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PubMed1998

Human papillomavirus oncoprotein E6 binds to the C-terminal region of human minichromosome maintenance 7 protein.

Oncoprotein E6 of the human papillomavirus (HPV) associated with cervical cancer (HPV-16 and -18) degrades tumor suppressor protein p53, but seems to have p53-independent transforming functions. We searched for other cellular targets for the N-terminal region of HPV-16 E6 using a yeast two-hybrid system. The E6 was found to bind to the C-terminal region of a human minichromosome maintenance 7 (hMCM7) protein, which is a component of replication licensing factors. The full-length hMCM7 translated in vitro was capable of binding to bacterially expressed E6. In yeast cells the E6s of the cancer-associated HPVs (HPV-16, -18, and -58) bound to hMCM7 more strongly than those of the HPVs associated with a benign tumor (HPV-6 and -11). Binding of E6 with hMCM7 may cause chromosomal abnormalities found in the human cells expressing E6s of oncogenic HPVs.

Binding Sites↗

PubMed2000

The MAT1 cyclin-dependent kinase-activating kinase (CAK) assembly/targeting factor interacts physically with the MCM7 DNA licensing factor.

MAT1 (ménage à trois1) functions as an assembly/targeting factor of CAK (cyclin-dependent kinase-activating kinase). In a search for MAT1-interacting proteins using yeast two-hybrid system, MCM7 (minichromosome maintenance 7), a member of a family of DNA licensing factors, was identified. The physical interaction between MAT1 and MCM7 was confirmed in vivo in yeast cells and verified with in vitro protein binding assays. Further studies showed the RING-finger motif of MAT1 is not required for the interaction with MCM7, while the C-terminal domain of MAT1 is indispensable. Immunoprecipitation of MCM7 in human osteosarcoma MG63 cells demonstrated that MCM7 associates with the CAK complex in vivo.

Amino Acid Motifs↗

PubMed2006

Expression of aromatase and 17beta-hydroxysteroid dehydrogenase types 1, 7 and 12 in breast cancer. An immunocytochemical study.

It is known that there is a local biosynthesis of estradiol (E2) in breast carcinoma. The steroidogenic enzymes involved in E2 formation are aromatase which transforms testosterone into E2 and androstenedione into estrone (E1) and reductive 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) which convert E1 into E2. Using immunocytochemistry, we have studied the expression of aromatase and the three reductive 17beta-HSDs 17beta-HSD types 1, 7 and 12 in 41 specimens of female human breast carcinoma and adjacent non-malignant tissues. These results were correlated with the estrogen receptor alpha (ERalpha) and beta (ERbeta), progesterone receptor, androgen receptor, CDC47 and c-erb B-2 expressions and with the tumor stages. Aromatase was found in 58%, 17beta-HSD type 7 in 47% and 17beta-HSD type 12 in 83% of the breast cancer specimens. The 17beta-HSD type 1 could be detected in only one tumor. A significant correlation was observed between the aromatase, 17beta-HSD type 7 and 17beta-HSD type 12 expression, as well as between each of the two enzymes 17beta-types 7 and 12 and the ERbeta expression. The expression of 17beta-HSD type 12 was significantly higher in breast carcinoma specimens than in normal tissue. There was also a significant association of CDC 47 expression with ERbeta, AR and 17beta-HSD type 12. The results indicate that aromatase, 17beta-HSD type 7 and 17beta-HSD type 12, but not 17beta-HSD type 1, are commonly expressed in human breast cancer. Moreover, the high expression of both 17beta-HSD type 12 and ERbeta in breast carcinoma cells may play a role in the development and/or progression of breast cancer.

17-Hydroxysteroid Dehydrogenases↗

PubMed2003

Comparative analysis of cervical cancer in women and in a human papillomavirus-transgenic mouse model: identification of minichromosome maintenance protein 7 as an informative biomarker for human cervical cancer.

Human papillomaviruses (HPVs), such as HPV-16, are associated with >99% of cervical cancers in women. Two viral oncogenes, E6 and E7, are selectively expressed in these cancers. K14E6 and K14E7 transgenic mouse strains, which express the HPV16 E6 or E7 gene in stratified squamous epithelia, display many acute and long-term phenotypes indicative of the oncogenic potential of E6 and E7 including epithelial hyperplasia, abrogation of normal DNA damage responses, and spontaneous skin tumors. When treated with estrogen, these HPV-16 transgenic mice develop a progressive disease leading to cervical cancer that shows many histopathological parallels to cervical cancer in women. In this study, we evaluated the cervical lesions that arise in these transgenic mice for the expression of biomarkers induced in human cervical cancer. These analyses, which showed close parallels in the timing and pattern of expression of cyclin E and Ki-67 in the mouse cervical disease compared with that in humans, provided further validation of this HPV-16 transgenic mouse model for human cervical cancer. We then used our mouse model to identify minichromosome maintenance protein 7 (MCM7), an E2F-induced cellular DNA replication factor, as a novel biomarker for cervical cancer. In both the mouse and human disease, strong, full thickness staining for MCM7 was seen selectively in the epithelium of high-grade intraepithelial lesions and in frank cancer. The uniform staining pattern and strong signal for MCM7 suggest that MCM7 may be a highly informative biomarker for cervical cancer.

Animals↗

PubMed2004

Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle.

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G(1) and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G(1). MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G(2), reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G(2), at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G(1) followed by a recruitment to multiple sites in chromatin subsequent to replication.

Binding Sites↗

PubMedDate not supplied

Immunohistochemical evaluation of minichromosome maintenance protein 7 in astrocytoma grading.

The minichromosome maintenance (MCM) proteins, which play an important role in eukaryotic DNA replication, represent a group of proteins that are currently under investigation as novel diagnostic tumor markers. Several studies have proved a greater reliability of MCM proteins to stain proliferating cells compared to Ki67 protein, a routinely used proliferation marker in histopathology. In the present study, the expressions of MCM7 and Ki67 were estimated in 66 primary human astrocytomas in relation to tumor grade (Grade I-IV, WHO). MCM7 significantly stained more nuclei compared to Ki67 in all the histopathological grades investigated. In addition, a stronger increase of the MCM7 labelling index, in relation to the tumor aggressiveness, was observed.

Astrocytoma↗

PubMed2001

Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures.

The minichromosome maintenance (Mcm) proteins 2-7 are required for both the initiation and elongation steps of chromosomal DNA replication. Previous studies have shown that the Mcm complex consisting of the Mcm 4, 6, and 7 proteins contains 3' to 5' DNA helicase activity with limited processivity (displacing duplex DNA regions up to 30 nt). In this report, we show that the presence of both 5' and 3' single-stranded tails in DNA helicase substrates is essential for the processive helicase activity of the Mcm complex. The presence of both 5' and 3' tails facilitated the formation of double heterohexameric complexes of Mcm4/6/7 on substrate DNA, which appeared to be essential for the processive helicase activity. The double heterohexameric complex of Mcm4/6/7, in the presence of a single-strand DNA binding protein, is capable of unwinding duplex DNA region of about 600 bp in length. These results support the hypothesis that the Mcm4/6/7 complex can function as a replication helicase.

Cell Cycle Proteins↗

PubMedDate not supplied

Minichromosome maintenance protein 7: a reliable tool for glioblastoma proliferation index.

At present there is increasing evidence concerning the value of minichromosome maintenance (MCM) protein expression as a novel indicator of proliferation. In the present study, 15 glioblastoma samples, classified according to WHO, were analysed to evaluate the expression of the principal proliferation markers. The samples examined were subdivided into 2 cytological subsets, small cell (SC) or multiforme cell (MC) glioblastoma, according to the predominant cell type defined in individual specimens. MCM7 detected more cells in the cycle than Ki67 and PCNA and all cases of SC glioblastoma, the most aggressive subset, displayed a significant increase of MCM7-stained nuclei versus those stained with Ki67. These results suggest that the cell cycle-associated proteins MCM are not only useful markers of proliferation, but also valid aids for diagnosis in cerebral glioblastoma.

Adult↗

PubMed2001

Nearest neighbour analysis of MCM protein complexes in Drosophila melanogaster.

The MCM proteins are a group of six proteins whose action is vital for DNA replication in eukaryotes. It has been suggested that they constitute the replicative helicase, with a subset of the proteins forming the catalytic helicase (MCM4,6,7) while the others have a loading or control function. In this paper we show that all six MCM proteins are present in equivalent amounts in soluble extracts and on chromatin. We have also analysed soluble and chromatin-associated MCM protein complexes under different conditions. This suggests that all six MCM proteins are always found in a complex with each other, although the interaction between the individual MCM proteins is not equivalent as stringent salt conditions are able to break the intact complex into a number of stable subcomplexes. These data contribute to the ongoing debate about the nature of MCM complexes, supporting the hypothesis that they act as a heterohexamer rather than as a number of different subcomplexes. Finally, using protein-protein cross-linking we have shown that MCM2 interacts directly with MCM5 and MCM6; MCM5 with MCM3 and MCM2; and MCM6 with MCM2 and MCM4. This provides the first direct information about specific subunit contacts in the MCM complex.

Animals↗

PubMed1998

E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box.

Ubiquitin ligases are generally assumed to play a major role in substrate recognition and thus provide specificity to a particular ubiquitin modification system. The multicopy maintenance protein (Mcm) 7 subunit of the replication licensing factor-M was identified as a substrate of the E3-ubiquitin ligase/E6-AP by its interaction with human papillomavirus-18E6. Mcm7 is ubiquitinated in vivo in both an E6-AP-dependent and -independent manner. E6-AP functions in these reactions independently of the viral oncogene E6. We show that recognition of Mcm7 by E6-AP is mediated by a homotypic interaction motif present in both proteins, called the L2G box. These findings served as the basis for the definition of substrate specificity for E6-AP. A small cluster of proteins whose function is intimately associated with the control of cell growth and/or proliferation contains the L2G box and is thereby implicated in an E6-AP and, by default, HPV-E6-dependent ubiquitination pathway.

Amino Acid Sequence↗

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↗

PubMed1999

The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum DeltaH contains DNA helicase activity.

Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance (MCM) complex, composed of MCM subunits 4, 6, and 7 [Ishimi, Y. (1997) J. Biol. Chem. 272, 24508-24513]. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum DeltaH (mth) genome contains a single open reading frame coding for an MCM protein. In this study we report the isolation of the mthMCM protein overexpressed in Escherichia coli. The purified recombinant protein was found to exist in both multimeric ( approximately 10(3) kDa) and monomeric (76 kDa) forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3'-to-5' ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to our understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication.

Adenosine Triphosphatases↗

PubMed2000

Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7.

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases of the Clb/Cdc28 family restrict the initiation of DNA replication to once per cell cycle by preventing the re-assembly of pre-replicative complexes (pre-RCs) at replication origins that have already initiated replication. This assembly involves the Cdc6-dependent loading of six minichromosome maintenance (Mcm) proteins, Mcm2-7, onto origins. How Clb/Cdc28 kinases prevent pre-RC assembly is not understood. RESULTS: In living cells, the Mcm proteins were found to colocalize in a cell-cycle-regulated manner. Mcm2-4, 6 and 7 were concentrated in the nucleus in G1 phase, gradually exported to the cytoplasm during S phase, and excluded from the nucleus by G2 and M phase. Tagging any single Mcm protein with the SV40 nuclear localization signal made all Mcm proteins constitutively nuclear. In the absence of functional Cdc6, Clb/Cdc28 kinases were necessary and sufficient for efficient net nuclear export of a fusion protein between Mcm7 and the green fluorescent protein (Mcm7-GFP), whereas inactivation of these kinases at the end of mitosis coincided with the net nuclear import of Mcm7-GFP. In contrast, in the presence of functional Cdc6, which loads Mcm proteins onto chromatin, S-phase progression as well as Clb/Cdc28 kinases was required for Mcm-GFP export. CONCLUSIONS: We propose that Clb/Cdc28 kinases prevent pre-RC reassembly in part by promoting the net nuclear export of Mcm proteins. We further propose that Mcm proteins become refractory to this regulation when they load onto chromatin and must be dislodged by DNA replication before they can be exported. Such an arrangement could ensure that Mcm proteins complete their replication function before they are removed from the nucleus.

Biological Transport↗

PubMed2006

Identification, molecular cloning, and cellular distribution of the rat homolog of minichromosome maintenance protein 7 (MCM7) in the rat testis.

As part of a program to decipher the rat testicular proteome, we studied spermatogonia and identified numerous proteins including the human homolog of the Minichromosome Maintenance Protein 7 (MCM7). MCM7 has been implicated in DNA replication in various species, but had not been detected in the testis. Here we describe the cellular distribution of MCM7 transcripts and protein, and their testicular ontogenetic expression. The full-length coding region of the rat MCM7 was also characterized. Northern blot analyses showed that MCM7 transcripts are more abundant in the testis than other organs and confirmed the presence of the 2.4 kb MCM7 transcript at all ages studied. Interestingly, two additional transcripts of 3.2 and 1.6 kb were found from 26 days post partum onwards, when spermatocytes and spermatids accumulate within the tubules. This was confirmed in isolated cell types: the three MCM7 transcripts were observed in meiotic and post-meiotic germ cells. The 3.2 kb isoform has an extended 5' untranslated region (UTR) and the 1.6 kb transcript is the result of alternative splicing of five exons. Western blot and immunohistochemistry experiments evidenced abundant MCM7 in proliferating gonocytes and Sertoli cells in the fetal testis. In the adult testis, an intense signal was observed in spermatogonia and primary spermatocytes. We conclude that the Mcm7 is one example of genes that are differently transcribed and translated in somatic and spermatogenetic cells in mammals. Further work is required to determine the roles of MCM7 in spermatogonia and germ lineage.

Adenosine Triphosphatases↗

PubMed1997

Immunolocalization of hCDC47 protein in normal and neoplastic human tissues and its relation to growth.

hCDC47 is a human member of the MCM family, which has been implicated in the regulatory machinery causing DNA to replicate once per cell cycle. We examined its protein expression and localization in normal human tissues, using immunostaining with polyclonal antibodies. Positive nuclei were found in the proliferative components of lymph nodes, bone marrow, epidermis and mucosa. Immunohistochemical analysis was also performed for 3 types of cutaneous keratinocytic tumor originating from same cell type but showing different grades of malignancy. In seborrheic keratosis, a benign condition, cells with hCDC47-positive nuclei were located in the outermost layers of the tumor lobules, while in Bowen's disease, carcinomas in situ and squamous-cell carcinomas, they were present throughout the lesions. The percentages of hCDC47-positive cells were 65.4% in squamous-cell carcinomas, 60.9% in Bowen's disease, 12.6% in seborrheic keratosis and 3.9% in normal epidermis (n = 5 in all cases). Further expansion of the analysis to include malignant tumors from several other organs revealed that all malignant lesions tested contained more nuclear hCDC47-positive cells than their normal counterparts. Our findings indicate that hCDC47 plays a role in normal and neoplastic cell growth in vivo and that hCDC47 immunolocalization could be used as an index of cell proliferation in tissue sections.

Carcinoma in Situ↗

PubMed1996

Inhibition of S-phase entry of human fibroblasts by an antisense oligomer against hCDC47.

The MCM protein family has been implicated in the regulatory machinery causing DNA to replicate only once in the S phase. Recently, we have identified human (h) CDC47, another member of the family, and demonstrated its unique S-phase nuclear association pattern implying participation in the regulation of DNA replication. In the present study, we applied an antisense strategy to assess the phenotype of human cells in which hCDC47 is repressed. As a result serum-induced accumulation of hCDC47 protein in human fibroblast cells was reduced by a specific antisense oligodeoxynucleotide and S-phase entry of such cells was significantly inhibited, indicating that hCDC47 protein is essential for DNA replication.

Base Sequence↗

PubMed2001

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein.

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.

Alleles↗

PubMed2006

Diagnostic and prognostic value of cell-cycle regulatory genes in malignant thyroid neoplasms.

BACKGROUND: Approximately 30% of patients with thyroid nodules have indeterminate or suspicious fine-needle aspiration (FNA) biopsy results. Because their risk of cancer is approximately 20%, these patients undergo thyroidectomy. We hypothesized that genes that regulate cell-cycle progression would be differentially expressed in malignant versus benign thyroid nodules and could serve as diagnostic markers and markers of disease aggressiveness. METHODS: We used a cDNA array with 96 cell-cycle regulatory genes to identify differentially expressed genes in pooled benign versus malignant thyroid neoplasms. Genes up- or down-regulated by more than 2-fold in malignant thyroid neoplasms were further evaluated by real-time quantitative polymerase chain reaction (PCR) in 95 patients with hyperplastic nodules (n = 19), follicular adenoma (n = 19), follicular thyroid cancer (n = 19), the follicular variant of papillary thyroid cancer (n = 19), and papillary thyroid cancer (n = 19). RESULTS: cDNA array analysis showed that cyclin B1, MCM5, MCM7, RAD9, ubiquitin C, CDK6, SKP2, and APAF1 were up-regulated in malignant thyroid neoplasms. Real-time quantitative PCR showed that MCM5, MCM7, and RAD9 mRNA expression were significantly higher in malignant than in benign thyroid neoplasms (< or = 0.0012). The combined use of MCM5, MCM7, and RAD9 mRNA expression had a sensitivity of 98.2% and a specificity of 65.7%. The level of MCM7 mRNA expression was higher in T4 than in T1, T2, and T3 differentiated thyroid cancers (P < 0.0127). CONCLUSIONS: MCM5, MCM7, and RAD9 are overexpressed in malignant thyroid neoplasms of follicular cell origin. These genes may be useful markers of malignant thyroid neoplasms as an adjunct to FNA biopsy. MCM7 mRNA expression is higher in locally invasive differentiated thyroid cancer.

Adenocarcinoma, Follicular↗