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PubMed2000

Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis.

Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.

Antibodies, Monoclonal↗
PubMed1996

Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication.

Members of the Mcm-protein family have recently been shown to be involved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-proteins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is detected throughout the nucleus in late G1 and early S, and at discrete subnuclear sites following further progression of S-phase. We use high resolution confocal microscopy to determine the subnuclear sites of chromatin-bound Mcm proteins in comparison to the sites of replicating DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not colocalise with replication foci, instead these proteins appear to coincide with subnuclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin at the time when this site is replicated. Consequently, early replicating sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later stages of S-phase. Furthermore, G2 nuclei and condensed chromatin in mitotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3. Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sites of DNA replication. Instead, they appear to be present only on unreplicated chromatin and are displaced from replicating chromatin, consistent with a role in monitoring unreplicated chromatin and ensuring only a single round of DNA replication per cell cycle.

Animals↗
PubMed2002

Prolonged arrest of mammalian cells at the G1/S boundary results in permanent S phase stasis.

Mammalian cells in culture normally enter a state of quiescence during G1 following suppression of cell cycle progression by senescence, contact inhibition or terminal differentiation signals. We find that mammalian fibroblasts enter cell cycle stasis at the onset of S phase upon release from prolonged arrest with the inhibitors of DNA replication, hydroxyurea or aphidicolin. During arrest typical S phase markers remain present, and G0/G1 inhibitory signals such as p21(WAF1) and p27 are absent. Cell cycle stasis occurs in T-antigen transformed cells, indicating that p53 and pRB inhibitory circuits are not involved. While no DNA replication is evident in arrested cells, nuclei isolated from these cells retain measurable competence for in vitro replication. MCM proteins are required to license replication origins, and are put in place in nuclei in G1 and excluded from chromatin by the end of replication to prevent rereplication of the genome. Strikingly, MCM proteins are strongly depleted from chromatin during prolonged S phase arrest, and their loss may underlie the observed cell cycle arrest. S phase stasis may thus be a 'trap' in which cells otherwise competent for S phase have lost a key component required for replication and thus can neither go forward nor retreat to G1 status.

Animals↗
PubMed2001

Stimulation of MCM3 gene expression in osteoblast by low level laser irradiation.

Biostimulatory effect of cell proliferation and bone formation by laser irradiation has been reported, however, very little is known about the molecular basis of mechanisms. We previously constructed the cDNA library of mouse osteoblastic cells (MC3T3-E1) which enhanced gene expression by laser irradiation using a subtracted gene cloning procedure. In the present study, we focused on a gene clone, designated as MCL-140, which exhibited the high homology of DNA sequence with mouse minichromosome maintenance (MCM) 3 gene. MCM3 is involved in the initiation of DNA replication as licensing factor in eukaryotic cells. Nucleotide sequence of MCL-140 insert was determined and assessed in the nucleic acid databases. The transcription level of MCL-140 was examined by Northern blot analysis. The DNA sequences of clone MCL-140 insert exhibited 96.2% homology with MCM 3 gene coding P1 protein. Higher MCM3 mRNA levels were observed in laser-irradiated cells compared to the levels in non-irradiated cells: furthermore, radiolabelled thymidine incorporation was increased by laser irradiation. These findings suggest that low-level laser irradiation may enhance DNA replication and play a role in stimulating proliferation of osteoblast through the enhancement of the MCM3 gene expression.

Animals↗
PubMed1995

MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells.

An intact nuclear membrane restricts DNA replication to only one round in each cell cycle, apparently by excluding an essential replication-licensing factor throughout interphase. A family of related yeast replication proteins, MCM2, 3 and 5 (also called, after cell-division cycle, CDC46), resemble licensing factor, entering the nucleus only during mitosis. We have cloned a Xenopus homologue of MCM3 (XMCM3) and raised antibodies against expressed protein. Immunodepletion of Xenopus egg extracts removes a complex of MCM2, 3 and 5 homologues and inhibits replication of Xenopus sperm nuclei or permeable G2 HeLa nuclei. However, G1 HeLa nuclei still replicate efficiently. Mock-depleted extracts replicate all three templates. XMCM3 accumulates in nuclei before replication but anti-XMCM3 staining decreases during replication. These results can explain why replicated nuclei are unable to reinitiate replication in a single cell cycle.

Amino Acid Sequence↗
PubMed1997

Unusual genome organisation in Entamoeba histolytica leads to two overlapping transcripts.

We have isolated homologs of the mini chromosome maintenance (MCM) gene family from the parasitic protozoan Entamoeba histolytica. The full length genomic and cDNA clones for the Eh MCM3 gene have been characterised. The Eh MCM3 gene is much smaller than the Saccharomyces cerevisiae MCM3 gene and other eukaryotic homologs of the MCM3/P1 family. The predicted Eh Mcm3 protein was 597 amino acids long and showed 37 and 46% positional identity with the Sc Mcm3 and the mouse P1 homologs respectively. While proceeding along the chromosome from the Eh MCM3 gene, we have identified a homolog (Eh PAK) of the murine p21 activated kinase (Rn KPAK), or S. cerevisiae STE20. Eh PAK lies 126 bp upstream of the Eh MCM3 gene. The predicted Eh p21 activated kinase protein was 459 amino-acids long and showed 33% positional identity with the murine p21 activated kinase and its yeast homolog Ste20. Analysis of cDNA and genomic sequences shows that the 3' untranslated region (UTR) of the Eh PAK mRNA and the 5' UTR of the Eh MCM3 mRNA are transcribed from a common 40 bp genomic segment. This is the first report of an amoeba gene being physically linked to a second gene such that their transcripts are overlapping and there is no non-transcribed intergenic region between the two genes. Primer extension studies have confirmed that unlike most E. histolytica genes, which have short 5' UTRs, the Eh MCM3 mRNA has a 126 bp long 5' UTR and the Eh PAK mRNA has a 265 bp long 5' UTR.

Amino Acid Sequence↗
PubMed2006

Proteomic analysis of the mouse mammary gland is a powerful tool to identify novel proteins that are differentially expressed during mammary development.

After lactation, the mouse mammary gland undergoes apoptosis and tissue remodelling as the gland reverts to its prepregnant state. This complex change was investigated using 2-DE. An integrated database was produced from lactation and involution proteomes. Forty-four molecular cluster indexes (MCIs) that showed altered expression from lactation to involution were selected for MS analysis. Of these, 32 gave protein annotations, 18 of which were unequivocal proteins. Selected proteins were then studied across all of development, including pregnancy, using data integrated from another proteome database. Two proteins, the RNA polymerase B transcription factor 3 (BTF3) and the minichromosome maintenance protein 3 (MCM3), although initially selected on the basis of the lactation/involution criteria, had expression profiles that indicated an additional role in mammary development and were further analysed. BTF3, a transcription factor previously not described in the mammary gland, was up-regulated strongly in pregnancy, indicating an involvement in alveolar growth. MCM3's expression was greatest in pregnancy and late involution, decreasing through lactation. Immunohistochemistry localised MCM3 to the mammary epithelium, where a greater proportion of cells stained than for the proliferation marker Ki67. MCM3 expression during lactation may identify cells that are licensed to repopulate the gland during cell loss in lactation and following involution.

Animals↗
PubMed1995

Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein.

The human protein P1 belongs to a newly discovered class of mammalian nuclear proteins with high sequence homology to yeast replication proteins. We present the entire amino acid sequence of the human protein P1 as predicted from the cDNA sequence, and show that P1 shares three central regions of high sequence similarity (about 75%) and a highly hydrophilic carboxy-terminal region with the yeast Mcm3 replication protein. The human genome most probably contains one P1 gene which is activated when HeLa cells progress to S phase, as shown by a several-fold increase in P1-specific mRNA. However, the amounts of P1 protein do not detectably change during this period, but P1 protein becomes phosphorylated at the beginning of S phase. In contrast to the yeast Mcm proteins, which disappear from nuclei after initiation of DNA replication, protein P1 remains in the nucleus during and after S phase. P1 is dispersed in mitotic cells and may be excluded from binding to chromosomes.

Amino Acid Sequence↗
PubMed2005

Minichromosome maintenance protein 3 elicits a cancer-restricted immune response in patients with brain malignancies and is a strong independent predictor of survival in patients with anaplastic astrocytoma.

PURPOSE: The identification of new molecular markers in astrocytic tumors may help to understand the biology of these tumors in more detail. Informative tumor markers may represent prognostic factors for response to therapy and outcome as well as potential targets for novel anticancer therapies. EXPERIMENTAL DESIGN: Tumor-associated antigens were identified by immunoscreening of a human glioma cDNA expression library with allogeneic sera from patients with diffuse astrocytoma (WHO grades 2-4). The expression of one of the identified antigens, the replication licensing factor minichromosome maintenance protein 3 (MCM3), was analyzed by immunohistochemistry in 142 primary and 27 recurrent astrocytomas (WHO grades 2-4). In addition, 98 serum specimens from patients with primary and secondary brain malignancies and 30 serum specimens from healthy controls were examined by serologic immunoscreening for immunoreactivity with MCM3. RESULTS: MCM3 is overexpressed in human astrocytic tumors and elicits a cancer-restricted humoral immune response in 9.3% (9 of 97) of patients with brain tumors (n = 95) and brain metastases (n = 2) but not in healthy controls. Expression of MCM3 in diffuse astrocytoma is significantly associated with age (P < 0.001), histologic grade (P < 0.001), time to recurrence (P = 0.01), and expression of the proliferation marker Ki-67 (P < 0.001) but not with sex (P = 0.800). Univariate and multivariate Cox regression analysis confirmed MCM3 expression as an independent predictor of poor outcome in astrocytoma patients (P < 0.001 for both). CONCLUSIONS: MCM3 may represent a glioma-associated antigen with significant prognostic role as well as have some potential as a target for cancer-directed therapy.

Adult↗
PubMed2004

Cancer-associated expression of minichromosome maintenance 3 gene in several human cancers and its involvement in tumorigenesis.

PURPOSE: The purpose of our study was to identify an unique gene that shows cancer-associated expression, evaluates its potential usefulness in cancer diagnosis, and characterizes its function related to human carcinogenesis. EXPERIMENTAL DESIGN: We used the differential display reverse transcription-PCR method with normal cervical, cervical cancer and metastatic tissues, and cervical cancer cell line to identify genes overexpressed in cancers. RESULTS: We identified a minichromosome maintenance 3 (MCM3) gene that was overexpressed in various human cancers, including leukemia, lymphoma, and carcinomas of the uterine cervix, colon, lung, stomach, kidney and breast, and malignant melanoma. Western blot and immunohistochemical analyses also revealed that MCM3 protein was elevated in most of human cancer tissues tested. We compared the MCM3 protein expression levels in human cancers with conventional proliferation markers, Ki-67 and proliferating cell nuclear antigen. MCM3 antibody was the most specific for multiple human cancers, whereas proliferating cell nuclear antigen was relatively less effective in specificity, and Ki-67 failed to detect several human cancers. The down-regulation of MCM3 protein level was examined under serum starvation in both normal and cancer cells. Interestingly, MCM3 protein was stable in MCF-7 breast cancer cells even up to 96 hours after serum starvation, whereas it was gradually degraded in normal BJ fibroblast cells. Nude mice who received injections of HEK 293 cells stably transfected with MCM3 formed tumors in 6 weeks. CONCLUSIONS: Our study indicates that determination of MCM3 expression level will facilitate the assessment of many different human malignancies in tumor diagnosis, and MCM3 is involved in multiple types of human carcino-genesis.

Animals↗
PubMed1999

Involvement of cyclin D1-cdk5 overexpression and MCM3 cleavage in bax-associated spontaneous apoptosis and differentiation in an A253 human head and neck carcinoma xenograft model.

Time-dependent ladder-type DNA fragmentation and morphological alterations consistent with apoptosis were observed among A253 human head and neck squamous cell carcinoma (HNSCC) cells in nude mice from 15 to 18 days after transplantation, without any drug treatment. No evidence of ladder-type DNA fragmentation was detected in A253 cells in vitro or in normal nude mouse tissues (skin and muscle). Our aim was to explore molecular factors associated with such spontaneous apoptosis. Bcl-2 protein expression decreased, while bax protein expression increased from day 9 after transplantation. Moreover, altered expression of bcl-2 and bax was accompanied by the increased proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Time-dependent dephosphorylation of Rb, followed by proteolytic cleavage, was also observed from day 9 after transplantation. The data indicate that the caspase-3 activation and cleavage of Rb protein may represent important steps in the regulation pathway of bax-mediated spontaneous apoptosis. Interestingly, the time-dependent activation of spontaneous apoptosis was almost simultaneous with the induction of differentiation and increased expression of several differentiation-associated regulatory proteins. An increased expression of cyclin D1 and cyclin-dependent kinase-5 (cdk5) was observed from day 9 after transplantation, whereas only slight alteration of cdk4 expression was found. The time-dependent activation of cyclin D1 and cdk5 preceded both the induction of ladder-type DNA fragmentation and increased keratin pearl formation. Furthermore, MCM3 was cleaved early in spontaneous apoptosis and differentiation. Our observations suggest the involvement of cyclin D1-cdk5 overexpression and MCM3 cleavage in bax-mediated spontaneous apoptosis and differentiation in A253 xenografts. P53 and WAF1 proteins were not expressed in the xenografts, indicating that the changes in the regulatory proteins during apoptosis and differentiation were not p53 or WAF1 dependent.

Animals↗
PubMed1994

DNA polymerase alpha associated protein P1, a murine homolog of yeast MCM3, changes its intranuclear distribution during the DNA synthetic period.

We isolated a murine gene for the DNA polymerase alpha associated protein P1, which shares high homology with the budding yeast MCM3 protein, which is a member of a protein family involved in the early event of DNA replication having a putative DNA-dependent ATPase motif. Using a polyclonal anti-P1 antibody raised against a beta-galactosidase-P1 fusion protein, we identified at least two forms of P1 protein in the nucleus of a mouse cell line, an underphosphorylated form that was associated with a particular nuclear structure and a hyperphosphorylated form loosely bound to the nucleus. During progression through S phase, P1 disappeared, first from the euchromatic region, then from the heterochromatic region, apparently in parallel with temporally ordered DNA replication. Thus, it is likely that the underphosphorylated P1 is dissociated from the nuclear structure after DNA replication by cell cycle-dependent phosphorylation. This is the first direct observation of a protein whose behavior is consistent with that of a hypothetical factor which restricts the chromatin to replicate once per cell cycle in higher eukaryotes.

Amino Acid Sequence↗
PubMed2001

The expression of Ki-67, MCM3, and p27 defines distinct subsets of proliferating, resting, and differentiated cells.

The mini-chromosome maintenance proteins (MCM), which are involved in the control of DNA replication, and the cyclin-dependent kinase inhibitors, such as p27/KIP1, represent two groups of proteins that are currently under investigation as diagnostic tumour markers. The expression of p27 and MCM3 was compared with the expression of the Ki-67 protein, an approved marker for proliferating cells, extensively used in histopathology and cancer research. The expression pattern of all three proteins was assessed on germinal centres and oral mucosa, which display a well-defined spatio-temporal organization. The expression of the p27 protein was closely related to differentiated cells, whereas MCM3 and Ki-67 were predominantly localized to the regions of proliferating cells. However, it is important to note that considerable numbers of cells that were growth-arrested, as confirmed by the absence of the Ki-67 protein, stained positive for the MCM3 protein. These results were verified in vitro using growth-arrested Swiss 3T3. The MCM3 protein is therefore expressed in cells that have ceased to proliferate, but are not terminally differentiated, according to the absence of p27 protein expression. In conclusion, a combined analysis of Ki-67, MCM3, and p27 protein expression may provide a more detailed insight into the cell proliferation and differentiation processes that determine individual tumour growth.

Animals↗
PubMed1998

Protein kinase inhibition in G2 causes mammalian Mcm proteins to reassociate with chromatin and restores ability to replicate.

Intact nuclei from G2-phase mammalian cells will replicate their DNA in Xenopus egg extract if they are preexposed to the protein kinase inhibitor 6-dimethylaminopurine in vivo (Coverley et al., Exp. Cell Res. 225, 294-300, 1996). Here, we demonstrate that this competence to rereplicate is accompanied by alterations in the subcellular distribution of the Mcm family of proteins, which are implicated in replication licensing (Hennessy et al., Genes Dev. 4, 2252-2263, 1990; Kubota et al., Cell 81, 601-609, 1995; and Chong et al., Nature 375, 418-421, 1995). All family members reassociate with chromatin in G2 cells and this correlates closely with regeneration of replication competence. Moreover, newly bound Mcm proteins are functional for replication because, unlike untreated G2 nuclei, replication of treated G2 nuclei in vitro occurs independent of the Xenopus Mcm protein complex. These observations show that the postreplicative state is actively maintained in G2 cells by a protein kinase(s) which regulates the behavior of Mcm family proteins.

Adenine↗
PubMed1998

Selective proteolysis of the nuclear replication factor MCM3 in apoptosis.

Cleavage of specific protein subsets is a key event in the execution of apoptosis. Protein degradation may serve for the structural alterations that result in cell self-destruction, but it may also function as a switch in the decisions between apoptosis and necrosis or apoptosis and cell proliferation. Here, we show that MCM3, but not other members of the Mcm family of replicative proteins, is cleaved early in several models of apoptosis. Cleavage of MCM3 can be prevented by caspase inhibitors, and it does not occur when cells are forced to undergo necrosis by energy deprivation. We propose that active destruction of MCM3 inactivates the Mcm complex and serves to prevent untimely DNA replication events during the execution of the cell death program.

Apoptosis↗
PubMed1998

Stability of the replicative Mcm3 protein in proliferating and differentiating human cells.

Mcm proteins are abundant nuclear proteins involved in the regulation of genome replication. Previous experiments had shown that levels of Mcm-specific mRNAs increase at the G1/S phase transition of the cell cycle, but that the amounts of Mcm proteins do not change much during the cell cycle. To learn more about the stability of an Mcm protein we performed experiments which showed that: (i) more than 60% of [35S]methionine pulse-labeled Mcm3 protein appears to be degraded during a 24-h chase in HeLa cells; (ii) the amount of Mcm3 protein significantly decreases during the differentiation of HL60 cells in vitro (whereas another replication-initiation protein, hOrc2, remains fairly constant); and (iii) according to immunohistochemical staining, Mcm3 protein is present in nuclei of cells in the proliferating zone of human epidermal tissue, but in decreasing amounts in nuclei of differentiating cells of the upper cell layers. Our interpretation is that Mcm3 protein is no longer synthesized after initiation of differentiation and slowly disappears at a half-life of approximately 24 h.

Cell Cycle Proteins↗
PubMed1995

Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor.

Replication licensing factor is thought to be involved in the strict control of the initiation of DNA replication in eukaryotes. We identified a 100 kDa protein as a candidate for the licensing factor in Xenopus egg extracts. This protein was required for replication; it bound to sperm DNA before the formation of nuclei and apparently dissociated from the nuclear DNA during the progression of replication without being transported into the nuclei. An immunologically homologous protein in HeLa cells behaved similarly to the Xenopus protein during the cell cycle. Cloning and sequencing of the cDNAs encoding the Xenopus and human proteins revealed that they are homologs of yeast Mcm3, a putative yeast DNA replication licensing factor.

Amino Acid Sequence↗