Immunolocalization Of Proliferating Cell Nuclear Antigen Research Articles

Integration of transcriptome and DNA methylome analysis reveals the molecular mechanism of taproot yield heterosis in radish (Raphanus sativus L.)

Heterosis, a crucial biological phenomenon, plays a vital role in determining the yield and quality of plants. Radish, an important root vegetable crop, exhibits notable heterosis in terms of root yield and quality. Nevertheless, the specific molecular mechanism behind the formation of heterosis in radish remains unclear. Herein, transcriptome and DNA methylome analyses were performed on F1 hybrids and parental lines. Expression level dominance (ELD) genes and allele-specific expression (ASEG) genes together significantly contribute to heterosis, primarily through energy metabolism and plant hormone signal transduction pathway. Additionally, an increase in the average methylation level in the F1 hybrids was observed compared to the parental lines. Interestingly, a negative correlation was found between the methylation level of differentially expressed genes (DEGs) in gene body regions and their expression levels in NAU-LB and the F1 hybrids. Conversely, a contrasting trend was observed between NAU-YH and the F1 hybrids. Furthermore, when the parental lines and the F1 hybrids were treated with 5-azacytidine, the hybrids were more sensitive to methylation inhibitors than their parents. A significant decrease was observed in root weight and total sugar content in the F1 hybrids compared to the control. Immunolocalization results indicated a significant decrease in the auxin content of the F1 hybrid under 5-azacytidine treatment. Proliferating cell nuclear antigen immunolocalization also revealed significant inhibition of vascular cambium activity in both the hybrids and parental lines. Notably, the expression profiles of a few differentially methylated DEGs including RsSUS1, RsSUC2a, RsIAA7, and RsIAA18, were significantly increased in the root of hybrids compared to their parents, suggesting a potential role for DNA methylation in yield heterosis. Collectively, these findings would provide valuable insight into the molecular mechanism underlying taproot yield heterosis and have the potential to facilitate the genetic improvement of taproot yield and quality in radish breeding programs.

Follicular development and morphological changes in the vaginal epithelium during the estrous cycle of Galea spixii.

The current study aimed to determine if characteristics observed in vaginal cytology during the estrous cycle of female SYT cavies corresponded with proliferation of the vaginal epithelium, characterized by proliferating cell nuclear antigen (PCNA) immunolocalization, and with follicular development at different phases of the estrous cycle. After determining estrous cycle phases by vaginal cytology, females were euthanized at metestrus, diestrus, proestrus, and estrus. Histological study of the vaginal epithelium and ovary were then performed. Immunohistochemistry for PCNA in vaginal tissue at each cycle phase was also performed. Superficial cornified cells and early post-ovulatory follicles were found at estrus. Few nuclei below the enucleate superficial cells were immunoreactive to PCNA. At metestrus, the vaginal epithelium underwent desquamation and lost the superficial cornified cells; basal and intermediate cells appeared, and the post-ovulatory follicle formed an early corpus luteum. No PCNA immunoreactivity was observed. At diestrus, the corpus luteum was developed, and the vaginal epithelium contained basal and intermediate cells. There was PCNA immunoreactivity in the cellular nucleus in the germinative stratum of the epithelium. Because of the growth and maturation of ovarian follicles, the vaginal epithelium suffered intense proliferation at proestrus. Vaginal cytology revealed large intermediate cells and nucleated and enucleated superficial cornified cells. In the ovary, mature follicles were present. More apparent immunoreactivity of PCNA in the germinative layer was found. In summary, we inferred that vaginal exfoliative findings matched the proliferation process of the vaginal epithelium. PCNA immunolocalization occurred as well as corresponding follicular development in the ovaries.

Vasoactive Intestinal Peptide Can Promote the Development of Neonatal Rat Primordial Follicles During In Vitro Culture1

Recruitment of primordial follicles is essential for female fertility. Some of the intraovarian growth factors involved in the initiation of primordial follicle growth have been identified, but the exact mechanisms regulating follicle activation are poorly understood. Strong evidence indicates that vasoactive intestinal peptide (VIP), a neuropeptide found in ovarian nerves, plays a role in the physiology of follicle development and function. The aim of the present study was to determine whether VIP might regulate the activation and growth of neonatal rat primordial follicles in an in vitro culture system. Ovaries from 4-day-old rats were cultured for 14 days in medium containing 10(-7) M VIP. At the end of the culture, the developmental stages and viability of the follicles were evaluated using histological sections. Immunohistochemistry studies for proliferating cell nuclear antigen (PCNA) were performed to assess the mitotic activity of granulosa cells. In addition, the expression level of kit ligand (KL) mRNA was examined after culture. Histology showed that primordial follicles could survive and start to grow in vitro. The proportion of primordial follicles was decreased and the proportion of early primary follicles increased after in vitro culture with VIP. Immunolocalization of PCNA showed that follicle growth was initiated after VIP treatment. The expression level of KL mRNA was increased in the VIP treatment group. Thus, VIP can promote primordial follicle development, possibly mediated in part through upregulating the expression of KL.

Immunolocalization of PCNA, Ki67, p27 and p57 in normal and dexamethasone-induced intrauterine growth restriction placental development in rat

Intrauterine growth restriction (IUGR) is a major clinical problem which causes perinatal morbidity and mortality. Although fetuses with IUGR form a heterogeneous group, a major etiological factor is abnormal placentation. Despite the fact that placental development requires the coordinated action of trophoblast proliferation and differentiation, there are few studies on cell cycle regulators, which play the main roles in the coordination of these events. Moreover it is still not determined how mechanisms of coordination of proliferation and differentiation are influenced by dexamethasone-induced IUGR in the placenta. The aim of the study was to investigate the spatial and temporal immunolocalization of proliferating cell nuclear antigen (PCNA), Ki67, p27 and p57 in normal and IUGR placental development in pregnant Wistar rats. The study demonstrated altered expressions of distinct cell cycle proteins and cyclin dependent kinase inhibitors (CKIs) in IUGR placental development compared to control placental development. We found reduced immunostaining of PCNA and Ki67 and increased immunostaining of p27 and p57 in the dexamethasone-induced IUGR placental development compared to control placental development. In conclusion, our data show that the cell populations in the placenta stain for a number of cell cycle related proteins and that these staining patterns change as a function of both gestational age and abnormal placentation.

Remodelling of adipose tissue during experimental omental adhesion formation

Peritoneal adhesions are fibrous bands of tissue connecting normally separated organs and frequently involve the fat-laden greater omentum. Remodelling of fibrin-rich exudate under reduced fibrinolytic conditions is thought to initiate adhesion formation following surgery. It is unclear whether adhesions that involve the omentum develop in a similar manner. To improve understanding of omental adhesion formation, adipose tissue distribution, cell proliferation and procollagen type I gene expression were investigated in a murine surgical model and in established omental adhesions from patients undergoing abdominal surgery. Experimental murine omental adhesions and human omental adhesions were analysed for signs of tissue remodelling using histology, immunohistochemistry and in situ hybridization. Murine omental tissue showed intense inflammation and reduced adipose tissue 3-7 days after surgery, but increased cellularity and collagen production. Adipose tissue remodelling was reversible with increased adipose tissue and decreased cell proliferation and procollagen type I gene expression, shown by proliferating cell nuclear antigen immunolocalization and in situ hybridization respectively. Human omental adhesions were heterogeneous, with varying amounts of fibrotic and adipose-rich regions, although most displayed proliferating and collagen-producing cells. Omental adhesions are not static scar tissue as traditionally thought, but undergo active adipose tissue remodelling over-time.

Immunolocalization of proliferating cell nuclear antigen (PCNA) in cynomolgus monkey (Macaca fascicularis) testes during postnatal development.

The age-related distribution of proliferating cell nuclear antigen (PCNA) in the testes of cynomolgus monkeys (Macaca fascicularis) during postnatal development was detected using light-microscopic immunohistochemistry. In neonatal testes, some PCNA-positive spermatogonia, Sertoli cells, peritubular cells, and Leydig cells were detected. In early infantile testes, only a few of these cell types were positive. In late infantile testes, the numbers of positive cells were greater than in the earlier developmental stages. In pubertal testes, the numbers of positive spermatogonia, spermatocytes, Sertoli cells, peritubular cells, and Leydig cells were considerably higher. In adult testes, a larger percentage of spermatogonia and spermatocytes was positive, and peritubular cells and Leydig cells were occasionally positive; secondary spermatocytes, spermatids, and Sertoli cells were not positive. We concluded that immunolocalization of PCNA can serve as a tool for studying proliferation status in developing testes of cynomolgus monkeys. A relatively low proliferative activity in early infantile testes and a remarkable increase of proliferative activity in pubertal testes correlate with the fluctuations of steroidogenic functions during postnatal development in cynomolgus monkeys.

The Effect of Age on Proliferating Cell Nuclear Antigen Expression in Oral Gingival Epithelium of Healthy and Inflamed Human Gingiva

Age-related changes in proliferative activity in human gingival epithelium are uncertain. Proliferating cell nuclear antigen (PCNA) is a nuclear protein associated with the cell cycle. Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues. The aims of this study were to investigate the localization of PCNA expression in oral gingival epithelium (OGE) and to define the age-related changes as to PCNA-proliferative index (PI) in inflamed as well as healthy gingiva. Mitotic index (MI) was also used as a conventional marker of cell proliferation. Additionally, the effect of aging upon the maximum epithelial thickness (MET) was determined. Twenty older (65 to 85 years) (study) and 20 middle-aged (35 to 45 years) (controls) subjects were included in the study. Biopsies were obtained both from healthy and inflamed gingiva. The expression of PCNA was evaluated in formalin-fixed, paraffin-embedded gingival samples using an immunoperoxidase technique and PC 10 monoclonal antibody to PCNA. Hematoxylin and eosin stained sections were used for the quantitative measurement of MI and MET. All the tissue sections contained positive staining cells for PCNA in the gingival epithelium. Although PCNA expression was observed both in the basal and suprabasal layers, it was more prominent in the suprabasal layers. PI in inflamed gingiva was significantly higher in the older group. However, no significant difference was observed between the study and control groups with respect to PI in healthy gingiva. When all the subjects taken into the study were analyzed as a single group, PI in the inflamed gingival samples were found to be increased with aging. Nevertheless, no age-related change was noted in MI and MET. In both the study and the control groups, PI, MI, and MET were found to be increased due to inflammation. Our data indicate that PCNA expression in inflamed gingiva is higher in older subjects. Furthermore, a significant correlation was noted between aging and PCNA expression in inflamed gingiva. As there is no increase in mucosal epithelial thickness despite increased proliferation, we speculate that the duration of the PCNA+ phase in cell cycle may be longer in older subjects. This study also implies that PCNA immunolocalization can be used as an index of the state of cell proliferation in both biological and pathological events of the gingiva and/or other mucosal tissues.

Transforming growth factor-alpha, epidermal growth factor receptor, and PCNA immunoexpression in uterine leiomyosarcomas and leiomyomas in B6C3F1 mice

The role of growth factors in the development of murine uterine mesenchymal tumors is unknown. In this study, immunohistochemical expression of transforming growth factor alpha (TGF-alpha) and its receptor epidermal growth factor (EGF-R) was assessed in spontaneous uterine leiomyomas and leiomyosarcomas in B6C3F1 mice. Cell proliferation, which has been induced by some growth factors, was evaluated by immunohistochemical detection of an endogenous marker of cell proliferation, proliferating cell nuclear antigen (PCNA). PCNA labeling indices were determined and compared to the intensity and distribution of TGF-alpha staining in sequential sections of control myometrium or tumor tissue. Results showed uterine leiomyosarcomas had positive cytoplasmic staining for TGF-alpha; however, all uterine leiomyomas evaluated were negative. Positive EGF-R staining was also observed in the uterine leiomyosarcomas, but not in the leiomyomas. EGF-R immunoexpression was detected primarily within the cytoplasm of the leiomyosarcoma cells, with occasional nuclear immunoreactivity. Immunohistochemical staining for PCNA was more intense and there were increased numbers of positively staining nuclei in the leiomyosarcomas compared to samples of control myometrium or leiomyomas. The mean labeling index for the uterine leiomyosarcomas (7.40%) was significantly (p < 0.01) higher than that of leiomyomas (0.29%) and control uterine myometrium (0.13%). We conclude, that TGF-alpha and its receptor, EGF-R, are expressed more intensely in uterine leiomyosarcomas, compared to leiomyomas in B6C3F1 mice. Immunoexpression of TGF-alpha may be an important biomarker of malignancy in uterine smooth muscle tumors in mice. Futhermore, TGF-alpha may play a critical role in increased proliferation of uterine smooth muscle tumor cells as suggested by increased immunolocalization of PCNA in rodent leiomyosarcomas expressing TGF-alpha, although other factors regulating cell replication can not be ruled out.

Epithelial proliferation in Barrett's esophagus by proliferating cell nuclear antigen immunolocalization

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase δ and is an absolute requirement for cellular proliferation. Specialized-type Barrett's columnar-lined esophagus (CLE) is associated with adenocarcinomatous change. In the present study, the cellular proliferation of three histological types of CLE was assessed by semi-quantitative evaluation of PCNA immunolocalization in 93 biopsy specimens from 45 patients using the murine monoclonal PC10. Statistical comparison was performed by the Mann-Whitney U test. Luminal surface cell labeling was uncommon in all histological types other than specialized CLE where 25 of the 43 biopsy specimens had at least occasional luminal surface cell labeling. Mean crypt labeling score of 4.06 for specialized type exceeded that for junctional (mean, 3.12; P < 0.001) and fundic types (mean, 1.6; P < 0.001). Gland cell PCNA staining scores for specialized-type CLE (mean, 3.18) exceeded that of junctional (mean, 1.97; P < 0.001) and fundic (mean, 1.04; P < 0.001). Summated PCNA scores for specialized-type, mean of 8.29, exceeded junctional mean score of 5.45 (P < 0.001) and fundic mean score of 2.76 (P < 0.001). PCNA immunolocalization reveals a high proportion of cells in cycle in the specialized-type CLE and expansion of the proliferative compartment, which may explain the association of specialized-type CLE with malignancy.

Proliferating cell nuclear antigen (PCNA)/cyclin expression during the cell cycle in normal and leukemic cells

Bivariate flow cytometric analysis of the cell proliferation-associated nuclear protein, identified as the “proliferating cell nuclear antigen” (PCNA)/cyclin and of nuclear DNA content, was performed in quiescent and mitogen-stimulated human peripheral blood lymphocytes, in EUE (human embryonic epithelium) cells, before and after a long-term exposure to a hypertonic (HT) medium, in 4 human leukemic cell lines and in fresh bone marrow (BM) cells from 10 patients with untreated acute non-lymphoblastic leukemia (ANLL). The PCNA/cyclin was detected using both an autoantibody extracted from sera of systemic lupus erythematosus patients and the recently produced mouse monoclonal antibody (MoAb) IgG, named 19F4. The distribution of cells in the different phases of the cycle and the percentage of S-phase cells were obtained in duplicate samples, by DNA flow cytometry (FCM) and by dual parameter FCM of DNA content and bromodeoxyuridine (BUDR) incorporation. In all cell types, the non-specific cytoplasmic background fluorescence was significantly lower with the MoAb compared to that obtained with the polyclonal Ab. The percentage of PCNA-positive cells (both with the autoantibody and the 19F4 MoAb) was always higher than that of S-phase cells by DNA FCM and of BUDR-labeled cells. The pattern of PCNA-expression in both normal proliferating cells and acute leukemia cells, showed that most G 0/G 1 cells did not express significant amounts of PCNA; an increase in PCNA immunofluorescence was found in late G 1 cells, and further increases were observed in S- and G 2-M phase cells. PCNA/cyclin, as revealed both with autoantibodies and with the 19F4 MoAb, is associated with all actively or potentially dividing (i.e. G 1, S and G 2-M) cells thus identifying the proliferative cellular compartment. Combined with the use of multiparameter FCM techniques, the PCNA immunolocalization offers a useful tool to study cell kinetics in normal and leukemic human cell populations.

Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: An index of cell proliferation with evidence of deregulated expression in some, neoplasms

Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein associated with the cell cycle. A monoclonal antibody, PC10, that recognizes a fixation and processing resistant epitope has been used to investigate its tissue distribution. Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues. PCNA immunoreactivity is induced in lectin stimulated peripheral blood mononuclear cells in parallel with bromodeoxyuridine incorporation and the number of cells with PCNA immunoreactivity is reduced by induction of differentiation in HL60 cells. In non-Hodgkin's lymphomas a linear relation between Ki67 and PCNA staining was demonstrated. These data suggest that in normal tissues and lymphoid neoplasms, PCNA immunolocalization can be used as an index of cell proliferation. However, in some forms of neoplasia, including breast and gastric cancer and in vitro cell lines, the simple relation between PCNA expression and cell proliferation is lost. In some breast and pancreatic tumours there is apparent deregulation of PCNA with increased expression in tissues adjacent to the tumours. The over-expression in some tumours and in adjacent morphologically normal tissue may represent autocrine or paracrine growth factor influence on PCNA gene expression.