Proliferating Cell Nuclear Antigen Gene Expression Research Articles

Age-dependent changes in the expression and localization of LYZL4, LYZL6 and PCNA during testicular development in the Ashidan yak

Lysozyme like 4 (LYZL4), lysozyme like 6 (LYZL6) and proliferating cell nuclear antigen (PCNA) are implicated in the regulation of testicular function, but there was no research reported available on the expression patterns of LYZL4, LYZL6 and PCNA genes at different developmental stages of yak testes. In this study, we used the qRT-PCR, western blotting and immunohistochemistry estimated the LYZL4, LYZL6 and PCNA gene expression and protein lo-calization at different developmental stages of yak testes. The qPCR results showed that the mRNA expression of LYZL4, LYZL6 and PCNA genes significantly increased with age in the testes of yaks. Western blot results showed that the protein abundance of LYZL4, LYZL6 and PCNA in yak testes was significantly higher after puberty than before puberty. Furthermore, the results of immunohistochemistry indicated that LYZL4, LYZL6 and PCNA may be involved in the regulation of spermatogonia proliferation and Leydig cell function in immature testis. In adult yak testes, LYZL4, LYZL6 and PCNA may involve in the development of round spermatids and primary spermatocytes during testicular development. Our results indicated that LYZL4, LYZL6 and PCNA may be involved in the development of Sertoli cells, Leydig cells and gonocytes in yak testes.

The PCNA GENE EXPRESSION AS A MARKER OF ALZHEIMER'S DISEASE IN THE BRAIN OF LONG-TAILED MACAQUES (Macaca fascicularis)

Early diagnosis of Alzheimer's is still difficult to do, thus it is important to carry out further research to find biomarkers that can be used to detect early Alzheimer's disease. Proliferating Cell Nuclear Antigen (PCNA) is known as a proliferation marker, which has the potential to detect neurogenesis in the brain. This study aimed to identify PCNA gene expression in the brain as a marker of Alzheimer’s disease in Macaca fascicularis. Macaca fascicularis was used in this study because of their similarities with humans in terms of their behavioral complexity, as well as high cognitive abilities and the formation of a pathological characteristic of Alzheimer's disease in the brain. This study used the brains of 7 monkeys in the hippocampus and cortical regions. Monkeys previously have been divided into old and adult age groups. The detection of PCNA gene expression was done using RT-qPCR method. The results showed the gene expression tended to be higher in the adult group and the hippocampus region, although based on statistical analysis showed no significant differences

Can PCNA and LIM15 gene expression levels predict sperm retrieval success in men with non-obstructive azoospermia?

Introduction and objectivesIt is necessary to be able to predict sperm retrieval before microdissection testicular sperm extraction (mTESE) in azoospermic men. This study established the importance of proliferating cell nuclear antigen (PCNA) and LIM15 gene expression levels in predicting the success of sperm retrieval by mTESE. Materials and methodsOne hundred and forty-three men who were diagnosed with non-obstructive azoospermia (NOA) were included in the study. Patients’ age, total testosterone and follicle stimulating hormone values, testicular volume and testicular histology were recorded by prospectively. PCNA and LIM15 gene expression levels were determined by real-time PCR in the materials from both ejaculate and testicular specimens. ResultsTestis volume and histology were the most important factors in predicting the sperm retrieval rate (SRR). The PCNA and LIM15 gene expression levels measured in testicular tissues and the LIM15 gene expression levels measured in ejaculate significantly correlated with the SRR in mTESE (p=0.038, p=0.022, and p=0.004, respectively). Although the PCNA gene expression level measured in ejaculate was higher in men with successful sperm retrieval, the difference was not statistically significant (p=0.061). According to the multivariate logistic regression analysis, testicular volume and LIM15 gene expression level in ejaculate were independent predictive parameters for sperm retrieval. ConclusionThe data showed that LIM15 gene expression level in ejaculate is a useful molecular marker to predict the SRR before mTESE.

Protective Effect of Hydroalcoholic Extract of Orange Peel on PCNA and FSH-R Gene Expression in Histological Damage and Oxidative Stress Due to Ovarian Torsion in Adult Rats

Objectives: The aim of this study was to evaluate the protective effect of the hydroalcoholic extract of orange peel on proliferating cell nuclear antigen (PCNA) and follicle-stimulating hormone receptor (FSH-R) gene expression in histological injuries and acid stress caused by ovarian torsion in adult rats. Materials and Methods: In this experimental study, 32 adult female rats were randomly divided into 4 groups. In group 1 (Sham), the abdominal wall was cut without applying torsion and in group 2, ovarian torsion was performed for 2 hours, followed by detorsion for 2 weeks. The hydro-alcoholic extract of orange peel was added to their diet for two weeks in group 3, followed by ovarian torsion for 2 hours and detorsion for 2 hours. Group 4 received the orange peel extract for two weeks and after then ovarian resection for the evaluation of histological damage and blood sampling to examine the serum level of antioxidant enzymes, as well as the expression of PCNA and FSH-R genes in the ovarian tissue. Results: Histological changes in the ovary tissue of rats showed that torsion and detorsion have destructive effects on the ovarian tissue, and torsion/detorsion led to a reduction in the expression of PCNA and FSH-R (P < 0.05). Based on biochemical and hormonal results, the ovarian torsion resulted in an imbalance in the oxidative stress markers and hormone profile of rats. Finally, the administration of the hydroalcoholic extract of orange peel due to its high antioxidant properties improves these effects. Conclusions: In general, administering an appropriate dose of the hydroalcoholic extract of orange peel for two consecutive weeks in the diet had a protective effect on the ovarian tissue at the risk of torsion/detorsion.

Evaluating the Effects of Different Concentrations of Human Follicular Fluid on Growth, Development, and PCNA Gene Expression of Mouse Ovarian Follicles

Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.

Effects of probiotics on apoptosis and proliferation of intestinal epithelial cells in rats with necrotizing enterocolitis

Objective To study the effect of compound Lactobacillus acidophilus on the apoptosis of intestinal epithelial cells and expression of proliferating cell nuclear antigen (PCNA) in rats with necrotizing enterocolitis, so as to provide some theoretical reference for the application of compound Lactobacillus acidophilus in the prevention and treatment of necrotizing enterocolitis(NEC) for newborn infants. Methods Thirty-six 48-hour old newborn SD rats were randomly divided into normal group, model group and intervention group, with 12 rats in each group.The NEC model was established by hypoxia and cold stimulation for 3 days, and the intervention group was given the compound Lactobacillus acidophilus at the same time; after the model was established, all the rats were sacrificed after 12 hours of fasting and their intestinal tissues were excised by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method to determine the condition of apoptosis and to detect expression of the PCNA gene in the intestinal tissues by fluorescent quantitative PCR. Results (1) The apoptosis rates of the intestinal tissues: in the model group, the normal group and the intervention group were (88.33±2.77)%, (4.75±0.75)% and (44.41±4.81)%, respectively, and the differences had statistical significance(all P<0.05). (2) The PCNA expression in the intestinal tissue by real-time PCR in the model group, normal group and intervention group were (9.28±3.26)×10-4, (15.35±1.91)×10-4 and (12.09±3.06)×10-4, respectively, and the differences had statistical significance(all P<0.05). Conclusions The apoptosis rate of intestinal epithelial cells increases and PCNA expression decreases when NEC occurs.Compound Lactobacillus acidophilus can reduce apoptosis, promote intestinal epithelial cell proliferation, and thus help to reduce the risk and severity of NEC in neonatal rats. Key words: Neonatal necrotizing enterocolitis; Apoptosis; Proliferating cell nuclear antigen; Compound Lactobacillus acidophilus

Toxic effect on the membrane system and cell proliferation of Prorocentrum donghaiense caused by the novel algicidal fungus Talaromyces purpurogenus YL13

A fungal strain YL13 with algicidal activity against the dinoflagellate Prorocentrum donghaiense was isolated from Wild Fox Island, Zhuhai, China, and identified as Talaromyces purpurogenus YL13 on the basis of 18S ribosomal RNA (rRNA) sequence. Strain YL13 exhibited algicidal activity through the mechanism of indirect attack, and its algicidal activity was improved from 80.3 to 96.8 % by optimization of environmental and nutrient factors with response surface methodology (RSM). Effects of strain YL13 on the membrane system and cell proliferating of P. donghaiense were investigated to elucidate the algicidal mechanism. The increase in both ATPase activities and malondialdehyde (MDA) contents suggested that the membrane in algal cells was damaged. Damage was observed in atomic force microscopy (AFM) images and the surface morphology of cells. Real-time PCR assay showed changes in the transcript abundance of proliferating cell nuclear antigen (PCNA) gene. The release of nucleic acids and changes of PCNA gene expression indicated that DNA synthesis was affected, and this influenced cell proliferation and the membrane system of P. donghaiense. The fungal supernatant might be potentially used as a bioagent for controlling harmful algae. Strain YL13 is the first record of a T. purpurogenus being algicidal to the harmful dinoflagellate P. donghaiense, and this is the first report to explore the mechanism of toxic effects on membrane system and cell proliferation of the marine T. purpurogenus against harmful P. donghaiense.

Ex vivo evaluation of the effect of photodynamic therapy on skin scars and striae distensae.

Skin scars and striae distensae (SD) are common dermal disorders with ill-defined treatment options. There is emerging clinical evidence for use of photodynamic therapy (PDT) in treating skin fibrosis. Therefore, the aim here was to investigate the effect of PDT on skin scars and SD in an exvivo model of human skin scarring. Photodynamic therapy, with 5ALA or MALA in addition to illumination with 40J/cm(2) of red light, was applied to striae alba, fine line, hypertrophic and keloid scars exvivo (n=18). General morphology was assessed by H&E, Herovici's and Weigert's differential staining. Apoptosis, proliferation, metalloproteinase 3 and tropoelastin expression were quantified immunohistochemically, and differential gene expression of proliferating cell nuclear antigen (PCNA), collagen (COL) type I and type III, matrix metalloproteinase 3 (MMP3) and tropoelastin (ELN) was assessed by real-time quantitative reverse transcription polymerase chain reaction. Apoptosis increased, which correlated with decreased proliferation and PCNA gene expression. Post-PDT, matrix components were found to be re-organised in both hypertrophic and keloid scars. COLI and COLIII gene expression levels decreased, whilst MMP3 and ELN increased significantly post-PDT compared to normal skin and untreated controls (P<0.05). However, no significant difference between 5ALA and MALA-PDT treatments was observed. Using our unique exvivo model, we show for the first time morphological and cellular effect of application of PDT, which correlates with the degree and severity of dermal fibrosis. In view of this, PDT may be ideal in targeting treatment of abnormal skin scarring.

Two Estrogen Response Element Sequences Near the PCNA Gene Are Not Responsible for Its Estrogen-Enhanced Expression in MCF7 Cells

BackgroundThe proliferating cell nuclear antigen (PCNA) is an essential component of DNA replication, cell cycle regulation, and epigenetic inheritance. High expression of PCNA is associated with poor prognosis in patients with breast cancer. The 5′-region of the PCNA gene contains two computationally-detected estrogen response element (ERE) sequences, one of which is evolutionarily conserved. Both of these sequences are of undocumented cis-regulatory function. We recently demonstrated that estradiol (E2) enhances PCNA mRNA expression in MCF7 breast cancer cells. MCF7 cells proliferate in response to E2.Methodology/Principal FindingsHere, we demonstrate that E2 rapidly enhanced PCNA mRNA and protein expression in a process that requires ERα as well as de novo protein synthesis. One of the two upstream ERE sequences was specifically bound by ERα-containing protein complexes, in vitro, in gel shift analysis. Yet, each ERE sequence, when cloned as a single copy, or when engineered as two tandem copies of the ERE-containing sequence, was not capable of activating a luciferase reporter construct in response to E2. In MCF7 cells, neither ERE-containing genomic region demonstrated E2-dependent recruitment of ERα by sensitive ChIP-PCR assays.Conclusion/SignificanceWe conclude that E2 enhances PCNA gene expression by an indirect process and that computational detection of EREs, even when evolutionarily conserved and when near E2-responsive genes, requires biochemical validation.

Human T-Cell Leukemia Virus Type 1 Tax Relieves Repression of Proliferating Cell Nuclear Antigen Gene Expression

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia. The transforming ability of Tax, the viral oncoprotein, is believed to depend on interactions with cell cycle regulators and on transactivation of genes that control cellular proliferation, including proliferating cell nuclear antigen (PCNA), a cofactor associated with DNA replication and repair. Tax associates with cellular transcription factors to alter their affinity for cognate DNA elements, leading to increased or decreased transcription from that promoter. Although it has been demonstrated that Tax transactivates the PCNA promoter, the mechanism of transcriptional activation is unknown. Here we report a cellular complex that binds specifically to a novel site within the minimal Tax-responsive element of the TATAA-less PCNA promoter. Mutation at this binding site or Tax expression inhibited complex formation and increased promoter activity, suggesting that the complex is a transcriptional repressor. The activation of PCNA gene expression by Tax and consequential decrease in nucleotide excision repair mediated by PCNA overexpression could contribute to the reduced DNA repair capacity and genomic instability observed in HTLV-1-infected cells.

Effects of Shock Waves on Tenocyte Proliferation and Extracellular Matrix Metabolism

The shock wave is an effective noninvasive modality for resolving various tendon pathologies. However, scientific rationale and mechanism of shock wave therapy remains limited. This study aims to investigate the effects of shock waves and their biochemical mechanisms on tenocyte proliferation and collagen synthesis. Tenocytes harvested from Achilles tendons of Sprague-Dawley rats were used in this study. Cell viability was assayed by trypan blue exclusion methods. The colorimetric assay was determined to evaluate the mitochondria activity of the tenocytes after shock wave exposure. Synthesis of collagen, nitric oxide (NO) and transforming growth factor-β1 (TGF-β1) were determined and their gene expression was also studied. The results showed that there was a dose-dependent impairment of cell viability observed in 0.36 mJ/mm 2 and 0.68 mJ/mm 2 stimulation. In the proliferation assay, low energy level with low impulses (0.36 mJ/mm 2 with 50 and 100 impulses) showed positive stimulatory effects, whereas the high energy level with high impulses (0.68 mJ/mm 2 with 250 and 500 impulses) had significant inhibitory effects. At 0.36 mJ/mm 2, 100 impulse shock waves treatment, up-regulation of proliferating cell nuclear antigen (PCNA) (at 6 and 24 h) and collagen type I, collagen type III and TGF-β1 gene expression (at 24 h) were observed; these were followed by the increases in NO production (at 24 h), TGF-β1 release (at 48 and 96 h) and collagen synthesis (at the 7th day). This study revealed that shock waves can stimulate tenocyte proliferation and collagen synthesis. The associated tenocyte proliferation is mediated by early up-regulation of PCNA and TGF-β1 gene expression, endogenous NO release and synthesis and TGF-β1 protein and then collagen synthesis. (E-mail: jssun@ym.edu.tw)

Urothelial sonic hedgehog signaling plays an important role in bladder smooth muscle formation

During bladder development, primitive mesenchyme differentiates into smooth muscle (SM) under the influence of urothelium. The gene(s) responsible for this process have not been elucidated. We propose that the Sonic hedgehog (Shh) signaling pathway is critical in bladder SM formation. Herein, we examine the role of the Shh-signaling pathway during SM differentiation in the embryonic mouse bladder. Genes in the Shh pathway and SM expression in mouse embryonic (E) bladders (E12.5, 13.5, and 14.5) were examined by immunohistochemistry (IHC), in situ hybridization, and reverse transcription polymerase chain reaction (RT-PCR). To examine the effects of disrupting Shh signaling, bladder tissues were isolated at E12.5 and E14.5, that is, before and after bladder SM induction. The embryonic bladders were cultured on membranes floating on medium with and without 10 μM of cyclopamine, an Shh inhibitor. After 3 days, SM expression was examined by assessing the following: SM α-actin (SMAA), SM γ-actin (SMGA), SM-myosin heavy chain (SM-MHC), Patched, GLI1, bone morphogenic protein 4 (BMP4), and proliferating cell nuclear antigen (PCNA) by IHC and RT-PCR. SM-related genes and proteins were not expressed in E12.5 mouse embryonic bladder before SM differentiation, but were expressed by E13.5 when SM differentiation was initiated. Shh was expressed in the urothelium in E12.5 bladders. Shh-related gene expression at E12.5 was significantly higher than at E14.5. In cyclopamine-exposed cultures of E12.5 tissue, SMAA, SMGA, GLI1, and BMP4 gene expression was significantly decreased compared with controls, but PCNA gene expression did not change. In cyclopamine-exposed E14.5 cultures, SMGA and SM-MHC gene expression did not change compared with controls. Using an in vitro embryonic bladder culture model, we were able to define the kinetics of SM- and Shh-related gene expression. Cyclopamine inhibited detrusor SM actin induction, but did not inhibit SM-MHC induction. SMAA and SMGA genes appear to be induced by Shh-signaling pathways, but the SM-MHC gene is not. Based on Shh expression by urothelium and the effects of Shh inhibition on bladder SM induction, we hypothesize that urothelial-derived Shh orchestrates induction of SM in the fetal mouse bladder.

BHLH-zip Transcription Factor Spz1 Mediates Mitogen-Activated Protein Kinase Cell Proliferation, Transformation, and Tumorigenesis

BHLH-zip proteins usually play important regulatory roles in cell growth and differentiation. In this study, we show that Spz1, a bHLH-zip transcription factor, acts downstream of mitogen-activated protein kinase (MAPK, extracellular signal-regulated kinase 1/2) to up-regulate cell proliferation and tumorigenesis. In addition, through an interaction with proliferating cell nuclear antigen (PCNA) promoter, Spz1 induced cell proliferation concomitant with an increase in PCNA gene expression. Spz1-transfected cells formed colony foci on soft agar and developed fibrosarcoma tumors in nude mice. MAPK directly interacted and phosphorylated Spz1 protein, which increased PCNA transcription and cell tumorigenic activities. Reduction of endogenous Spz1 expression via RNA interference decreased cell proliferation in p19 embryonic carcinoma cells. High levels of Spz1 expression were detected in murine tumor cell lines and tumor samples of both human and Spz1 transgenic mice. Thus, Spz1 may act as a proto-oncogene, participating in the MAPK signal pathway, and be a potential therapeutic target in the treatment of Ras-induced tumors.

Induction of p53-dependent Activation of the Human Proliferating Cell Nuclear Antigen Gene in Chromatin by Ionizing Radiation

A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.

Trans-cellular proliferating cell nuclear antigen gene activation in cerebral vascular smooth muscle by endothelial oxidative injury in vivo.

This study was undertaken to assess the role of vascular smooth muscle cell (VSMC) Ca2+ channels and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in gene regulation after oxidative endothelial injury (OEI). OEI was produced by infusion of Na fluorescein (NaFluo) photoactivated by UV light immediately before intravenous injection. Posterior cerebral arteries were studied using immunofluorescence imaging, Western blotting, or patch clamping of isolated cells. After infusion of photoactivated NaFluo, but not NaFluo, (1) superoxide dismutase-1 (SOD-1) was upregulated in endothelium, consistent with oxidant stress; (2) the fraction of VSMC nuclei labeled for proliferating cell nuclear antigen (PCNA) increased 7-fold at 6 hours, preceded by a several-fold increase in nuclear phospho-cAMP-response element binding protein, with PCNA upregulation prevented by pretreatment with polyethylene glycol (PEG)-SOD; (3) in VSMCs, phospho-CaMKII increased 20-fold 5 minutes after OEI, with a 2-fold increase in peak Ca2+ channel currents; and (4) changes in cAMP-response element binding protein and PCNA were blocked by systemic administration of lipophilic (nifedipine) or hydrophilic (amlodipine) 1,4-dihydropyridine Ca2+ channel blockers, the calmodulin inhibitor trifluoperazine, or the CaMKII inhibitor KN-93, with none of these agents preventing SOD-1 upregulation in endothelium. Activation of VSMC Ca2+ channels and CaMKII is a key early signaling event required for upregulation of PCNA gene expression in VSMCs after oxidative injury to endothelium.

Down-Regulation of Proliferating Cell Nuclear Antigen Gene Expression Occurs during Cell Cycle Arrest Induced by Human Fecal Water in Colonic HT-29 Cells

Cancer is a disease in which the cell cycle is altered, and the elucidation of the mechanisms by which constituents of human fecal water influence the cell cycle can lead to noninvasive measurement of colon cancer risk. The purpose of the present study was to investigate the effect of human fecal water on HT-29 cell-cycle progression with sodium selenite as a reference for comparison. Both human fecal water (2.5-5.0%) and selenite (3-4 micro mol/L) significantly inhibited cell growth. Cell-cycle analysis revealed that human fecal water decreased the proportion of S + G2 phase cells and increased that of G1 phase cells. In contrast, selenite decreased G1 phase cells and increased proportions of S and G2 phase cells. Both 5% human fecal water and 4 micro mol/L selenite greatly increased the mRNA level of the cyclin-dependent kinase inhibitor gene p21(waf1). Interestingly, the mRNA levels of cyclin A and proliferating cell nuclear antigen (PCNA) were dramatically decreased by 69 and 62%, respectively, in HT-29 cells treated with fecal water but not selenite. In contrast, the mRNA level of DNA damage-inducible transcript 1, gadd45, was significantly increased by 2.28-fold in HT-29 cells treated with selenite but not fecal water. Furthermore, a PCNA gene promoter was cloned into a luciferase reporter construct and its activity was significantly reduced in a dose-dependent manner in cells treated with fecal water but not selenite. Collectively, these results suggest that human fecal water and selenite can differentially induce growth arrest genes, and that PCNA gene expression is uniquely and highly sensitive to human fecal water.

Spatial repression of PCNA by p53 during kidney development.

Transcriptional repression is a key mechanism for the spatial specification of gene expression and cell fate determination. During kidney development, proliferating cell nuclear antigen (PCNA) is expressed in the nephrogenic zone and is downregulated rapidly as renal epithelial cells enter terminal differentiation and acquire functional characteristics. Our laboratory reported that the transcription factor p53 stimulates the terminal differentiation of renal epithelial cells by means of transcriptional activation of renal function genes (Saifudeen Z, Dipp S, and El-Dahr SS. J Clin Invest 109: 1021-1030, 2002). Because p53-induced growth arrest correlates with downregulation of PCNA gene expression, we examined the impact of p53 inactivation on PCNA expression in mice and evaluated the effect of p53 on PCNA transcription. Immunohistochemistry revealed that the transition from nephrogenesis to terminal epithelial cell differentiation correlates with accumulation of the transcription factor p53. Importantly, the spatially restricted pattern of PCNA expression is disrupted in kidneys of p53-deficient pups, in which there was a redistribution of PCNA expression into the differentiation zone (without a change in total kidney PCNA content) and distortion of the tubular architecture. Electrophoretic mobility shift assays revealed that the binding of kidney nuclear extracts to the p53 response elements in human and rat PCNA promoters is developmentally regulated. Transient transfection assays performed in p53-deficient HeLa cells revealed that exogenous p53 strongly represses transcription from human PCNA promoter-reporter constructs. Interestingly, deletion of the p53-binding site confers enhanced responsiveness to p53-mediated repression, suggesting that transcriptional repression of PCNA by p53 is achieved by a mechanism other than direct DNA binding. On the basis of these results, we propose the hypothesis that p53-mediated transcriptional repression plays a role in the spatial restriction of PCNA gene expression during normal renal development.

Suppression of DNA Repair by HTLV Type 1 Tax Correlates with Taxtrans-Activation of Proliferating Cell Nuclear Antigen Gene Expression

The human T cell leukemia virus type 1 (HTLV-1) viral oncoprotein Tax acts as a transcriptional trans-activator affecting viral as well as cellular gene expression. To understand how Tax induces transformation, the consequences of its ability to alter expression of cellular genes must be examined. We have previously demonstrated that Tax activates expression of the cellular gene, proliferating cell nuclear antigen (PCNA), and that Tax suppresses DNA repair. In this study we tested the ability of previously described Tax mutants to activate PCNA gene expression and their ability to interfere with DNA repair. The results revealed a strong correlation between Tax trans-activation of PCNA gene expression and its ability to inhibit DNA repair via the nucleotide excision repair (NER) pathway. Thus, a consequence of activated PCNA gene expression appears to be reduced DNA repair capacity. These effects of Tax are likely to play important roles in its transforming activity.

Neonatal ovine uterine development involves alterations in expression of receptors for estrogen, progesterone, and prolactin.

Effects of age on uterine histoarchitecture, cell proliferation, and hormone receptor expression were determined for neonatal ewe lambs from birth (Postnatal Day [PND] 0) to PND 56. Uteri were histologically evaluated and proliferating cell nuclear antigen (PCNA), estrogen receptor alpha (ER-alpha), progesterone receptor (PR), and prolactin receptor (PRL-R) expression were characterized by in situ hybridization (ISH), immunohistochemistry, or both. The most striking feature of neonatal uterine development was the genesis and development of glands in the intercaruncular areas of endometrium. After birth, endometrial glandular epithelium (GE) budded and differentiated into the underlying stroma from the luminal epithelium (LE) between PNDs 1 and 7. Between PNDs 14 and 56, extensive coiling and branching morphogenesis of nascent endometrial glands occurred. By PND 56, the uterine wall appeared to be histoarchitecturally mature. At birth, nuclear PCNA protein was strongly detected in LE. Between PNDs 7 and 56, high levels of PCNA, ER-alpha, and PR gene expression were detected in both nascent and developing GE. Higher levels of PCNA and ER-alpha expression were detected in GE at the tips of developing glands as well as in the surrounding stroma. Progesterone was below detectable limits in serum. Serum estradiol-17beta levels were high on PND 1, increased from PNDs 14 to 28, and declined from PND 42 to PND 56. Serum PRL levels increased from PNDs 1 to 14 and declined thereafter. Using ISH and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, expression of mRNAs for short and long forms of the ovine PRL-R were first detected in nascent GE on PND 7 and increased between PNDs 7 and 56 in proliferating and differentiating GE. These results indicate that 1) uterine gland genesis is initiated between PNDs 1 and 7 after birth and is essentially completed by PND 56; 2) neonatal uterine morphogenesis involves temporal and spatial alterations in cell proliferation and ER-alpha, PR, and PRL-R gene expression; 3) PRL-R expression is a unique marker of GE differentiation and proliferation; and 4) serum estradiol-17beta and PRL levels increase during the onset of GE tubular branching morphogenesis. Results support the hypothesis that neonatal ovine uterine development involves epithelial PRL-R and ER-alpha activation to stimulate and maintain endometrial gland genesis and branching morphogenesis.

Single nucleotide polymorphism analyses of the human proliferating cell nuclear antigen (pCNA) and flap endonuclease (FEN1) genes.

The products of proliferating cell nuclear antigen (PCNA) and flap endonuclease (FEN1) genes are multifunctional proteins that are involved in DNA replication and damage repair. Yeast models suggest association of mutant forms of PCNA and FEN1 with genomic instability. In our study, we have determined the single nucleotide polymorphisms in human PCNA and FEN1 genes. We sequenced the coding region and adjacent noncoding region of both the PCNA and FEN1 genes in 120 alleles (60 individuals). In the PCNA gene, we detected 9 sequence variants with Hardy-Weinberg allele frequency ranging from 0.008 to 0.088. No polymorphism was detected in the FEN1 gene. The sequence variants in the PCNA gene included 7 intronic single nucleotide polymorphisms (SNP) and 2 synonymous exonic SNPs. All the intronic SNPs were located in introns 1 and 4, which contain several regulatory elements involved in the control of PCNA gene expression. Six of the 7 intronic SNPs showed complete linkage disequilibrium. We confirmed this allelic linkage disequilibrium by allele-specific PCR sequencing. We genotyped 117 additional individuals belonging to 3 population subgroups using the PCR-RFLP method. Finally, to see if the detected polymorphisms are associated with any cancer type, we genotyped cases with melanomas (37 cases), breast cancers (118 cases) and lung cancers (100 cases). We did not find statistical difference in frequency of polymorphism in any cancer type compared with healthy controls, although in breast cancer the frequency was low. Our results suggest that the coding regions of the PCNA and FEN1 genes are highly conserved when compared with other DNA repair genes. The potential of some of the detected intronic polymorphisms to effect regulation of the PCNA gene expression remains to be determined.