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Results for “Minichromosome Maintenance Complex Component 2”

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PubMed2003

Surface expression of minichromosome maintenance proteins provides a novel method for detecting patients at risk for developing adenocarcinoma in Barrett's esophagus.

PURPOSE: The purpose is to determine whether a novel cell cycle marker, minichromosome maintenance protein 2 (Mcm2), predicted esophageal adenocarcinoma (AC) risk in Barrett's esophagus (BE) and whether this could be used in combination with a surface sampling method. EXPERIMENTAL DESIGN: Archival specimens [30 normal squamous esophagus (NE), 20 gastric antrum (GA), 13 duodenum (D2), 62 BE +/- dysplasia, and 16 (AC)] were stained for Mcm2. Sequential biopsies from nine patients who developed AC during surveillance were compared with 18 matched controls who did not progress. Prospective endoscopic cytological brushings (61 NE, 90 BE +/- dysplasia, and 11 AC) were scored as Mcm2 positive or negative. RESULTS: Mcm2 was not expressed on the luminal surface of NE, GA, and D2. In BE, the percentage of surface cells expressing Mcm2 correlated highly with the degree of dysplasia (P < 0.0001). In patients who developed AC, biopsies before dysplasia had higher Mcm2 expression than the matched control patients (mean, 28.4 and 3.4% positive cells, respectively, P < 0.0001). In the prospective cohort, the histopathological diagnosis of dysplasia or AC and the Mcm2-positive brushings were concordant in 91% of the patients (P < 0.005), and the results correlated with the frequency of cases with surface expression of Mcm2 in the retrospective study (P < 0.0001). CONCLUSIONS: Surface expression of Mcm2 can be used to detect dysplasia and AC, as well as patients with BE at risk for subsequent development of dysplasia and AC. A brushing technique combined with Mcm2 staining has the potential to be exploited in surveillance and screening protocols.

Adenocarcinoma↗

PubMed2003

Prognostic value of MCM2 immunoreactivity in stage T1 transitional cell carcinoma of the bladder.

OBJECTIVE: Due to the heterogeneous biologic behavior of stage T1 bladder carcinomas, there is a need for new markers allowing to assess the prognosis more accurately. To our knowledge, there are no reports on studies investigating minichromosome maintenance protein 2 (MCM2) expression in bladder carcinomas. Thus, we investigated the prognostic value of MCM2 immunoreactivity in stage T1 bladder tumors. METHODS: Fifty-four tumors were analyzed using Biochip microarrays. Also p53 and Ki67 antigen expression were examined. Immunohistochemical scores were compared with the clinical outcome. RESULTS: During a median follow-up of 43 months, tumor recurrence was registered in 43 and progression to stage T2 in 19 patients. Kaplan-Meier curves demonstrated that high-level MCM2 expression was significantly associated with early tumor recurrence when using a cutoff of 60% (p=0.0035 by log-rank test), and with early tumor progression when using a cutoff of 20% (p=0.0454). There was no relationship (p=0.604) between MCM2 and p53, but a tendentious relationship (p=0.082) between MCM2 and Ki67 antigen expression. MCM2 (p=0.006), Ki67 antigen (p=0.035) and p53 expression (p=0.049) as well as tumor grade (p=0.026) and age (p=0.025) were found significantly associated with recurrence-free survival by univariate Cox regression analysis, among which only Ki67 antigen expression (p=0.015) and age (p=0.019) proved to be of independent predictive value by multivariate analysis. Concerning tumor progression, MCM2 expression was identified as the only predictive parameter by log-rank test, but it was not of independent predictive value by multivariate analysis (p=0.101). CONCLUSION: Our data suggest that MCM2 expression may bear some prognostic relevance in stage T1 bladder carcinomas.

Adult↗

PubMed2005

Mcm2, Geminin, and KI67 define proliferative state and are prognostic markers in renal cell carcinoma.

PURPOSE: The origin licensing factors minichromosome maintenance 2 (Mcm2) and Geminin have recently been identified as critical regulators of growth and differentiation. Here we have investigated the regulation of these licensing factors together with Ki67 to further elucidate the cell cycle kinetics of renal cell carcinoma (RCC). Furthermore, we have examined the role of Ki67, Mcm2, and Geminin in disease-free survival after nephrectomy in patients with localized RCC. EXPERIMENTAL DESIGN: Tissue sections from 176 radical nephrectomy specimens were immunohistochemically stained with Mcm2, Geminin, and Ki67 antibodies. Labeling indices (LI) for these markers were compared with clinicopathologic parameters (median follow-up 44 months). RESULTS: In RCC, Mcm2 is expressed at much higher levels than Ki-67 and Geminin, respectively [medians 41.6%, 7.3%, and 3.5% (P < 0.001)] and was most closely linked to tumor grade (P < 0.001). For each marker, Kaplan-Meier survival curves provided strong evidence that increased expression is associated with reduced disease-free survival time (P < 0.001). Additionally, an Mcm2-Ki67 LI identified a unique licensed but nonproliferating population of tumor cells that increased significantly with tumor grade (P = 0.004) and was also of prognostic value (P = 0.01). On multivariate analysis, grade, vascular invasion, capsular invasion, Ki67 LI >12%, and age were found to be independent prognostic markers. CONCLUSIONS: Although Ki67 is identified as an independent prognostic marker, semiquantitative assessment is difficult due to the very low proliferative fraction identified by this marker. In contrast, Mcm2 identifies an increased growth fraction that is closely linked to grade, provides prognostic information, and is amenable to semiquantitative analysis in routine pathologic assessment.

Biomarkers, Tumor↗

PubMed2004

Telomerase expression and proliferative activity suggest a stem cell role for thyroid solid cell nests.

Solid cell nests of the human thyroid gland are composed of main cells and C cells. In order to investigate the putative stem cell nature of the role for solid cell nests, we evaluated the histological features, and the immunohistochemical expression of p63, bcl-2, telomerase catalytic subunit, and two proliferative markers (Ki-67 and minichromosome maintenance protein 2), in a series of 24 cases of solid cell nests. Proliferative indices were determined in (a) solid cell nests, (b) thyroid follicular cells in the vicinity of solid cell nests within a low-power field, and (c) distant thyroid tissue, at a distance of at least three low-power fields from solid cell nests. In 15 cases of solid cell nests (62.5%), mixed follicles were observed; papillary formations were observed in four cases (16.6%), and ciliated cells were observed in the lining of microcysts associated with two cases (8.3%). Salivary gland-type tissue, cartilage islands, adipose and fibrous tissues, and small nerves were also associated with some cases of solid cell nests. We observed that the main cells of the solid cell nests express consistently telomerase, although at lower levels than p63, and show strong cytoplasmic immunoreactivity for bcl-2, which is associated with an increased differentiation potential. We also observed that despite their relative low proliferative index, main cells of the solid cell nests display higher proliferation than follicular cells in the vicinity and follicular cells in more distant thyroid tissue. We conclude that main cells of the solid cell nests apparently harbor the minimal properties of a stem cell phenotype (capacity for both self-renewal, conferred by telomerase activity, and differentiation to one or more than one type of specialized cells, given by the high expression of p63 and bcl-2) and may thus represent a pool of stem cells of the adult thyroid.

Adult↗

PubMed2006

Replication competence and senescence in central and peripheral human corneal endothelium.

PURPOSE: To compare replication competence and senescence in human corneal endothelial cells (HCECs) between the central and peripheral areas and between younger and older donors. METHODS: Human corneas were obtained from the eye bank and separated into two groups: young (younger than 30 years) and old (older than 50 years). Corneas were cut in quarters and a 2-mm scrape wound was created in the endothelium from the periphery to the center. Unwounded endothelium acted as a negative control. Corneal pieces were incubated for 24, 36, 48, 60, 72, 84, and 96 hours in medium containing 8% fetal bovine serum (FBS) plus additional growth factors. Tissue was fixed, immunostained for minichromosome maintenance (MCM)-2, a marker of replication competence, and mounted in medium containing propidium iodide (PI) to visualize all nuclei. Fluorescence microscope images were used to count PI-stained and MCM2-positive HCECs in three 100-microm2 areas within the central and peripheral wound area. Results are expressed as mean number of cells/100 microm2. Senescent HCECs in ex vivo corneas were identified by staining for senescence-associated beta-galactosidase activity (SA-beta-Gal). Whole corneas were cut in quarters and incubated in staining solution containing SA-beta-Gal at pH 6.0. The number of cells stained for SA-beta-Gal and the grade of SA-beta-Gal intensity in three 100-microm2 areas were averaged for the central and peripheral areas from each donor. For all studies, results were compared between central and peripheral cornea and between younger and older donors. RESULTS: In both age groups (n = 4/group), cells repopulated the wound area in a time-dependent manner. In corneas from older donors, significantly fewer HCECs migrated into the wound bed in the central cornea than in the periphery. At each time point, the density of cells in the central wound area was lower in corneas from older donors than from younger donors. In both age groups, the mean percentage of MCM2-positive cells increased with time until wound healing. In both age groups, more MCM2-positive cells were present in the wounded area of the peripheral than of the central cornea. At 36, 48, 60, and 72 hours after wounding, the percentage of MCM2-positive cells in the central or peripheral area of older corneas was significantly less than in the corresponding region in younger corneas. No MCM2-positive staining was observed in unwounded areas at any time point. HCECs in corneas from younger donors (n = 4) showed little to no SA-beta-Gal activity in either the central or peripheral area. SA-beta-Gal activity was easily detectable in corneas from older donors (n = 4) and a significantly higher percentage of central HCECs showed strong SA-beta-Gal activity compared with HCECs in the periphery. CONCLUSIONS: In ex vivo corneas, HCECs from the peripheral area retain higher replication competence, regardless of donor age. HCECs in the central area of corneas from older donors retain replicative competence, but the relative percentage of cells that are competent to replicate is significantly lower than in the periphery or in the central area of corneas from younger donors. This reduction in replicative competence negatively correlates with the observed increase in the population of central HCECs exhibiting senescence-like characteristics.

Adolescent↗

PubMed2000

An MCM2-related gene is expressed in proliferating cells of intact and regenerating planarians.

The minichromosome maintenance (MCM2-7) gene family encodes conserved proteins, which are essential for DNA replication licensing in eukaryotes. They are abundant in proliferating cells, and specific MCM transcripts undergo cell cycle-dependent oscillations. Here we report the characterization of a planarian MCM2 homologue, DjMCM2, which represents the first molecular marker for detecting proliferating cells in planarians. DjMCM2-expressing cells are broadly distributed in the mesenchymal space of the body, with the exception of the cephalic region, and are preferentially accumulated in the peripheral area of the dorso-lateral mesenchyme, along the anteroposterior axis. During regeneration, no DjMCM2 transcripts are observed within the blastema, according to the current view that this structure is not a proliferation site in planarians. Spatio-temporal changes in DjMCM2 RNA expression pattern in the stump parallel blastema growth, coordinately with the orientation of the cut. X-ray irradiation results in the disappearance of DjMCM2 expression, thus confirming that these transcripts are detected specifically in proliferating cells, visualized as neoblasts by in situ hybridization in dissociated cells. In addition to neoblasts, rare large DjMCM2-expressing cells are observed in macerates of tissues excised just below the wound, suggesting that cell types other than neoblasts may be sporadically recruited for proliferation in planarians.

Amino Acid Sequence↗

PubMed1992

Chromosome detection by in situ hybridization in cancer cell populations which were flow cytometrically sorted after immunolabeling.

We examined the feasibility of performing non-radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly-l-lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling.

Antibodies, Monoclonal↗

PubMed2005

Distinct patterns of MCM protein binding in nuclei of S phase and rereplicating SV40-infected monkey kidney cells.

BACKGROUND: Simian Virus 40 (SV40) infection of growth-arrested monkey kidney cells stimulates S phase entry and the continued synthesis of both viral and cellular DNA. Infected cells can attain total DNA contents as high as DNA Index, DI = 5.0-6.0 (10-12C), with host cell DNA representing 70-80% of the total. In this study, SV40-infected and uninfected control cells were compared to determine whether continued DNA replication beyond DI = 2.0 was associated with rebinding of the minichromosome maintenance (MCM) hexamer, the putative replicative helicase, to chromatin. METHOD: Laser scanning cytometry was used to measure the total expression per cell and the chromatin/matrix-association of two MCM subunits in relation to DNA content. RESULTS: MCM2 and MCM3 proteins that were associated with the chromatin/matrix fraction in G1 phase of both uninfected and SV40-infected cells were gradually released during progression through S phase. However, in SV40-infected cells that progressed beyond DI = 2.0, chromatin/matrix-associated MCM2 and MCM3 remained at the low levels observed at the end of S phase. Rereplication was not preceded by an obvious rebinding of MCM proteins to chromatin, as was observed in G1 phase. CONCLUSIONS: The rereplication of host cell DNA in the absence of the reassociation of MCM proteins with chromatin indicates that SV40 infection induces a novel mechanism of licensing cellular DNA replication.

Animals↗

PubMed2003

A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer.

An immunohistochemical method for assessing cell-cycle phase distribution in colorectal resection specimens would enable phase data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in colorectal cancer. In contrast to flow cytometry, an immunohistochemical method would also allow the phase distribution to be examined within morphologically heterogeneous regions of neoplasms. Paraffin sections of normal colon (n = 25), colonic adenoma (n = 15), and colonic adenocarcinoma (n = 30) were analysed by immunohistochemistry using antibodies against markers of cell-cycle entry, Mcm-2 and Ki67, and putative markers of the cell-cycle phase, cyclins D1 and E (putative markers of G1 phase), cyclin A (S phase), cytoplasmic cyclin B1 (G2 phase), and phosphohistone H3 (M phase). The phase specificity of each marker was assessed by examining the degree of co-expression of adjacent phase markers using double-antibody fluorescence confocal microscopy and by comparison with flow cytometric analysis performed on adjacent tissue sections. The S-phase specificity of detectable cyclin A was also assessed in combination with in situ DNA replication using fluorescence confocal microscopy. All cells expressing phase markers co-expressed Mcm-2. Adjacent phase markers were not significantly co-expressed, confirming the relative specificity of these markers in tissue sections of colon. Cell-cycle phase distribution, calculated by immunohistochemistry, compared well with phase analyses obtained by flow cytometry. No cells expressed cyclin A in the absence of active DNA replication. The S-phase labelling index, as defined by detectable cyclin A expression, showed a positive correlation with the Mcm-2 labelling index and increased in the progression from normal colon to adenocarcinoma. In conclusion, a combination of these cell-cycle phase markers can be used to calculate the distribution of cells throughout each phase of the cell cycle in colorectal tissue sections. Detectable cyclin A can be used as a surrogate marker of S phase and may be of value in predicting prognosis and response to adjuvant therapy.

Adenocarcinoma↗

PubMed2005

DNA replication licensing in peripheral B-cell lymphoma.

Peripheral B-cell lymphomas representing 90% of lymphoid neoplasms are divided into low- and high-growth fraction lymphomas. Here we investigate regulation of DNA replication licensing during B-cell lymphomagenesis. Combined analysis of origin licensing factors Mcm2 and geminin with the proliferation marker Ki67 in SLL/CLL, MCL, DLBCL and Burkitt lymphoma reveals for the first time the precise cell cycle state of these entities. Given that tight Mcm2 downregulation defines the quiescent state (G0) and that both high- and low-growth fraction lymphomas express Mcm2, the data demonstrate that neoplastic lymphocytes of SLL/CLL and MCL reside in an "in-cycle" G1 state and not in G0 as previously thought. Absence of the S/G2/M phase marker geminin in SLL/CLL and MCL further indicates failure of cell cycle progression in these tumours. In contrast, the high-growth fraction lymphomas DLBCL and Burkitt lymphoma exhibit differential expression of geminin, with the geminin/Ki67 ratio increasing for more aggressive neoplasms in keeping with a shortened G1 phase and thus representing an important discriminator for differential diagnosis. These data provide new insights into abrogation of cell cycle control during B cell lymphomagenesis and suggest that combined analysis of origin licensing factors may contribute to improved treatment decisions and prognosis in haematopoietic malignancies.

Biomarkers, Tumor↗

PubMed1999

Identification, characterization and chromosomal localization of the cognate human and murine DBF4 genes.

The kinase Dbf4p/Cdc7p is required for the G1/S phase transition during the cell cycle and plays a direct role in the activation of individual origins of replication in Saccharomyces cerevisiae. Here, we report the identification and characterization of mouse and human cDNAs whose products are related in sequence to Saccharomyces cerevisiae DBF4 cDNA. Both mammalian Dbf4 proteins contain a putative site for phosphorylation by CDK, PEST protease cleavage sites, nuclear localization signals and a short-looped zinc finger-like domain. Transcription of MmDBF4 is suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. Upon replenishment of the medium, transcript levels increase during progression through G1, peaking as cells enter S phase. MmDbf4p interacts physically with Cdc7p and Mcm2p in vivo. Using fluorescence in situ hybridization (FISH), the human DBF4 gene was localized to chromosome 7 (q21.3), whereas FISH mapped the murine counterpart to band A2 on chromosome 5. The results of chromosome mapping indicate that in both mouse and human the gene is present as a single copy. The structural conservation between Dbf4-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes.

3T3 Cells↗

PubMed1996

Identification of two Xenopus laevis genes, xMCM2 and xCDC46, with sequence homology to MCM genes involved in DNA replication.

The Schizosaccharomyces pombe genes, nda1 and nda4, are essential for the normal regulation of DNA replication and belong to the MCM gene family. This gene family includes Saccharomyces cerevisiae MCM2, MCM3, MCM5/CDC46 and CDC47, S. pombe nda1, nda4, cdc21 and mis5, and genes encoding human BM28, P1MCM3 and P1.1MCM3 and mouse P1MCM3, most of which are considered to be required for the initiation of DNA replication. We isolated two homologues of the MCM genes, xMCM2 and xCDC46, from a Xenopus laevis cDNA library using the polymerase chain reaction (PCR) method. The predicted amino acid (aa) sequences of xMCM2 and xCDC46 are most similar to those of human BM28 (78% identity) and S. pombe Nda4 (48% identity), respectively. By Western blot analysis using anti-xMCM2 and anti-xCDC46 polyclonal antibodies (Ab) raised against glutathione S-transferase (GST)::xMCM2 or GST::xCDC46 fusion proteins, xMCM2 and xCDC46 were identified as 120- and 95-kDa proteins, respectively. When either xMCM2 or xCDC46 was immunoprecipitated with the specific Ab, the other was also co-precipitated. These results suggest that xMCM2 and xCDC46 physically interact with each other.

Animals↗

PubMed2004

ORC-associated replication factors as biomarkers for cancer.

Early detection and treatment of cancer are of central importance to improving patient prognoses. Traditional biomarkers of cell proliferation, such as Ki-67 and PCNA, have had a mixed clinical track record, proving to be good indicators of certain types of cancers but of limited use for many others. Recently, human counterparts of replication factors originally identified in budding yeast have shown great promise as new cancer biomarkers. Each of these factors has been shown to interact with the origin recognition complex (ORC) in yeast, and each has an essential role in the initiation of DNA replication. Studies with minichromosome maintenance (MCM) family proteins show that their levels are upregulated in tumor cells and are much better indicators of a wide variety of cancers than traditional biomarkers. Similarly encouraging results have been obtained in preliminary studies examining Cdc6 protein and Cdc7 kinase transcript levels in normal and cancerous cells.

Animals↗

PubMed2006

An immunohistochemical study of p16(INK4a) expression in multistep thyroid tumourigenesis.

Normal human thyroid follicular epithelial cells exhibit a very low proliferative rate which in vitro is dramatically increased by RAS oncogene activation, resulting in clones displaying a phenotype consistent with that of a ras-induced follicular adenoma in vivo. Eventual spontaneous cessation of growth of these clones is closely correlated with increasing expression of the tumour suppressor gene p16(INK4a), suggesting that p16 may limit clonal expansion in this tumour model. We therefore hypothesised that p16 expression would also increase in vivo in follicular adenomas, and further that escape from growth control in follicular cancers would be accompanied by loss of p16 expression. This was tested using tissue microarrays, representing multiple stages of thyroid tumourigenesis. Whereas the majority of normal thyroids showed no immunostaining, p16 protein was readily detectable in follicular adenomas. Unexpectedly, however, p16 expression was also observed in follicular and papillary carcinomas. Poorly differentiated (insular) carcinomas showed either very intense staining, or a complete loss of staining. We conclude that loss of p16 is not necessary for malignant transformation in thyroid follicular cells, but that it may form one of two or more events needed for progression to more aggressive forms of thyroid cancer.

Adenoma↗

PubMed2004

Pairwise interactions of the six human MCM protein subunits.

The eukaryotic minichromosome maintenance (MCM) proteins have six subunits, Mcm2 to 7p. Together they play essential roles in the initiation and elongation of DNA replication, and the human MCM proteins present attractive targets for potential anticancer drugs. The six MCM subunits interact and form a ring-shaped heterohexameric complex containing one of each subunit in a variety of eukaryotes, and subcomplexes have also been observed. However, the architecture of the human MCM heterohexameric complex is still unknown. We systematically studied pairwise interactions of individual human MCM subunits by using the yeast two-hybrid system and in vivo protein-protein crosslinking with a non-cleavable crosslinker in human cells followed by co-immunoprecipitation. In the yeast two-hybrid assays, we revealed multiple binary interactions among the six human MCM proteins, and a subset of these interactions was also detected as direct interactions in human cells. Based on our results, we propose a model for the architecture of the human MCM protein heterohexameric complex. We also propose models for the structures of subcomplexes. Thus, this study may serve as a foundation for understanding the overall architecture and function of eukaryotic MCM protein complexes and as clues for developing anticancer drugs targeted to the human MCM proteins.

Cell Cycle Proteins↗

PubMed2005

Expression of Ki67, PCNA and the chromosome replication licensing protein Mcm2 in glial cells of the ageing human hippocampus increases with the burden of Alzheimer-type pathology.

Cell-cycle mechanisms may be aberrantly reactivated in the ageing brain and associated with the development of pathology, including Alzheimer's disease. Activation of cell-cycle mechanisms in glia has, however, been little studied. Our aim was to determine whether expression of a marker for chromosomal replication licensing, Mcm2, occurs in glia of the ageing hippocampus, and to compare its expression to that of Ki67 and PCNA. Blocks of hippocampus were obtained from 19 elderly brains derived from the MRC-CFAS neuropathology cohort, which included a spectrum of Alzheimer-type pathology, semi-quantified using the Braak scoring system for neurofibrillary tangles. Mcm2, PCNA and Ki67 were detected immunohistochemically. Expression of Mcm2, Ki67 and PCNA was observed in glial cells and neurons, with a trend to increased expression in association with higher burdens of Alzheimer-type pathology. Mcm2 expression in glial cells showed a significant linear trend across Braak stages (P = 0.043). This study demonstrates that grey and white matter glial cells show expression of cell-cycle markers in the ageing brain and that re-licensing for chromosomal replication is a component of the mechanisms activated. A quantitative relationship to the burden of Alzheimer-type pathology suggests that cell-cycle re-entry in glial cells may be important in the pathogenesis of age-related neurodegeneration.

Aged↗

PubMed2005

Repression of DNA replication licensing in quiescence is independent of geminin and may define the cell cycle state of progenitor cells.

The DNA replication (or origin) licensing machinery ensures precise duplication of the genome and contributes to the regulation of proliferative capacity in metazoa. Using an in vitro fibroblast model system coupled to a cell-free DNA replication assay, we have studied regulation of the origin licensing pathway during exit from and re-entry into the mitotic cell cycle. We show that in the quiescent state (G0) loss of proliferative capacity is achieved in part through down-regulation of the replication licensing factors Cdc6 and Mcm2-7. The origin licensing repressor geminin is absent in quiescent fibroblasts, suggesting that this powerful inhibitor of the licensing machinery is not required to suppress proliferative capacity in G0. Geminin expression is induced at a late stage in the G0-S transition post pre-RC assembly. Ectopic geminin can block re-acquisition of DNA replication competence during re-entry into the cell cycle, indicating that geminin levels must be tightly down-regulated for escape from G0. Analysis of geminin levels in thyroid shows that geminin expression is suppressed in anatomical compartments/tissues harbouring quiescent cells, confirming our in vitro data. Spatio-temporal control of geminin expression may therefore be of particular relevance for multi-potential stem cells which cycle infrequently. In support of this hypothesis, we have identified a unique population of cells in the putative stem cell niche of intestinal epithelium that are unlicensed and lack geminin expression, a prerequisite for successful re-entry into cycle. Our data argue that the prolonged cell cycle times observed for intestinal stem cells could be due to exit of progenitor cells from cycle into an unlicensed "out-of-cycle" state, a powerful mechanism by which rapidly proliferating tissues may resist genotoxic insult.

3T3 Cells↗

PubMed2004

Cyclins and proliferation markers in early squamous cervical carcinoma.

OBJECTIVES: To examine the prognostic significance of the protein expression of cyclin E, cyclin A and cyclin D3, and of the proliferation markers Ki-67 and BM28 in early squamous cervical carcinoma (SCC). METHODS: Tissue blocks from 221 patients who underwent radical hysterectomy and bilateral lymphadenectomy at our institution for stage IB SCC between 1987 and 1993 were available for this study. Immunohistochemistry using monoclonal antibodies against cyclin E, cyclin A, cyclin D3, Ki-67 and BM28 was used to examine protein expression. Ten patients who underwent hysterectomy for uterine prolapse served as controls. RESULTS: Cyclin E, cyclin A, Ki-67 and BM28 expression was increased in SCC and high expression was observed in 81.5% (180/221), 35% (78/221), 25.5% (56/221) and 92% (204/221) of tumors, respectively. Cyclin D3 was decreased in SCC and low expression was found in 50.5% (111/220) of tumors. In univariate analysis, all classical clinicopathological parameters but none of the investigated proteins were associated with prognosis. In multivariate analysis, only deep stromal invasion was independently related to survival. CONCLUSION: Cyclin E, cyclin A and cyclin D3 and the proliferation markers Ki-67 and BM28 are not independently associated with prognosis in stage IB SCC.

Adult↗