a homologous region to Map80 that is associated with MCM3, a protein essential for DNA replication. Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also up-regulated in the GC area. These results suggest that the up-regulation of GANP might participate in the development of Ag-driven B cells in GCs through its interaction with MCM3.

Acetyltransferases↗
PubMed1995

Interactions of human nuclear proteins P1Mcm3 and P1Cdc46.

Human nuclear proteins P1Mcm3 and P1Cdc46 have high sequence similarities with the corresponding yeast proteins known to be required for the initiation of genome replication. Nuclei of proliferating HeLa cells contain relatively high amounts of P1Mcm3 (about 10(6) molecules/nucleus) of which only a small fraction is bound to a nuclear structure, most probably chromatin. At 0.5 M NaCl, the structure-bound nuclear protein can be partially solubilized as a dimer composed of P1Mcm3 and the related protein P1Cdc46. However, most protein P1Mcm3 is not bound to a nuclear structure and appears in the nucleoplasm. About 10% of protein P1Mcm3 in the soluble fraction is free and uncomplexed, and the remaining P1Mcm3 forms stable complexes with protein P1Cdc46. These P1Mcm3/Cdc46 complexes occur as dimers and in high-molecular-mass complexes (approximately 500 kDa). The high-molecular-mass complexes dissociate in 0.5 M NaCl and release P1Mcm3/Cdc46 dimers. It has frequently been proposed that the Mcm proteins may function as licensing factors for genome replication. Our data imply that the active form of an Mcm protein is not a monomer, but a protein complex that includes an Mcm3/Cdc46 dimer. DNA polymerase alpha is not a component of this complex.

Amino Acid Sequence↗
PubMed1996

The role of MCM/P1 proteins in the licensing of DNA replication.

The DNA replication licensing system ensures that eukaryotic chromosomes replicate precisely once per cell cycle. A central component of the licensing system, RLF-M, has recently been shown to consist of a complex of Mcm/P1 proteins. This result allows us to integrate data about the MCM/P1 family obtained in different eukaryotes, ranging from yeast to man, into a general picture of the way that chromosome replication is controlled.

Cell Cycle Proteins↗
PubMed1999

The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin.

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1] [2]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3] [4] [5] [6] [7]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) [8] [9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9] [10] [11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.

Animals↗
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↗
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↗
PubMed2003

Improved detection of hepatocyte proliferation using antibody to the pre-replication complex: an association with hepatic fibrosis and viral replication in chronic hepatitis C virus infection.

To test the hypothesis that hepatitis C virus (HCV) might induce hepatocyte proliferation directly, thereby predisposing HCV carriers to cirrhosis and hepatocellular carcinoma, we have used a new method to identify proliferating hepatocytes, employing a novel monoclonal antibody to minichromosome maintenance (Mcm) proteins, essential components of the pre-replication complex. Antibody to Ki-67, a conventional marker of cell division, was also studied. Eighty-seven patients with chronic HCV infection and a broad spectrum of histological change were studied. Proliferation was observed rarely in hepatocytes from normal liver from healthy controls (always less than 0.01%). However, proliferating hepatocytes were detected in all HCV-infected patients and the proportion of hepatocytes expressing Mcm-2 (3-40%) always exceeded that expressing Ki-67 (1-14%) and correlated positively with increasing stage of fibrosis (P = 0.0001) and viral replication (P = 0.0004). There were weaker but significant associations between the proportion of hepatocytes expressing Mcm-2 and inflammatory indices including interface hepatitis, portal tract inflammation, lobular inflammation and steatosis. There was no association between the proportion of hepatocytes expressing Mcm-2 and age, gender or past alcohol consumption, but there was a weak association with current consumption of alcohol (P = 0.0067). The proportion of Ki-67 hepatocytes did not correlate with any clinical, laboratory or histological parameter. Mcm-2 was also detected in bile duct cells, sinusoidal lining cells and infiltrating lymphocytes, but at low frequency. These data indicate first, that Mcm-2 is a more sensitive marker of hepatocyte proliferation than Ki-67, second that many hepatocytes in chronic HCV infection have entered the cell cycle and third, suggest that interference with the hepatocyte cell cycle might be an alternative approach to therapy.

Adult↗
PubMed2006

Effect of acid suppression on molecular predictors for esophageal cancer.

BACKGROUND: Gastroesophageal reflux disease is a risk factor for the development of Barrett's esophagus and esophageal adenocarcinoma. The effect of antireflux therapy on the incidence of esophageal adenocarcinoma is unknown. Acid exposure in vitro induces hyperproliferation via a cyclooxygenase-2 (COX-2) dependent mechanism. Epidemiologic and animal studies suggest that COX inhibitors decrease the incidence of esophageal adenocarcinoma. AIM: To study the differential effect of complete compared with incomplete acid suppression on proliferation, apoptosis, and COX-2. PATIENTS AND METHODS: Fifty-one patients with Barrett's esophagus who underwent pH monitoring were divided into two groups according to their DeMeester score: 32 acid-suppressed patients (group 1) and 19 patients with abnormally high acid exposure (group 2). Slides from biopsies taken 3 months before and 4 and 12 months after pH monitoring were stained for Mcm2, COX-2, c-myc, and cleaved caspase-3 (marker of apoptosis). RESULTS: There was no evidence of a difference between the two groups in terms of age, gender ratio, medication, dysplasia status, and the expression levels of any marker before pH monitoring. In group 1, Mcm2 expression decreased in the luminal surface and throughout the tissue 12 months after monitoring when compared with the two previous time points (P < 0.05). The levels of COX-2 increased overtime (P < 0.01 in group 1, not significant in group 2). There was no correlation between Mcm2 and COX-2 expression. Acid suppression had no effect on c-myc or apoptosis. CONCLUSION: Long-term acid suppression reduces proliferation in Barrett's esophagus samples but has no advantageous effect on c-myc, apoptosis, or COX-2.

Adenocarcinoma↗