Extracorporeal Shock Waves Increase Markers of Cellular Proliferation in Bronchial Epithelium and in Primary Bronchial Fibroblasts of COPD Patients.

Chronic obstructive pulmonary disease (COPD) is due to structural changes and narrowing of small airways and parenchymal destruction (loss of the alveolar attachment as a result of pulmonary emphysema), which all lead to airflow limitation. Extracorporeal shock waves (ESW) increase cell proliferation and differentiation of connective tissue fibroblasts. To date no studies are available on ESW treatment of human bronchial fibroblasts and epithelial cells from COPD and control subjects. We obtained primary bronchial fibroblasts from bronchial biopsies of 3 patients with mild/moderate COPD and 3 control smokers with normal lung function. 16HBE cells were also studied. Cells were treated with a piezoelectric shock wave generator at low energy (0.3 mJ/mm2, 500 pulses). After treatment, viability was evaluated and cells were recultured and followed up for 4, 24, 48, and 72 h. Cell growth (WST-1 test) was assessed, and proliferation markers were analyzed by qRT-PCR in cell lysates and by ELISA tests in cell supernatants and cell lysates. After ESW treatment, we observed a significant increase of cell proliferation in all cell types. C-Kit (CD117) mRNA was significantly increased in 16HBE cells at 4 h. Protein levels were significantly increased for c-Kit (CD117) at 4 h in 16HBE (p < 0.0001) and at 24 h in COPD-fibroblasts (p = 0.037); for PCNA at 4 h in 16HBE (p = 0.046); for Thy1 (CD90) at 24 and 72 h in CS-fibroblasts (p = 0.031 and p = 0.041); for TGFß1 at 72 h in CS-fibroblasts (p = 0.038); for procollagen-1 at 4 h in COPD-fibroblasts (p = 0.020); and for NF-κB-p65 at 4 and 24 h in 16HBE (p = 0.015 and p = 0.0002). In the peripheral lung tissue of a representative COPD patient, alveolar type II epithelial cells (TTF-1+) coexpressing c-Kit (CD117) and PCNA were occasionally observed. These data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in 16HBE cells and primary bronchial fibroblasts of COPD and control smoking subjects.

Hepatocyte regeneration after partial liver irradiation in rats.

Liver has a strong potential for regeneration after physical, biological or chemical injury, but no data have been reported so far on liver regeneration in response to irradiation injury. The present experiment in rats was designed to clarify whether partial liver irradiation could induce and stimulate the unirradiated part of liver to regenerate. The left-half of rat liver was irradiated with a single dose of 25Gy. Liver tissues from the irradiated and unirradiated liver, and blood sample were collected at different time points after irradiation. Radiation injury was evaluated by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, histopathologic features and trichrome stain. The hepatic regeneration was assessed by serum hepatic growth factor (HGF), mitotic index and proliferation cell nuclear antigen (PCNA) immunohistochemical stain. Expression of transforming growth factor-beta1 (TGF-beta1) by immunohistochemistry assay was also performed. Results showed that 25Gy of single-dose irradiation produced severe hepatic injury in the irradiated liver, and the unirradiated liver had been stimulated to regenerate, demonstrated by significant increases of serum HGF 30 days after irradiation, and increase of mitotic index and the number of PCNA-positive hepatocytes 60 days after irradiation. TGF-beta1 was strongly and uniformly expressed in the irradiated liver 90-day-post-irradiation, and it was also expressed slightly in unirradiated liver region. In summary, partial liver irradiation could stimulate the unirradiated liver to regenerate, and the role of TGF-beta1 in hepatic injury and proliferation needs further investigation.

The use of light-emitting diodes to stimulate mitochondrial function and liver regeneration of partially hepatectomized rats.

The biostimulating effect of laser radiation has been observed in many areas of Medicine. However, there are still several questions to be answered, among them the importance of light coherence in the stimulatory process. In the present study, we used light-emitting diodes (LED) to promote the stimulation of liver regeneration after partial hepatectomy in rats. Fourteen male Wistar rats weighing 200-250 g were submitted to partial hepatectomy (70%) followed by LED light irradiation (630 nm) of the remaining part of the liver at two doses, i.e., 10 (N = 7) and 140 (N = 7) J/cm(2). A group irradiated with laser, 590 nm (N = 7, 15 J/cm(2)) was performed for the study of proliferating cell nuclear antigen-labeling index. Data are reported as mean +/- SEM. Statistical comparisons of the groups were performed by analysis of variance for parametric measurements followed by the Bonferroni post-test, with the level of significance set at P < 0.05. Respiratory mitochondrial activity was increased in the irradiated groups (states 3 and 4; P < 0.05), with better results for the group exposed to the lower LED dose (10 J/cm(2)). The proliferating cell nuclear antigen-labeling index, by immunohistochemical staining, was similar for both LED-exposed groups (P > 0.05) and higher than for the control group (P < 0.05). The cell proliferation index obtained with LED and laser were similar (P > 0.05). In conclusion, the present results suggest that LED irradiation promotes biological stimulatory effects during the early stage of liver regeneration and that LED is as effective as laser light, independent of the coherence, divergence and cromaticity.

The use of light-emitting diodes to stimulate mitochondrial function and liver regeneration of partially hepatectomized rats

The biostimulating effect of laser radiation has been observed in many areas of Medicine. However, there are still several questions to be answered, among them the importance of light coherence in the stimulatory process. In the present study, we used light-emitting diodes (LED) to promote the stimulation of liver regeneration after partial hepatectomy in rats. Fourteen male Wistar rats weighing 200-250 g were submitted to partial hepatectomy (70 percent) followed by LED light irradiation (630 nm) of the remaining part of the liver at two doses, i.e., 10 (N = 7) and 140 (N = 7) J/cm². A group irradiated with laser, 590 nm (N = 7, 15 J/cm²) was performed for the study of proliferating cell nuclear antigen-labeling index. Data are reported as mean ± SEM. Statistical comparisons of the groups were performed by analysis of variance for parametric measurements followed by the Bonferroni post-test, with the level of significance set at P < 0.05. Respiratory mitochondrial activity was increased in the irradiated groups (states 3 and 4; P < 0.05), with better results for the group exposed to the lower LED dose (10 J/cm²). The proliferating cell nuclear antigen-labeling index, by immunohistochemical staining, was similar for both LED-exposed groups (P > 0.05) and higher than for the control group (P < 0.05). The cell proliferation index obtained with LED and laser were similar (P > 0.05). In conclusion, the present results suggest that LED irradiation promotes biological stimulatory effects during the early stage of liver regeneration and that LED is as effective as laser light, independent of the coherence, divergence and cromaticity.

An animal study of the effects on p16 and PCNA expression of repeated treatment with high-energy laser and intense pulsed light exposure.

BACKGROUND AND OBJECTIVE: Non-ablative skin rejuvenation treatments that involve the use of laser/light sources together with cooling devices have gained much popularity in recent years due to the lack of down time that is associated with them. One important but neglected issue is long-term safety. Does the repeated use of non-ablative skin rejuvenation lead to photoaging? Are we creating another sun-bed phenomenon? Recently, we performed an in vitro study to examine the effect of sub-lethal QS 755 nm lasers on the expression of p16INK4a on melanoma cell lines, and found that sub-lethal laser damage could increase DNA damage, which led to an increase in p16 expression. Our objective was to assess the cutaneous effect of repeated exposure to high-energy lasers and intense pulsed light sources on male Institute of Cancer Research (ICR) mice. STUDY DESIGN/MATERIALS AND METHODS: Twenty-eight male ICR mice were divided into four groups. Other than the control group, all groups received either laser (585 nm pulsed dye laser or 1,320 nm Nd:YAG laser) or intense pulsed light (IPL) treatment. All four groups were anesthetized with a mixture of Hypnorm/Dormicum before treatment. The animals were irradiated twice a week for 6 months. Signs of toxicity such as mortality and weight loss were checked once a week. Skin tumor formation was evidenced by lesions of greater than 1 mm in diameter that persisted for 2 weeks. At the end of the 6 months, the expression of proliferating cell nuclear antigen (PCNA) and p16 in the mouse skin was determined by immunohistochemical staining and immunoblotting using specific monoclonal antibodies for mouse PCNA and p16. The results were expressed as mean +/- standard error of the mean (SEM). Statistical difference was assessed by multiple ANOVA. A P-value of <0.05 was considered to be significant. RESULTS: At the end of the 6 months, none of the animals had developed any signs of toxicity such as mortality or weight lost. There was no evidence of tumor formation. There were significant elevations of p16 and PCNA in all treated groups as compared to the control group (ANOVA P < 0.05). This particularly applied to the group that was treated with the 1,320 nm Nd:YAG laser. CONCLUSION: The repeated use of high-energy laser and intense pulsed light source did not cause any toxicity in mice. The changes in p16 and PCNA imply that further studies are necessary to consider the implications of repeated exposure to longer wavelength radiation in human skin.

Reproducibility and expression of skin biomarkers in sun-damaged skin and actinic keratoses.

OBJECTIVES: To explore p53 and proliferating cell nuclear antigen (PCNA) expression and polyamine content as biomarkers in skin cancer chemoprevention trials, we evaluated their expression in early stages of UV-induced squamous cell tumorigenesis. METHODS: Biopsies were collected from three groups: 78 subjects with sun damage on forearms, 33 with actinic keratosis (AK) on forearms, and 32 with previous squamous cell carcinoma. Participants with sun damage were randomized to sunscreen or no sunscreen. RESULTS: We found significant differences in p53 and polyamines in forearms from the sun-damaged group (11.5 +/- 1.2% for p53, 65.5 +/- 1.9 nmol/g for putrescine, and 187.7 +/- 3.3 nmol/g for spermidine) compared with the group with sun damage plus AK (20.9 +/- 2.3% for p53, P = 0.0001; 81.7 +/- 3.9 nmol/g for putrescine, P = 0.0001; 209.4 +/- 8.2 nmol/g for spermidine, P < 0.06). PCNA was not different. When lesion histology was considered, there was a stepwise significant increase in p53 in biopsies without characteristics of AK compared with early AK (P = 0.02) and AK (P = 0.0006) and a similar pattern for PCNA with the only significant difference between early AK and AK. There was a stepwise increase in putrescine and spermidine in normal, sun-damaged forearm, forearm from subjects with AK, and the AK lesion itself (P < 0.0001). No significant differences in p53 or polyamines were seen in 3-month biopsies or, as a result of sunscreen use, although PCNA in the sun-damaged group not using sunscreen decreased significantly. CONCLUSIONS: p53 expression and polyamines in skin were elevated in early stages of skin tumorigenesis and were not affected by sunscreen, adding validity to their use as biomarkers in skin cancer chemoprevention trials.

PCNA is a cofactor for Cdt1 degradation by CUL4/DDB1-mediated N-terminal ubiquitination.

Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage.

Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage.

Most types of DNA damage block replication fork progression during DNA synthesis because replicative DNA polymerases are unable to accommodate altered DNA bases in their active sites. To overcome this block, eukaryotic cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. In particular, TLS by DNA polymerase eta (poleta) is the major pathway for bypassing UV photoproducts. How the cell switches from replicative to TLS polymerase at the site of blocked forks is unknown. We show that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein. Monoubiquitinated PCNA but not unmodified PCNA specifically interacts with poleta, and we have identified two motifs in poleta that are involved in this interaction. Our findings provide an attractive mechanism by which monoubiquitination of PCNA might mediate the polymerase switch.

Functional alterations in immature cultured rat hippocampal neurons after sustained exposure to static magnetic fields.

In cultured rat hippocampal neurons, gradual increases were seen in the expression of microtubule-associated protein-2 (MAP-2), neuronal nuclei (NeuN) and growth-associated protein-43 (GAP-43), in proportion to increased duration, up to 9 days in vitro (DIV). Sustained exposure to static magnetic fields at 100 mT for up to 9 DIV significantly decreased expression of MAP-2 and NeuN in cultured rat hippocampal neurons without markedly affecting GAP-43 expression. Although a significant increase was seen in the expression of glial fibrillary acidic protein (GFAP) in hippocampal neuronal preparations cultured for 6-9 DIV under sustained magnetism, GFAP and proliferating cell nuclear antigen expression were not affected markedly in cultured astrocytes prepared from rat hippocampus and neocortex, irrespective of cellular maturity. No significant alteration was seen in cell survivability of hippocampal neurons or astrocytes cultured under sustained magnetism. In hippocampal neurons cultured for 3 DIV under sustained magnetism, marked mRNA expression was seen for N-methyl-D-aspartate (NMDA) receptor subunits, NR1, NR2A-2C, NR2D, and NR3A. In addition, significant potentiation of the ability of NMDA to increase intracellular free Ca(2+) ions was observed. Differential display analysis revealed a significant decrease in mRNA expression for the transcription factor ALF1 in response to sustained magnetism for 3 DIV. These results suggest that sustained exposure to static magnetic fields may affect cellular functionality and maturity in immature cultured rat hippocampal neurons through modulation of expression of particular NMDA receptor subunits.

Observation of multiple isoforms and specific proteolysis patterns of proliferating cell nuclear antigen in the context of cell cycle compartments and sample preparations.

The proliferating cell nuclear antigen (PCNA) is an essential component for eukaryotic chromosomal DNA replication and repair. PCNA forms a homotrimer ring, which may function as a DNA sliding clamp for DNA polymerases and, possibly, a docking station for other replication- and repair-related proteins. Several reports have suggested the existence of different PCNA isoforms. Here we confirm, using high resolution two-dimensional electrophoresis with narrow pH ranges, the existence of three PCNA isoforms in both Chinese hamster and human breast cancer cells. Among the three isoforms, M or main form is the dominant one throughout the cell cycle while the relative amounts of the minor components A (acidic) and B (basic) forms appear to vary during the cell cycle. We also observed that a specific pattern of PCNA proteolysis occurred during isoelectric focusing in spite of high urea (8 M) and detergent (2% 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate), which was largely inhibited by the proteosome inhibitor MG132 or boiling. Interestingly, the proteolysis pattern was mainly observed with samples isolated from cells in S and G2 phases. A similar but much lower level of PCNA proteolysis also occurred in vivo within the nuclei of the cells in S phase. Taken together, our data are consistent with the idea that the existence of the different isoforms and specific proteolysis of PCNA are relevant to its functions in vivo.

Spectral response for laser enhancement in hepatic regeneration for hepatectomized rats.

BACKGROUND AND OBJECTIVES: The low intensity laser therapy (LILT) has been widely used in all medical fields due to its therapeutic effects in reparative process, pain relief, and biostimulation. Even though there is a therapeutic window of wavelengths for clinical application, little has been done concerning the frequency spectrum response to biological effects. In this work, we investigate the dependence of different wavelengths irradiation in the enhancement of the tissue regeneration after partial hepatectomy in Wistar rats. STUDY DESIGN/MATERIALS AND METHODS: The proliferating cell nuclear antigen (PCNA) labeling index and the respiratory control (oxygen consumption in extracted mitochondria) were the tests used to evaluate the liver regeneration after laser irradiation with different wavelengths. RESULTS AND CONCLUSIONS: The results show a correlated spectral response that can be explained based on the combined effect of light penetration on biological tissues and the biomolecular excitation efficiency for each wavelength used.

The roles of p53, DNA repair, and oxidative stress in ultraviolet C induction of proliferating cell nuclear antigen expression.

The expression of proliferating cell nuclear antigen (PCNA) promoter was moderately induced in UV-irradiated, quiescent human and rodent cells. The induction was independent of tumor suppressor gene p53, because the PCNA expression was UV-inducible in the subclones of human fibroblasts in which the activity of p53 was abrogated by human papilloma virus E6. Furthermore, the induction did not depend on DNA repair, since PCNA was UV inducible in UVL-10 and xrs-6 cells, in which nucleotide excision repair and double-stranded repair, respectively, are largely compromised. However, the induction was inhibited by antioxidant N-acetylcysteine. The role of oxidative stress observed here is consistent with the previous finding that the proximal AP-1 site is critical to the UV inducibility of PCNA promoter.

Proliferating cell nuclear antigen positive cells in the hippocampal subgranular zone decline after irradiation in a rodent model.

Four-week-old ICR mice were systemically exposed to 18Gy X-rays. Afterwards, the expression of proliferating cell nuclear antigen (PCNA) in the cerebrum was observed with time using Western blot analysis and immunohistochemical staining. As a result, PCNA-positive cells were observed in the subgranular zone (SGZ) of the hippocampus and subventricular zone (SVZ) of the lateral ventricles in unirradiated mice. The number of PCNA-positive cells decreased with time in all zones after irradiation, but the decrease was more marked in the hippocampal SGZ. We think that PCNA-positive cells are stem cells. The selective vulnerability to radiation in the hippocampus is considered to be attributed to the fact that stem cells in the SGZ selectively undergo radiation-induced apoptosis.

Relationship between cyclin D1 expression and poor radioresponse of murine carcinomas.

PURPOSE: We recently reported that overexpression of epidermal growth factor receptor (EGFR) positively correlated with radioresistance of murine carcinomas. Because cyclin D1 is a downstream sensor of EGFR activation, the present study investigated whether a relationship exists between the extent of cyclin D1 expression and in vivo radiocurability of murine tumors. We further investigated the influence of radiation on cyclin D1 expression and the expression of p27, an inhibitor of the cyclin D1 downstream pathway, as well as the relationship of these molecular determinants to cell proliferation and induced apoptosis in tumors exposed to radiation. METHODS AND MATERIALS: Cyclin D1 expression was assayed in nine carcinomas syngeneic to C3Hf/Kam mice using Western blot analysis. These tumors greatly differed in their radioresponse as assessed by TCD(50). The expression of cyclin D1 and p27 proteins was determined by Western blotting. Cell proliferative activity in tumors was determined by proliferating cell nuclear antigen (PCNA) immunochemistry. The effect of irradiation on the expression of cyclin D1 or p27 proteins and on PCNA positivity was determined in the radiosensitive OCa-I and in the radioresistant SCC-VII tumors. RESULTS: Cyclin D1 expression varied among tumors by 40-fold, and its magnitude positively correlated with poorer tumor radioresponse (higher TCD(50) values). The level of cyclin D1 expression paralleled that of EGFR. A 15-Gy dose reduced constitutive expression of cyclin D1 in the radiosensitive OCa-I tumors, but had no influence on expression of cyclin D1 in the radioresistant SCC-VII tumors. In contrast, 15 Gy increased the expression of p27 in radiosensitive tumors and reduced it in radioresistant tumors. Radiation induced no significant apoptosis or change in the percentage of PCNA-positive (proliferating) cells in SCC-VII tumors with high cyclin D1 levels, but it induced significant apoptosis and a decrease in the percentage of proliferating cells in OCa-I tumors with low cyclin D1 expression. CONCLUSION: Our findings show a positive correlation between cyclin D1 expression and tumor radioresistance. The expression of cyclin D1 and p27 was modified by radiation and was associated with cellular response to radiation, but this depended on the pretreatment level of cyclin D1 expression. These findings may have important clinical implications: The pretreatment assessment of cyclin D1 expression could serve as a useful predictor of radiotherapy outcome and assist in selecting an effective treatment modality.

Sensitivity to low-level radiation in radiosensitive "wasted" mice.

Mice homozygous for the autosomal recessive "wasted" mutation (wst/wst) have abnormalities in T lymphocytes and in the anterior motor neuron cells of the spinal cord, leading to sensitivity to low doses of ionizing radiation, hind limb paralysis, and immunodeficiency. This defect results in a failure to gain weight by 20 days of age and death by 28 days. The wasted mutation (previously mapped to mouse chromosome 2) is shown to be a 3-bp deletion in a T cell-specific (and perhaps motor-neuron-specific) regulatory region (promoter) of the proliferating cell nuclear antigen (PCNA) gene on mouse chromosome 2. A regulatory element is also shown to be important in PCNA expression in T lymphocytes and motor neuron cells affected by the 3-bp deletions in the PCNA promoter. The model is as follows. Absence of PCNA expression in the thymuses (and motor neurons) of wasted mice causes cellular apoptosis. This absence of expression is mediated by a positive transfactor that can bind to the wild-type but not to the wasted mutant PCNA promoter. The bound protein induces late expression of PCNA in T lymphocytes and prevents onset of radiation sensitivity in the cells.

Influence of estrogen, antiestrogen and UV-light on the balance between proliferation and apoptosis in MCF-7 breast adenocarcinoma cells culture.

Studies of the mechanism of actions of estrogen, antiestrogen and physical factors may provide clues to an understanding of breast cancer growth and/or regression regulation and thus identify novel targets for therapeutic intervention. Defective control of apoptosis appears to play a central role in the pathogenesis of neoplasia. Conversely, cancer therapy and ionizing radiation can induce cancer cell death by apoptosis and/or necrosis. bcl-2 gene and p-53 gene products have been both linked to programmed cell death pathways. We have analyzed the effect of estradiol, tamoxifen and UV exposure on the induction of apoptosis, expression of p53 and bcl-2 gene products as well as the proliferative activity (expressed as [3H]thymidine incorporation and PCNA and MPM2 antigens involvement) in MCF7. It has been found that estradiol increases the speed of cell cycle in MCF7 and acts as antiapoptotic factor. Tamoxifen has multiple influence on the rate of growth of cancer cells: depends on estrogen receptor (ER), conducts reduction of proliferation rate; depends on ER and other mechanisms conducts to suppressions of Bcl-2 protein expression and induction of cell death through apoptotic pathway. Estradiol prevents the apoptotic influence of tamoxifen probably by enhancement of Bcl-2 protein expression and does not prevent the inhibition of proliferation rate. The irradiation with UV induces apoptosis by over-expression of p53 and down-regulation of bcl-2 gene.

Schizosaccharomyces pombe replication and repair proteins: proliferating cell nuclear antigen (PCNA).

Schizosaccharomyces pombe has a cell cycle progression with distinctive phases that serves as a perfect model system for investigating DNA replication and repair of eukaryotic cells. Here, we use proliferating cell nuclear antigen (PCNA) of S. pombe to demonstrate how the function of this protein in both DNA replication and repair can be assessed by genetic and biochemical approaches. We describe a method of introducing site-specific mutations into the fission yeast PCNA gene pcn1(+). The in vivo effects of these pcn1 mutants in a strain with a null pcn1 background are described and their in vitro biochemical properties are characterized. Mutants described here are those that are defective in enhancing processivity of DNA polymerase delta, show temperature-sensitive growth, and have increased sensitivity to hydroxyurea (HU), UV and gamma irradiation, and methyl methanesulfonate (MMS). Three mutants that show reduced growth rate in vivo and decreased capacity to enhance polymerase delta DNA synthetic activity and processivity in vitro-pcn1-1, pcn1-5, and pcn1-26-are described as examples of using a genetic approach to identify the biochemical function of replication proteins. One cold-sensitive growth allele, pcn1-3, that has a recessive cold-sensitive cdc phenotype and shows sensitivity to HU and UV and gamma irradiation is used as an example of using the genetic approach to reveal the function of replication proteins in repair. The power of combining both biochemical and genetic disciplines is emphasized. Methods for site-directed mutagenesis, in vitro analysis of mutant proteins, and in vivo characterization of mutants in response to UV or gamma irradiation, MMS, HU, and temperature, as well as genetic epistasis are described. Locations of functionally significant residues on the PCNA tertiary structure are summarized.

The C-terminal region of Schizosaccaromyces pombe proliferating cell nuclear antigen is essential for DNA polymerase activity.

Proliferating cell nuclear antigen (PCNA), the processivity factor (sliding clamp) of DNA polymerases (Pols), plays essential roles in DNA metabolism. In this report, we examined the functional role of the C-terminal region of Schizosaccaromyces pombe PCNA both in vitro and in vivo. The deletion or Ala substitution of the last 9 aa (252-260A), as well as Ala replacement of only 4 aa (252-255A) at the C terminus, failed to substitute for the wild-type PCNA protein for cell growth in S. pombe. Two other PCNA mutant proteins, A251V and K253E, exhibited cold-sensitive phenotypes. Several yeast strains harboring mutations, including those at the acidic C-terminal region, showed elevated sensitivity to DNA damage. The ability of the mutant PCNA proteins to stimulate DNA synthesis by Poldelta and Polepsilon also was studied in vitro. The mutant proteins that did not support cell growth and a mutant protein containing a single amino acid substitution at position 252, where Pro is replaced by Ala, stimulated Poldelta and Polepsilon activities poorly. All mutant PCNA proteins, however, were assembled around DNA by the clamp loader, replication factor C, efficiently. Thus, the C-terminal region of PCNA is important for interactions with both Poldelta and Polepsilon and for cell survival after DNA damage. The C terminus of sliding clamps from other organisms has been shown to be important for clamp loading as well as polymerase interactions. The relationship between the conserved sequence in this region in different organisms is discussed.

UV inducibility of rat proliferating cell nuclear antigen gene promoter.

Proliferating cell nuclear antigen (PCNA), also known as a cofactor of DNA polymerase delta, is required for eukaryotic cell DNA synthesis and nucleotide excision repair. Expression of PCNA gene is growth-regulated and UV inducible. In our previous study, we have observed that the rat PCNA promoter has the serum responsiveness. In this study, we demonstrate its UV inducibility in CHO.K1 cells. The UV induction of the rat PCNA promoter activity was dose-dependent in the cells synchronized at different phases. In addition, the sequences of the promoter responsible for the UV inducibility were delimited to the region between nucleotides -70 and +125, which contains an AP-1 site and a downstream proximal ATF/CRE site. While mutation of the AP-1 site abrogated the UV inducibility, mutation of the ATF/CRE site enhanced the UV inducibility, suggesting that the two sites play different roles in the UV induction of the promoter. In addition, the role of p53 in the UV induction of rat PCNA promoter was investigated. We found that exogenous p53 was unable to mimic the UV irradiation to induce rat PCNA promoter and that the UV induction of the rat PCNA promoter was seen in p53 deficient cells. Therefore, it is unlikely that the UV induction of the rat PCNA promoter is p53 dependent.

[The immunohistochemical determination of p53 and of proliferating cell nuclear antigen in the epithelial nuclei of benign prostatic hyperplasia following the accident at the Chernobyl Atomic Electric Power Station]. / Imunohistokhimichne vyznachennia p53 ta proliferatyvnoho klitynnoho iadernohoantyhena v iadrakh epiteliiu dobroiakisnoï hiperplaziï peredmikhurovoï zalozy pislia avariï na ChAES.

With the purpose of studying into the morphogenesis and proliferous activity of the prostatic epithelium under a long-term exposure to low doses of ionizing radiation there have been conducted comparative histological and immunohistochemical (expression of p53 and proliferous cellular nuclear antigen-PCNA) investigations designed to study benign prostatic hyperplasia in patients living in those Ukraine territories affected by radionuclide contamination (group III), residents of Kiev (group II), and patients having been operated on before the Chernobyl accident, having constituted the control group I. It has been found out that the incidence of prostatic intraepithelial neoplasia (PIN), the level of nuclear expression of proteins p53 (in the PIN epithelium) and PCNA (in the epithelium of both benign prostatic hyperplasia and PIN) of patients in groups II and III are by far higher as compared with those in group I. The stroma of benign prostatic hyperplasia in patients of groups II and III was clearly different from that in the control group in that the former was characterized by apparent phenomena of hyalinosis, sclerosis, fibrosis, and extensive inflammatory infiltration, which changes can be explained by a long-term systematic exposure of prostatic tissue to low doses of ionizing radiation.