The effect of probucol on intimal thickening of autologous vein grafts in hyperlipidemic rabbit

OBJECTIVE: To define whether there is any relation between the iron status of patients with hepatitis C virus (HCV) chronic liver disease and their response to interferon therapy. DESIGN: To evaluate the long-term response to 1 year of interferon therapy with addition of phlebotomies after 3 months of treatment if at that time alanine aminotransferase (ALT) had not normalized in a group of patients with HCV-positive chronic liver disease whose iron status had been characterized. SETTING: A northern Italian hospital. PARTICIPANTS: Fifty-eight anti-HCV-positive patients (four HCV-RNA negative) with biopsy proven chronic hepatitis and no evidence of iron overload as indicated by normal transferrin saturation at the time of enrollment in the study. INTERVENTION: Three times a week intramuscular injection of alpha interferon 3 MU for 1 year with addition of phlebotomies (350 ml/week) till iron depletion if after 3 months of interferon therapy ALT had not normalized. RESULTS: A long-term response was observed in 19 of the 52 patients who completed the treatment, four HCV-RNA negative and 15 positive. The four RNA-negative and seven of the 15 RNA-positive long-term responders had been treated with interferon alone, and the other eight also with phlebotomies. At univariate analysis only HCV genotype, gamma-glutamyltranspeptidase and liver iron concentration were significantly associated with response whereas sinusoidal iron deposition was of borderline significance. No association was found with sex, age, duration of disease, histology, Knodell score, transferrin saturation %, serum ferritin, hepatocytic iron score, and portal iron score. HCV-RNA serum levels, measured in 29 patients, did not correlate with response. At multivariate analysis liver iron concentration was still significant and one unit reduction of liver iron concentration (natural logarithm transformed) was associated with 2.95 odds ratio of response. CONCLUSION: These results indicate that iron in the liver is more closely related to response to interferon than the other variables considered, including HCV characteristics.     

Luminal foam cell accumulation is associated with smooth muscle cell death in the intimal thickening of human saphenous vein grafts

BACKGROUND: Occlusion of saphenous vein grafts is a major problem after coronary artery bypass graft surgery. Diffuse intimal thickening develops in all implanted aortocoronary saphenous vein grafts within 6 months to 1 year. In some regions of the thickened intima, foam cells accumulate along the luminal margin. This particular morphology resembles the morphology of unstable atherosclerotic plaques as they occur in coronary arteries. In the present study, we focused on the possible topographic relation between luminal foam cell accumulation and cell death of smooth muscle cells (SMCs) within the adjacent thickened intima. METHODS AND RESULTS: Segments of occluded and suboccluded implanted human aortocoronary saphenous vein grafts were obtained during reintervention coronary artery bypass graft surgery in 30 patients. In the regions of the vein grafts with luminal foam cell accumulation, the percentage of SMC alpha-actin immunoreactive area of the superficial intimal thickening was 6 +/- 1.4%, which was different from the 17.6 +/- 2.3% of the deep intimal thickening. A strong negative correlation between the number of foam cell nuclei and the percentage of SMC alpha-actin immunoreactive area in the adjacent superficial intimal thickening was present (r = -.77, P < .001). Within the superficial intimal thickening, cytoplasmic and DNA fragmentation could be detected, which points to apoptotic cell death. A fraction of the cytoplasmic fragments fitted the ultrastructural characteristics of matrix vesicles and showed pronounced calcium and phosphorus accumulation as demonstrated with the use of x-ray microanalysis. CONCLUSIONS: The close spatial relation among foam cell accumulation, pronounced intimal SMC loss, and cell death suggests the presence of a foam cell-derived factor that can induce cell death in the SMC population of the intimal thickening. The depletion of the intimal SMC population could promote plaque rupture and thrombotic complications in the grafts.     

Determination of the epitope of an inhibitory antibody to proliferating cell nuclear antigen

We identified an expansion of the CAG trinucleotide repeat in the coding region of the Machado-Joseph disease gene in 7 of 24 American families diagnosed with autosomal dominant ataxia. All affected individuals were heterozygous for an expanded allele that ranged from 67 to more than 200 CAG repeats, whereas the normal allele had 14 to 33 repeats. In contrast to the Azorean-Portuguese origins of Machado-Joseph disease, the two largest American families were of German and Dutch-African descent. Clinical, pathologic, and genetic evaluations suggest that American families with spinocerebellar ataxia type 3 differ from those with Machado-Joseph disease by their ethnic origins, predominant spinopontine atrophy, lack of dystonic features, and larger CAG repeat expansion.     

Proliferative potential of meningiomas evaluated by proliferating cell nuclear antigen expression

Meningiomas are principally benign in nature. Some meningiomas, however, grow fast or recur even after total removal. The biological behavior of meningiomas often can not be predicted from conventional histopathological studies. A monoclonal antibody against proliferating cell nuclear antigen (PCNA) was used to investigate the usefulness of the PCNA index as a parameter to estimate the proliferative activity of meningiomas. Fifty-two meningiomas were examined. The mean PCNA index of recurrent meningiomas (3.37 +/- 0.92%) was significantly higher than that of non-recurrent meningiomas (1.12 +/- 0.51%) (p < 0.005). The PCNA indices of recurrent cases were all higher than 2.0%. A semilog linear regression analysis between tumor doubling time and PCNA index showed a significant correlation (r = 0.90, p < 0.05). An inverse linear correlation between PCNA index and interval to recurrence was observed (r = 0.62, p < 0.05). A good linear correlation was also shown between PCNA index and BUdR labeling index (r = 0.88, p < 0.01). The results of this study suggest that, providing the methods of tissue processing, immunostaining and counting of positive nuclei are unified, the PCNA index is a useful parameter for estimating the biological behavior of meningiomas.     

Expression of proliferating cell nuclear antigen in corneas kept in long term culture

PURPOSE: To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. METHOD: Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. RESULT: Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. CONCLUSION: Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.     

Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.     

p53-mediated regulation of proliferating cell nuclear antigen expression in cells exposed to ionizing radiation

BACKGROUND: The complications associated with anterior craniofacial resections for benign and malignant tumors were reviewed in 104 patients treated between January 1981 and June 1996. METHODS: Information regarding patient characteristics, histologic type, history of prior therapy, extent of the disease, extent of surgical procedure, and type of reconstruction were entered in a microcomputer database. To better understand and stage postoperative complications, we divided them into early (<14 days) and late (>14 days) according to the time of presentation, into major and minor depending on the morbidity potential of complication, and into local and systemic ones. Comparison between risk factors associated with complications was made using chi-square analysis with Yates' correction for continuity. Survival analysis was performed using the Kaplan-Meier product limit method. RESULTS: There were 8 (7.6%) postoperative deaths, with only 1 occurring from systemic complications. Complications occurred in 53 (48.6%) patients. Local major complications occurred in 49 (45%) patients, local minor in 29 (26.6%), and systemic in 11 (10%). Early complications occurred in 40 (38.5%) patients and late complications in 13 (12.5%) patients. These complications developed during a period ranging from 1 day to 5 months. More than one complication occurred in a number of patients. Bacterial contamination leading to local septic complications was the principal cause of morbidity, accounting for 54.7% (29/53) of complications. Major complications included meningitis in 8 patients associated with cerebrospinal fluid leak in 7, cerebral abscess in 2, sepsis in 1, and subdural hemorrhage in 1, all of which resulted in death except for one case. The extent of the craniofacial resection (p = .011) was the most important factor associated with major complications. Invasion of the dura and the type of reconstruction of the anterior skull base were the most important factors related to cerebrospinal fluid leakage (p = .048 and p = .032) and meningitis (p = .011). CONCLUSION: Contemporary surgical approaches and methods of reconstruction have enabled skull base surgeons to extend their cranial base resections and increase the 5-year survival rates of patients. Nevertheless, significant complications persist. Knowledge and high index of suspicion together with early recognition of these complications are essential for effective management of patients undergoing craniofacial resection. The factors related to major complications found in this study stressed the need to develop more effective methods to prevent contamination of intracranial structures.     

A unique form of proliferating cell nuclear antigen is present in malignant breast cells

Despite extensive research efforts to identify unique molecular alterations in breast cancer, to date, no characteristic has emerged that correlates exclusively with malignancy. Recently, it has been shown that the multiprotein DNA replication complex (synthesome) from breast cancer cells has a significantly decreased replication fidelity compared to that of nonmalignant breast cells. Proliferating cell nuclear antigen (PCNA) functions in DNA replication and DNA repair and is a component of the synthesome. Using two-dimensional PAGE analysis, we have identified a novel form of PCNA in malignant breast cells. This unique form is not the result of a genetic alteration, as demonstrated by DNA sequence analysis of the PCNA gene from malignant and nonmalignant breast cells. This novel form is most likely the result of an alteration in the post-translational modification of PCNA in malignant breast cells. These findings are significant in that it is now possible to link changes in the fidelity of DNA replication with a specific alteration of a component of the DNA synthetic apparatus of breast cancer cells. The novel form of PCNA may prove to be a new signature for malignant breast cells.     

Chimeric DNA-RNA hammerhead ribozyme to proliferating cell nuclear antigen reduces stent-induced stenosis in a porcine coronary model

BACKGROUND: Stent-induced coronary restenosis is a major clinical and public health problem. Proliferating cell nuclear antigen (PCNA) is an important regulator of cell division, and blocking of its expression after angioplasty may limit intimal proliferation. METHODS AND RESULTS: We cloned the porcine PCNA gene and constructed a chimeric hammerhead ribozyme to a segment of the gene with human homology. In vitro studies with both cultured porcine and human vascular smooth muscle cells demonstrated uptake of ribozyme within the nucleus and significant inhibition of cellular proliferation. The ribozyme was then delivered locally into pig coronaries in a stent model. At 30 days, histomorphometric analysis showed neointimal thickness of 0.51+/-0.20 mm in the ribozyme group versus 0.71+/-0.27 and 0.66+/-0.25 mm in stent controls and scrambled ribozyme control, respectively (P=0.002, P=0.03). Quantitative angiographic analysis showed late loss of 1.4+/-0.5 mm for ribozyme versus 1.9+/-0.4 and 2.0+/-0.4 mm for the controls (P=0.05 and P=0. 02). CONCLUSIONS: Chimeric hammerhead ribozyme to PCNA inhibits smooth muscle cell proliferation in vitro and reduces both histomorphometric and angiographic restenosis in the porcine coronary stent model when delivered locally.     

Proliferative activity of gastric cancer assessed by immunostaining for proliferating cell nuclear antigen

The growth activity of 107 gastric carcinomas was assessed by immunohistochemical staining for formalin-fixed, paraffin-embedded tissue with a monoclonal antibody against proliferating cell nuclear antigen (PCNA). When the tumor doubling times (Tds) of 10 patients were estimated from the serum levels of carcinoembryonic antigen and carbohydrate antigen 19-9, there was an inverse correlation between the Tds and PCNA labeling index (LI) at P = 0.055. Flow-cytometric analysis was carried out by double staining for PCNA and DNA using fresh materials from 14 patients. The PCNA-positive cell fraction revealed by flow cytometry showed a good linear correlation with PCNA LI in routinely stained tissue. The LI of well-differentiated adenocarcinoma was significantly higher than that of the poorly differentiated type. When the LI was analyzed in well- or poorly differentiated adenocarcinoma, the value was significantly higher in the well-differentiated type with hepatic metastasis and in the poorly differentiated type with lymph node metastasis.     

Replication factor C interacts with the C-terminal side of proliferating cell nuclear antigen

Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and repair. It is a molecular matchmaker required for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and, thus, for PCNA-dependent DNA elongation by DNA polymerases delta and epsilon. To elucidate the mode of RF-C binding to the PCNA clamp, modified forms of human PCNA were used that could be 32P-labeled in vitro either at the C or the N terminus. Using a kinase protection assay, we show that the heteropentameric calf thymus RF-C was able to protect the C-terminal region but not the N-terminal region of human PCNA from phosphorylation, suggesting that RF-C interacts with the PCNA face at which the C termini are located (C-side). A similar protection profile was obtained with the recently identified PCNA binding region (residues 478-712), but not with the DNA binding region (residues 366-477), of the human RF-C large subunit (Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messner, H., Khastilba, S., Hübscher, U., and Fotedar, A., (1996) EMBO J., 15, 4423-4433). Furthermore, we show that the RF-C 36 kDa subunit of human RF-C could interact independently with the C-side of PCNA. The RF-C large subunit from a third species, namely Drosophila melanogaster, interacted similarly with the modified human PCNA, indicating that the interaction between RF-C and PCNA is conserved through eukaryotic evolution.     

Molecular genetic and biochemical analysis of Brassica napus proliferating cell nuclear antigen function

A cDNA encoding the proliferating cell nuclear antigen (PCNA) from Brassica napus (oilseed rape) was shown to complement the lethal deletion mutation in the PCNA gene (delta POL30) of Saccharomyces cerevisiae. We provide unequivocal evidence that the B. napus PCNA can perform all the essential functions of the yeast PCNA in DNA replication, although some species-specific differences may exist. In addition, the B. napus PCNA expressed as a fusion polypeptide with glutathione S-transferase (GST) was shown to stimulate the activity and processivity of two delta-like DNA polymerases from wheat in vitro. These experiments provide direct biochemical evidence that the B. napus PCNA may function as an auxiliary factor in plant cell DNA replication.     

Trisomy 21 placentas: histopathological and immunohistochemical findings using proliferating cell nuclear antigen

OBJECTIVE: The cause of growth retardation in trisomy 21 and other autosomal trisomies is not known, but may be the result of defective cell proliferation, slowing of the cell cycle, or placental structural abnormalities. Abnormalities of the fetal cell cycle may be reflected in placental growth and can be detected using proliferating cell nuclear antigen (PCNA). METHODS: Twelve second-trimester and six third-trimester trisomy 21 placentas were examined histopathologically and stained immunohistochemically using antibodies to PCNA. Normal age-matched placentas were used as controls. RESULTS: The second-trimester trisomy 21 placentas all exhibited many large irregular hypovascular villi. The third-trimester trisomy 21 placentas showed two patterns: (i) many large, irregular hypovascular villi, and (ii) relatively normal-appearing villi with only a few abnormal villi and focal hypervascularity. PCNA staining was significantly greater in second-trimester placentas when compared to third-trimester placentas for both trisomy 21 and controls. There was no significant difference in PCNA staining in trisomy 21 placentas when compared to the normal age-matched controls. CONCLUSIONS: PCNA staining indicates no significant differences in proliferation between normal and trisomy 21 placentas. Trisomy 21 placentas show villus abnormalities, including hypovascularity.     

Expression of proliferating cell nuclear antigen (PCNA) in premalignant lesions of laryngeal epithelium

It is important to study the proliferative activity of cells in the premalignant lesions of laryngeal epithelium. Using PC-10, an antibody to PCNA and a standard immunohistochemical staining, we examined 11 cases of simple hyperplasia of epithelium (SHE), 32 cases of atypical hyperplasia of epithelium (AHE) and 42 cases of laryngeal squamous cell carcinoma (LSCC) for expression of PCNA, a protein associated with DNA polymerase delta and DNA replication. The results revealed that the PCNA indices in SHE, AHE and LSCC were 9.57%, 27.33% and 68.05%, respectively. The PCNA indices were 13.79%, 30.84% and 39.94%, in the mild, moderate and severe AHE, respectively. There were significant differences among the SHE, AHE and LSCC. A good correlation was found among different degrees of AHE. The pattern and location of PCNA positive cells in the intraepithelium had diagnostic importance. PCNA might provide a useful tool for studying cell proliferation in situ under normal and pathological conditions.     

Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.     

Marimastat inhibits neointimal thickening in a model of human vein graft stenosis

BACKGROUND: There is now accumulating evidence that matrix metalloproteinases (MMPs), the physiological mediators of matrix deposition and degradation, play an important role in the development of intimal hyperplasia following arterial bypass. This study investigated the effect of marimastat, an orally active specific MMP inhibitor, on neointima formation in cultured human saphenous vein. METHODS: Segments of human saphenous vein obtained from ten patients undergoing arterial bypass surgery were cultured for 14 days in serum-supplemented RPMI medium (controls) or in control medium supplemented with marimastat at three different concentrations (treatment groups). Following culture, half of each segment was prepared for histological examination and MMPs were extracted from the other half for gelatin zymography. RESULTS: Marimastat inhibited neointimal thickening in a concentration-dependent manner; inhibition was significant at 10(-5) and 10(-6) mol/l (P=0.006). This observation was paralleled by a significant reduction in the levels of MMP-2 and MMP-9 in the tissues. CONCLUSION: Marimastat significantly reduced neointimal thickening in this laboratory model. MMP inhibitors may offer a potential therapeutic strategy in the prevention of intimal hyperplasia.