Proliferating cell nuclear antigen (PCNA) is a sliding clamp that acts as a central co-ordinator for mismatch repair as well as DNA replication. Loss of Elg1, the major subunit of the PCNA unloader complex, causes over-accumulation of PCNA on DNA and also increases mutation rate, but it has been unclear if the two effects are linked. In this study, I showed that timely control of PCNA retention on DNA by Elg1 replication factor C-like complex (Elg1-RLC) ensures correct mismatch repair. Although premature unloading of PCNA generally increases mutation rate, PCNA mutants PCNA-R14E and PCNA-D150E that spontaneously fall off DNA attenuate the mutator phenotype of elg1Δ. In contrast, PCNA-D21K that accumulates on DNA due to enhanced electrostatic PCNA-DNA interactions exacerbates the elg1Δ mutator phenotype. Next, I addressed how accumulation of PCNA on DNA increases mutation rate. Epistasis analysis suggests that PCNA over-accumulation on DNA predominantly prevents the Msh2-Msh6-dependent and Exo1-independent mismatch repair pathways. In elg1Δ, over-retained PCNA hyper-recruits the Msh2-Msh6 mismatch recognition complex through its PCNA-interacting peptide motif, causing accumulation of mismatch repair intermediates. The results suggest that PCNA retention controlled by the Elg1-RLC is critical for efficient mismatch repair: PCNA needs to be on DNA long enough to enable mismatch repair, but if it is retained too long it interferes with downstream repair steps.

In the normal ectocervix, mitoses are rare and are usually confined to the basal layers. In contrast, they occur more frequently in cervical intraepithelial neoplasia (CIN) and are seen at higher levels, suggesting that CIN may be associated with a progressive dysfunction in proliferative activity of cervical cells. The objective of this study was to use proliferating cell nuclear antigen (PCNA) immunohistochemistry to examine the proliferative activity of cervical epithelial cells in CIN lesions. Sixty- eight cervical biopsies were examined; 20 were totally benign, 14 had CIN I, 21 CIN II, and 13 CIN III. In benign epithelia, PCNA staining was usually confined to the basal layers, whereas in CIN the staining was seen at progressively higher levels of the epithelium. There was a statistically significant correlation between the CIN grade and the highest level of PCNA staining (PCNA grade, r = 0.746, P < 0.001). In addition, the PCNA grade showed significant correlation with the highest level at which mitoses were seen (mitosis grade, r = 0.706, P < 0.001), and a strong direct correlation between the mitosis and CIN grades was also observed (r = 0.955, P < 0.001). These data demonstrate that (1) PCNA immunoreactivity in neoplastic cervical epithelium is different from that seen in the normal cervix, suggesting that CIN is associated with a dysfunctional proliferation of cervical epithelium, (2) that there is a significant correlation between the PCNA grade and CIN grades, and (3) the 'mitosis grades' have a strong correlation with the CIN grades.

by Chung Hoi Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 92-106). / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iii / ACKNOWLEDGMENT --- p.v / Chapter 1. --- Introduction / Chapter 1.1 --- Angiogenesis in general --- p.1 / Chapter 1.1.1 --- The concept of angiogenesis --- p.1 / Chapter 1.1.2 --- The process of angiogenesis --- p.1 / Chapter 1.2 --- Measurement of angiogenesis --- p.3 / Chapter 1.2.1 --- In vivo assays --- p.3 / Chapter 1.2.2 --- In vitro assays --- p.5 / Chapter 1.3 --- Angiogenic factors --- p.6 / Chapter 1.4 --- Angiogenesis in the female reproductive system --- p.7 / Chapter 1.5 --- Evidence of hormonally-regulated angiogenesis in endocrine tissues --- p.10 / Chapter 1.5.1 --- Ovary --- p.10 / Chapter 1.5.2 --- Thyroid --- p.11 / Chapter 1.6 --- Angiogenesis in the testis --- p.12 / Chapter 1.6.1 --- Structure of testicular vasculature --- p.12 / Chapter 1.6.2 --- Angiogenic factors in the testis --- p.13 / Chapter 1.6.3 --- Vascular effects of hCG/LH in the testis --- p.17 / Chapter 1.6.4 --- Postnatal development of testicular vasculature --- p.17 / Chapter 1.7 --- Aims of the present study --- p.19 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Animals --- p.20 / Chapter 2.2 --- Experimental design --- p.20 / Chapter 2.2.1 --- Testicular angiogenesis in adult rats - hormonal stimulation by hCG --- p.20 / Chapter 2.2.1.1 --- Changes with time after hCG treatment --- p.20 / Chapter 2.2.1.2 --- Effect of Leydig cell depletion --- p.22 / Chapter 2.2.1.3 --- Effect of Leydig cell suppression by subcutaneous testosterone-filled silastic implants --- p.22 / Chapter 2.2.1.4 --- Effect of testicular macrophage activation --- p.24 / Chapter 2.2.1.5 --- Effect of testicular macrophage depletion --- p.26 / Chapter 2.2.2 --- Developmental changes in testicular angiogenesis --- p.29 / Chapter 2.3 --- Perfusion of testes with fixative or Indian Ink --- p.29 / Chapter 2.4 --- Processing of the testes for histological sections --- p.30 / Chapter 2.5 --- Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) --- p.31 / Chapter 2.6 --- Immunohistochemical staining for vascular endothelial growth factor --- p.32 / Chapter 2.7 --- Quantification of PCNA-positive endothelial cells --- p.33 / Chapter 2.8 --- Quantification of blood vessel density --- p.34 / Chapter 2.9 --- Estimation of intertubular area in testis section --- p.35 / Chapter 2.10 --- Preparation of liposome-entrapped dichloromethylene diphosphonate (Cl2MDP-lp) --- p.38 / Chapter 2.11 --- Radioimmunoassay of serum tsetosterone --- p.38 / Chapter 2.12 --- Statistical analyses --- p.40 / Chapter 3. --- Results / Chapter 3.1 --- hCG-induced increase in endothelial cell proliferation in adult rat testes --- p.41 / Chapter 3.1.1 --- Testicular histology --- p.41 / Chapter 3.1.2 --- Changes in the number of PCNA-positive endothelial cells --- p.41 / Chapter 3.1.3 --- Changes in blood vessel density --- p.44 / Chapter 3.1.4 --- Changes in testis weight and serum testosterone concentration --- p.44 / Chapter 3.2 --- Effect of Leydig cell depletion by ethane dimethane sulphonate (EDS) on hCG-induced endothelial cell proliferation in adult rat testes --- p.48 / Chapter 3.2.1 --- Testicular histology --- p.48 / Chapter 3.2.2 --- Changes in the number of PCNA-positive endothelial cells --- p.48 / Chapter 3.2.3 --- Changes in serum testosterone concentration and testis weight --- p.52 / Chapter 3.3 --- Effect ofLeydig cell suppression by testosterone-filled subcutaneous silastic implants on hCG-induced endothelial cell proliferation in adult rat testes --- p.54 / Chapter 3.3.1 --- "Changes in serum testosterone concentration, testis weight, and testicular intertubular area" --- p.54 / Chapter 3.3.2 --- Changes in the number of PCNA-positive endothelial cells --- p.58 / Chapter 3.3.3 --- Changes in the level of vascular endothelial growth factor (VEGF) immunoreactivity in the testis --- p.60 / Chapter 3.4 --- Effect of testicular macrophage activation by polystyrene latex beads on hCG-induced endothelial cell proliferation in adult rat testes --- p.60 / Chapter 3.4.1 --- Testicular histology --- p.60 / Chapter 3.4.2 --- Changes in the number of PCNA-positive endothelial cells --- p.63 / Chapter 3.4.3 --- Changes in testis weight and serum testosterone concentration --- p.65 / Chapter 3.5 --- Effect of testicular macrophage depletion by liposome-entrapped C12MDP treatment on hCG-induced endothelial cell proliferation in adult rat testes --- p.67 / Chapter 3.5.1 --- Testicular histology --- p.68 / Chapter 3.5.2 --- Changes in the number of PCNA-positive endothelial cells --- p.68 / Chapter 3.5.3 --- Changes in testis weight and serum testosterone --- p.72 / Chapter 3.6 --- Endothelial cell proliferation in rat testes during postnatal development --- p.74 / Chapter 3.6.1 --- Changes in the number of PCNA-positive endothelial cells --- p.74 / Chapter 3.6.2 --- Changes in blood vessel density --- p.74 / Chapter 3.6.3 --- Changes in testis weight and intertubular area of the testes --- p.77 / Chapter 4. --- Discussion / Chapter 4.1 --- hCG-induced endothelial cell proliferation and changes in blood vessel density --- p.79 / Chapter 4.2 --- Role of Leydig cells in hCG-induced endothelial cell proliferation in adult rat testes --- p.82 / Chapter 4.3 --- Role of testicular macrophages in hCG-induced endothelial cell proliferation in adult rat testes --- p.86 / Chapter 4.4 --- Testicular angiogenesis during postnatal development --- p.88 / Chapter 5. --- References --- p.92

Abstract The maintenance of integrity of the genome is essential for a cell. DNA repair and faithful DNA replication ensure the stability of the genome. DNA polymerases (pols) are the enzymes that synthesise DNA, a process important both in DNA replication and repair. In DNA replication DNA polymerases duplicate the genome during S phase prior to cell division. Pols α, δ, and ε are implicated in chromosomal DNA replication, but their exact function in replication is not yet completely clear. The mechanisms of different repair pathways and proteins involved are not yet completely characterised either. The deeper understanding of DNA repair and replication mechanisms is crucial for our understanding on the function of the cell. The mechanism of repair of DNA double strand breaks (DSBs) by non-homologous end joining (NHEJ) was studied with an in vitro assay. DNA polymerase activity was found to be involved in NHEJ and important in stabilising DNA ends. Antibodies against pol α, but not pol β or ε, decreased NHEJ significantly, which indicates the involvement of pol α in NHEJ. In addition, the removal of proliferating cell nuclear antigen (PCNA) slightly decreased NHEJ activity. The division of labour between pols α, δ, and ε during DNA replication was studied. Results from UV-crosslinking, chromatin association, replication in isolated nuclei, and immunoelectron microscopy (IEM) studies showed that there are temporal differences between the activities and localisations of the pols during S phase. Pol α was active throughout S phase, pol ε was more active at early S phase, whereas the activity of pol δ increased as S phase advanced. These results suggest that pols δ and ε function independently during DNA replication. Pol ε could be crosslinked to nascent RNA, and this labelling was not linked to DNA replication, but rather to transcription. Immunoprecipitation studies indicated that pol ε, but not pols α and δ, associated with RNA polymerase II (RNA pol II). Only the hyperphosphorylated, transcriptionally active RNA pol II was found to associate with pol ε. A large proportion of pol ε and RNA pol II colocalised in cells as determined with immunoelectron microscopy. The interaction between pol ε and RNA pol II suggests that they are involved in a global regulation of transcription and DNA replication.

Abstract Automated quantitative computer-assisted morphometric analysis of immunohistochemical expression of markers of neoplastic development and progression in experimentally induced and in human neoplasms showed very high sensitivity and reproducibility, allowing analysis of large numbers of cell and tissue components. Totals of 26 million pixels, 25,000 cells and 1500 vessels were examined, with a sensitivity exceeding 99% and reproducibility exceeding 99%. The total expression of proliferating cell nuclear antigen (PCNA) and p53 increased consistently during 7H-dibenz[c, g] carbazole (DBC)-induced formation of dysplasias and squamous cell carcinomas (SCC:s) in hamster lung. In dysplasia, nuclear size and PCNA staining intensity increased; in SCC:s nuclear size decreased. In a retrospective study on archival material of human laryngeal squamous cell carcinomas, the occurrence and location of PCNA-positive cells were specifically related to the degree of differentiation. In SCC:s nuclear size decreased, while shape alterations and PCNA staining intensity increased in relation to degree of malignancy. In DBC-induced respiratory carcinogenesis increased collagen matrix synthesis occurred prior to neoplasm development. Among squamous cell carcinomas, in well-differentiated tumors, collagen deposition increased, as did fiber size, in moderately differentiated tumors collagen synthesis and the deposition of new collagen decreased. The increase in transforming growth factor beta expression in differentiated cells and in the matrix was isoform-specific. Increased angiogenesis in laryngeal tumor development occurred in preneoplastic states and in SCC: s, inversely related to the degree of differentiation. In well-differentiated neoplasms the vessels were lying in the direction of the BM, in moderately differentiated neoplasms vessels were lying in the direction of tumor invasion and in poorly differentiated neoplasms irregular, partly abnormal vessels intermixed with tumor cells. Small regular vessels predominated in benign conditions and large, irregular vessels in malignant conditions. Experimental models provided the advantage of examining homogenous, well-characterized neoplasm progression without interfering with the process. Morphometric methods provided detailed information on large numbers of cells, useful for studies of tumor behavior and with potential clinical applications.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G(1) cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Purpose: Meningiomas are common intracranial tumors, often well controlled with surgical resection alone. While the efficacy of radiation therapy in improving local control and progression-free survival is well documented, prognostic data substantiate factors that are predictive of poor local control following definitive radiation therapy. PCNA is a DNA polymerase expressed at the highest levels in the S-phase, the most resistant portion of the cell cycle to ionizing radiation in vitro. We investigated the possible correlation between the levels of PCNA expression and the clinical outcome of patients treated with definitive radiation therapy. Methods and Materials: Archival tissue was collected from 33 cases of meningioma treated at our institution for definitive radiation therapy between 1970 and 1990. Age-matched normal meningeal tissue and asymptomatic meningiomas removed at autopsy served as tissue controls. A standard ABC immumoperoxidase technique employing antibodies to PCNA, PC-10 (Dako, California) was used to stain specimen slides for PCNA. PCNA index was defined as the number of positive nuclei per 10 high-power fields at 400x magnification. Two independent observers scored the slides without prior knowledge of the cases at hand. Results: Patients with high PCNA index were less likely to be controlled by therapeutic radiation (p < 0.001, Kaplan- Meier). All patients with a PCNA index greater that 25 failed radiation therapy. Using multivariate analyses, malignant (but not atypical), histology and PCNA index were significant predictors of progression following radiation therapy (p < 0.05, log rank). Conclusion: PCNA index may be a useful adjunct to more standard histopathologic criteria in the determination of meningioma local control and progression-free survival following therapeutic irradiation. Data on a more expanded population evaluated on a prospective basis will be needed before such criteria are routinely employed in the clinical setting.

Several cDNAs isolated from brains of diapausing pupae of the flesh fly, Sarcophaga crassipalpis, show expression patterns unique to diapause. To isolate such cDNAs a diapause pupal brain cDNA library was screened by using an elimination hybridization technique, and cDNAs that did not hybridize with cDNA probes constructed from the RNA of nondiapausing pupae were selected for further screening. The 95 clones that did not hybridize in the initial library screen were selected for further characterization. These clones were then screened against diapause and nondiapause pupal poly(A)+ Northern blots. The secondary screen identified 4 diapause-up-regulated clones, 7 diapause-down-regulated clones, 8 clones expressed equally in both diapause and nondiapause, and 75 clones without detectable expression. The diapause- up-regulated and down-regulated clones were further characterized by partial DNA sequencing and identity searches by using GenBank. Identities between our cloned cDNAs and other genes included those linked to cell cycle progression, stress responses, and DNA repair processes. The results suggest that insect diapause is not merely a shutdown of gene expression but is a unique, developmental pathway characterized by the expression of a novel set of genes.

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