DNA replication licensing and human cell proliferation
Kai Stoeber1,2,3,
Thea D. Tlsty4,
Lisa Happerfield1,
Geraldine A. Thomas5,
Sergei Romanov4,
Lynda Bobrow1,
E. Dillwyn Williams5 and
Gareth H. Williams1,*
1 Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
2 Wellcome/CRC Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
3 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
4 Department of Pathology, University of California San Francisco, School of Medicine, CA 94143-0506, USA
5 Thyroid Carcinogenesis Group, University of Cambridge, Strangeways Research Laboratory, UK
View larger version (23K):
[in a new window]
|
Fig. 1. Assembly of the pre-replicative complex renders chromatin competent for replication. In early G1 phase, the origin recognition complex (ORC) recruits Cdc6, which in turn promotes loading of MCM proteins onto chromatin. Activation of the Cdc7/Dbf4 kinase and S phase-promoting cyclin-dependent kinases (CDKs) induces a conformational change in the MCM complex that is required for unwinding of origin DNA and recruits Cdc45 to the pre-RC. Initiation of DNA replication occurs when replication protein A (RPA) and DNA polymerase  -primase are recruited to the unwound replication origin.
|
|
View larger version (54K):
[in a new window]
|
Fig. 2. Regulation of ORC, Cdc6 and MCM proteins in cycling human cells and following reversible withdrawal into the quiescent state. (A) Immunoblot analysis of Orc2, Cdc6 and Mcm5 in chromatin-bound and soluble fractions of HeLa S3 cells during the indicated times after release from nocodazole arrest in metaphase (left panels) or double thymidine block at G1/S (right panels). Cell cycle synchronisation was verified by flow cytometry of isolated nuclei. Cells began to enter S phase 10 hours (*) after release from nocodazole arrest, and the majority of cells reached G2 phase by 8 hours ( ) after release from a double thymidine block. The soluble fraction contains cytoplasmic and soluble nuclear proteins. (B) Immunoblot analysis of ORC, Cdc6 and MCM proteins in total cell extracts of asynchronously proliferating (AS) and contact-inhibited (G0) WI38 and NIH3T3 cells. Entry into quiescence was confirmed by BrdU labelling and antibody staining of asynchronously proliferating and contact-inhibited cultures (left panels; merged images of FITC (BrdU) and TOTO-3 (DNA) channels). (C) MCM protein expression in liver, an example of a stable tissue. The upper and lower panels show indirect immunoperoxidase staining of liver biopsies obtained from two patients undergoing liver transplantation with an anti-Mcm2 MAb (x160).
|
|
View larger version (99K):
[in a new window]
|
Fig. 3. MCM protein expression in differentiating and terminally differentiated tissues. (A,B) Indirect immunoperoxidase staining of colon with an anti-Mcm2 MAb. (A) The majority of Mcm2-expressing cells are located in the lower third of the crypt, which corresponds to the proliferative zone of the mucosa (x87). B, Notably, it appears to be the anatomical location corresponding to the transit amplifying population of cells that shows the highest levels of Mcm2 expression. The proportion of Mcm2-positive cells and the levels of Mcm2 expression decline in the middle third of the crypt, becoming undetectable in surface terminally differentiated cells (x156). (C) Indirect immunoperoxidase staining of adult brain with an anti-Mcm5 rabbit PAb. Neurones and glial cells lack Mcm5 expression (x170). (D) Hematoxylin and Eosin stained medulloblastoma showing sheets of undifferentiated mononuclear tumour cells containing well demarcated islands of glial differentiation (x68). (E,F) Indirect immunoperoxidase staining of medulloblastoma with an anti-neurofilament (NF) PAb (E) and anti-Mcm5 rabbit PAb (F) (x68). Mcm5 expression is strictly confined to the undifferentiated tumour cells with downregulation occurring in areas of differentiation.
|
|
View larger version (67K):
[in a new window]
|
Fig. 4. (A) Phase-contrast microscopy and immunofluorescence staining of primary human breast fibroblast (HMF48) and epithelial (HMEC48) cells derived from reduction mammoplasty specimens with anti-Mcm2 MAb. Nuclei are counterstained with DNA stain 4,6-diamidino-2-phenylindole (DAPI). (B) Immunoblots of Mcm2 and ß-actin in total cell extracts from isogenic sets of human breast fibroblasts (HMF48) and epithelial cells (HMEC48). HMF and HMEC growth curves and BrdU labelling indices indicate the proliferative state. (C) Immunoblot analysis of ORC, Cdc6, MCM and p21 proteins in total cell extracts of asynchronously proliferating (AS) and replicative senescent (SEN) WI38 HDF. (D) Giant cell tumour of bone (B) stained with Haematoxylin and Eosin (left panel) and with rabbit anti-Mcm5 polyclonal antibody (indirect immunoperoxidase staining, right panel) (x130). The tumour is composed of multinucleate giant cells (G) regularly dispersed in a background of small mononuclear (M) neoplastic cells. Mcm5 expression is restricted to the mononuclear neoplastic cell population.
|
|
View larger version (26K):
[in a new window]
|
Fig. 5. (A) Cell-free DNA replication system. Nuclei (N) prepared from G1 phase WI38 fibroblasts, synchronised by release from quiescence (G0), initiate a single round of semi-conservative DNA replication in cytosolic extracts (C) from S phase HeLa cells substituted with ribonucleoside and deoxyribonucleoside triphosphates (NTPs, dNTPs) and an energy regeneration system (creatine phosphate (CP) and phosphocreatine kinase (CK)). Nuclei are stained with propidium iodide to reveal DNA (red) and with fluorescein-streptavidin (green) to detect biotin-16-dUTP incorporation resulting from in vitro DNA synthesis (Stoeber et al., 1998). Results are expressed as the percentage of nuclei replicating and summarised in the histograms (mean+s.d.). Substitution of nuclear templates and/or extracts with subcellular components from quiescent (G0), terminally differentiated (not shown) or replicative senescent (SEN) cells provides a functional assay for analysis of the mechanisms that establish and/or maintain loss of replicative capacity in ‘out-of-cycle’ cells (B-D). (B) In vitro analysis of the replicative capacity of WI38 G1, quiescent and replicative senescent nuclear templates in either physiological buffer supporting elongation or S phase cytosol. Initiation of DNA replication in vitro is restricted to G1 phase nuclei. (C) In vitro analysis of the replicative capacity of WI38 G1 nuclear templates in quiescent, replicative senescent and S phase cytosolic extracts. S phase cytosolic extracts, but not quiescent or replicative senescent extracts, support efficient in vitro replication. (D) In vitro analysis of the replicative capacity of WI38 G1 nuclear templates in elongation buffer (EB), S phase cytosolic extracts and after titration of senescent cytosolic or nuclear extract into S phase cytosol. Titration of senescent nuclear extract but not nuclear extraction buffer (Buffer) alone resulted in striking inhibition of DNA replication initiation in vitro. Note that different proportions of S phase contaminants in WI38 G1 nuclear preparations in (B, 15%) and (D, <1%) relate to the use of two different batches of G1 nuclei.
|
|
View larger version (26K):
[in a new window]
|
Fig. 6. Three ways out of the cell cycle. The replication initiation factors ORC, Cdc6 and MCM are present throughout all phases of the proliferative cell cycle. In contrast, the Cdc6 and MCM components of the replication initiation pathway are downregulated in quiescent, terminally differentiated and replicative senescent ‘out-of-cycle’ states. Thus, Cdc6 and MCM proteins comply with the theoretical definition of a proliferation marker.
|
|
View larger version (142K):
[in a new window]
|
Fig. 7. Indirect immunoperoxidase staining of prostate and breast with anti-Ki67 and anti-Mcm2 monoclonal antibodies. (A,B) In prostate, only a small number of glandular epithelial cells express Mcm2 (A) and Ki67 (B), which are restricted to the basal cell population (x158). (C) A high proportion of mammary luminal epithelial acinar cells in resting pre-menopausal breast show Mcm2 expression (x158). (D) In contrast to Mcm2, Ki67 is only expressed in a small number of mammary luminal epithelial acinar cells in pre-menopausal breast (x158). (E) In breast tissue from pregnant females the proportion of Mcm2 expressing acinar cells increases and now also includes the outer myoepithlial cell population including some specialised stromal cells (x158). (F) In breast tissue from lactating females the majority of mammary acinar cells lack Mcm2 expression (x158).
|
|
© The Company of Biologists Ltd 2001
