Proliferating Cell Nuclear Antigen Content Research Articles

Sorafenib suppresses proliferation rate of fibroblast-like synoviocytes through the arrest of cell cycle in experimental adjuvant arthritis.

Rheumatoid arthritis, a recurrent incendiary autoimmune joint syndrome, features by prominent synovial hyperplasia. Fibroblast-like synoviocytes are the executive components in the pathogenesis of rheumatoid arthritis. It is generally accepted that excessive proliferation and reduced apoptosis of fibroblast-like synoviocytes lead to synovial hyperplasia. Our previously studies found that sorafenib could inhibit adjuvant arthritis in rats and induced adjuvant arthritis fibroblast-like synoviocytes apoptosis. Presently, we aim to investigate the inhibitory effect with mechanisms of action of sorafenib on adjuvant arthritis fibroblast-like synoviocytes proliferation. Cell counting kit-8 and flow cytometry detection were conducted to monitor FLSs proliferation and cell cycle. Western blotting and qPCR assays were performed to detect P21, P53, CDK4, CyclinD1 and proliferating cell nuclear antigen content levels. Sorafenib significantly inhibited adjuvant arthritis fibroblast-like synoviocytes proliferation with an IC50 value of 4 µmol/L by a concentration-dependent pattern, which accompanies by G1 cell cycle arrest. Also, sorafenib significantly decreased the levels of P21, CyclinD1, CDK4 and proliferating cell nuclear antigen, as well as up-regulated P53 expression in adjuvant arthritis fibroblast-like synoviocytes. Sorafenib could inhibit adjuvant arthritis fibroblast-like synoviocytes proliferation via arresting G1/S cell cycle progression, which was partially through CDK4/CyclinD1-mediated pathway, as well as up-regulating P53 and down-regulating proliferating cell nuclear antigen expressions. These results suggest that sorafenib may provide a new paradigm for rheumatoid arthritis treatment.

Sea buckthorn (Hippophae rhamnoides L.) oil enhances proliferation, adipocytes differentiation and insulin sensitivity in 3T3-L1 cells.

The objective of this study is to investigate the effects of sea buckthorn oil (SBO) on proliferation, adipogenic differentiation and insulin sensitivity of 3T3-L1 cells. Results showed that SBO increased cell proliferation ability, accompanied by up-regulated proliferating cell nuclear antigen content (p < 0.05) and p38 activity (p < 0.05). SBO also promoted adipogenesis and enhanced adipogenic transcriptional factors expression. Mitochondrial biogenesis related gene expressions were elevated in SBO treated cells (p < 0.05). Of note, SBO also increased glucose uptake and glucose transporter 4 abundance (p < 0.05). Cells treated with SBO exhibited greater phosphorylated insulin receptor substrate 1 (p < 0.05), phosphorylated-Akt (p < 0.05) and phosphorylated AMP-activated protein kinase (p < 0.01) contents. When taken together, these results suggest that SBO promotes 3T3-L1 cells proliferation, adipogenesis and insulin sensitivity.

Significance of the expression of proliferating cell nuclear antigen and P53 in the regeneration process of an atrophic parotid gland

This research aims to further explore the expression and significance of proliferating cell nuclear antigen (PCNA) and P53 in regenerating rat atrophy parotid gland from the gene and protein levels. One hundred and two Wistar rats were randomly divided into experimental and control groups; the former group's duct was ligated and then released respectively in 7 (Group A) and 14 days (Group B). Fresh parotid specimens were obtained at 0, 3, 5, 7, 10, 14, 21, and 28 days after being released. Hematoxylin-eosin staining method was used to observe the morphological changes of the parotid gland. The significance of P53 and PCNA in two groups was resolved by real-time fluorescence quantitative polymerase china reaction and Western blot. Acinar cells aoptosis and duct cells proliferation occurred when the occlusion of the parotid duct was reversed on days 7 and 14. The expression of P53 was higher than that of PCNA, and they reached the peak at the third and fifth days after groups A and B regenerated, respectively. This finding was significantly different compared with the control (P<0.01). P53 and PCNA contents decreased gradually; acinar and duct gradually returned to normal morphology; PCNA and P53 contents gradually close to the normal control group. After ligating the parotid duct, P53 was highly expressed, and induced parotid gland atrophy. Mean-while, PCNA was highly expressed, which then decreased inducing gland recovery.

Increase of Palmitic Acid Concentration Impairs Endothelial Progenitor Cell and Bone Marrow–Derived Progenitor Cell Bioavailability

Metabolic profiling of plasma nonesterified fatty acids discovered that palmitic acid (PA), a natural peroxisome proliferator–activated receptor γ (PPARγ) ligand, is a reliable type 2 diabetes biomarker. We investigated whether and how PA diabetic (d-PA) concentrations affected endothelial progenitor cell (EPC) and bone marrow–derived hematopoietic cell (BM-HC) biology. PA physiologic (n-PA) and d-PA concentrations were used. Proliferating cell nuclear antigen content and signal transducer and activator of transcription 5 (STAT5), PPARγ, cyclin D1, and p21Waf expression were evaluated. Small interfering RNA technology, gene reporter luciferase assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, and coimmunoprecipitation were exploited. In vivo studies and migration assays were also performed. d-PA, unlike n-PA or physiological and diabetic oleic and stearic acid concentrations, impaired EPC migration and EPC/BM-HC proliferation through a PPARγ-mediated STAT5 transcription inhibition. This event did not prevent the formation of a STAT5/PPARγ transcriptional complex but was crucial for gene targeting, as p21Waf gene promoter, unlike cyclin D1, was the STAT5/PPARγ transcriptional target. Similar molecular events could be detected in EPCs isolated from type 2 diabetic patients. By expressing a constitutively activated STAT5 form, we demonstrated that STAT5 content is crucial for gene targeting and EPC fate. Finally, we also provide in vivo data that d-PA–mediated EPC dysfunction could be rescued by PPARγ blockade. These data provide first insights on how mechanistically d-PA drives EPC/BM-HC dysfunction in diabetes.

The role of histamine in human mammary carcinogenesis: H3 and H4 receptors as potential therapeutic targets for breast cancer treatment.

There is increasing evidence that describes a histamine role in normal and cancer cell proliferation. To better understand the importance of histamine in breast cancer development, the expression of histamine H3 (H3R) and H4 (H4R) receptors and their association with proliferating cell nuclear antigen (PCNA), histidine decarboxylase (HDC) and histamine content were explored in mammary biopsies. Additionally, we investigated whether H3R and H4R were implicated in the biological responses triggered by histamine in MDA-MB-231 breast cancer cells. The expression levels of H3R, H4R, PCNA, HDC and histamine content were determined by immunohistochemistry in 40 benign and malignant lesions. MDA-MB-231 cells proliferation (clonogenic assay and BrdU incorporation) and cell cycle distribution (flow cytometry) were evaluated upon treatment with histamine, H3R and H4R agonists and antagonists. Apoptosis was determined by Annexin staining and TUNEL assay. Cell migration was assessed by transwell system. Results indicate that H3R was detected in 67% (10/15) of benign lesions and in almost all carcinomas (24/25), being the level of its expression significantly higher in carcinomas (P=0.0016). The non-tumoral breast tissue surrounding carcinomas revealed a lower H3R expression compared to the tumor cells. Only 13% (2/15) of the benign lesions expressed H4R compared to 44% (11/25) of the carcinomas. Interestingly, H3R expression was correlated in carcinomas with the expression of HDC and PCNA (P

Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen

A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.

Stimulation of TM3 Leydig cell proliferation via GABAA receptors: A new role for testicular GABA

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABAA and GABAB receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABAA receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABAA receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABAA receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABAA agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABAA antagonist bicuculline, implying a role for GABAA receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABAA receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.

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.

Effects of chronic exposure to simulated microgravity on skeletal muscle cell proliferation and differentiation.

Cell culture models that mimic long-term exposure to microgravity provide important insights into the cellular biological adaptations of human skeletal muscle to long-term residence in space. We developed insert scaffolding for the NASA-designed rotating cell culture system (RCCS) in order to study the effects of time-averaged microgravity on the proliferation and differentiation of anchorage-dependent skeletal muscle myocytes. We hypothesized that prolonged microgravity exposure would result in the retardation of myocyte differentiation. Microgravity exposure in the RCCS resulted in increased cellular proliferation. Despite shifting to media conditions promoting cellular differentiation, 5 d later, there was an increase in cell number of approximately 62%, increases in total cellular protein (52%), and cellular proliferating cell nuclear antigen (PCNA) content (2.7 times control), and only a modest (insignificant) decrease (10%) in sarcomeric myosin protein expression. We grew cells in an inverted orientation on membrane inserts. Changes in cell number and PCNA content were the converse to those observed for cells in the RCCS. We also grew cells on inserts at unit gravity with constant mixing. Mixing accounted for part, but not all, of the effects of microgravity exposure on skeletal muscle cell cultures (53% of the RCCS effect on PCNA at 4-6 d). In summary, the mechanical effects of simulated microgravity exposure in the RCCS resulted in the maintenance of cellular proliferation, manifested as increases in cell number and expression of PCNA relative to control conditions, with only a modest reciprocal inhibition of cellular differentiation. Therefore, this model provides conditions wherein cellular differentiation and proliferation appear to be uncoupled.

Prognostic Implications of PCNA Expression and DNA Contents in Squamous Cell Carcinoma of Larynx

Objective To investigate Proliferating cell nuclear antigen(PCNA) and DNA index(DI) correlation to biological characteristics of laryngeal carcinoma and their values of clinical prognosis.Methods Immunohistochemical staining was used to detect the expression of PCNA in laryngeal cancer and normal tissue,and with MIPS I image analysis system DNA contents of cancer cell were measured and made out DNA index.Results PCNA positive rate was 100% in laryngeal carcinoma,and it was negative in all 10 normal laryngeal mucosa specimens( P 0 01).The positive expression of PCNA and DNA contents in the laryngeal carcinoma were increased with the differentiation of tumors lower( P 0 05).With the increasing PCNA positive expression and DI,the prognosis of the patients got poorer( P 0 05).Conclusion PCNA and DI were simultaneously detected can be used as the prediction of tumor malignancy and prognosis.

Expression of p53, c-erbB-2, PCNA and DAN content in lung carcinoma

The expression of p 53 protein, c-erbB-2 oncoprotein, proliferating cell nuclear antigen (PCNA) were studied by the streptavidin peroxidase conjugated (S-P) immunohistochemical method and DNA content in situ was tested, in order to explore the significance of p 53 , c-erbB-2, PCNA in primary lung carcinoma.

Discordant hepatic expression of the cell division control enzyme p34cdc2 kinase, proliferating cell nuclear antigen, p53 tumor suppressor protein, and p21Waf1 cyclin-dependent kinase inhibitory protein after WY14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) dosing to rats.

The hepatocarcinogen and peroxisome proliferator WY14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) was examined for its ability to induce changes in the intracellular protein expression of hepatic p34cdc2 kinase (CDK1), proliferating cell nuclear antigen (PCNA), p53 tumor suppressor protein, and p21Waf1 CDK inhibiting protein. Young adult male rats were administered 45 mg-kg/day WY14,643 intraperitoneally for 1, 2, 3, 4, or 5 days or fed diets containing 0% or 0.08% WY14,643 for 1, 2, 3, or 4 weeks. WY14,643 dosing increased concentrations of hepatic proteins of 34- and 37-kDa molecular mass, which were identified through immunoprecipitation as CDK1 and PCNA, respectively. Gel filtration of the hepatic S9 fractions determined by enzyme-linked immunosorbent assay confirmed the increased expression of CDK1 and PCNA immunoreactivity in livers from WY14,643-treated rats. Also, gel filtration revealed that the native CDK1 and PCNA in hepatic S9 from WY14,643-treated rats chromatographed as a major peak with an apparent molecular mass of 70 and 76 kDa, respectively. Immunoblotting of the 70-kDa fraction with anti-CDK1 revealed a single band of molecular mass of 34 kDa. Thus, the CDK1 in the major immunoreactive peak of WY14,643-treated rat liver S9 seems to exist as a heterodimer or homodimer. Immunohistochemistry of formalin-fixed liver demonstrated a cytosolic localization of immunoreactive CDK1 and nuclear localization of immunoreactive PCNA in proliferating cells of WY14,643-treated rat livers. WY14,643 increased hepatic CDK1 content by 1.9-6.3-fold through postdosing days 1-5. Hepatic PCNA content was increased 1.9-5-fold over the same period. In the 4-week feeding study, CDK1 and PCNA expression were increased at all weekly time points by an average of 15-50-fold, respectively. Furthermore, the dietary administration of 0.08% WY14,643 resulted in sustained, overexpression of hepatic p53 tumor suppressor protein from week 1 through week 4 and of p21Waf1 CDK inhibitory protein from week 3 to week 4.

Age-related changes in proliferating cell nuclear antigen levels

To clarify the effect of aging on rat liver regeneration, we compared proliferating cell nuclear antigen (PCNA) levels in control and regenerating livers from young and aged rats 48 h after partial hepatectomy. The nucleoplasm and cytoplasm from regenerating livers of 2-month and 24 month-old rats were fractionated by phosphocellulose column chromatography, aliquots of fractions were transferred to nitrocellulose filters and the amounts of PCNA in each fraction were measured by an immunostaining method. Two forms of PCNA, L type (eluted at low concentrations of KCl) and H type (eluted at high KCl concentrations) were observed in the nucleoplasm from both control and regenerating young rat liver. On the other hand, the cytoplasm contained P type (eluted in the pass-through fraction), L type and H type PCNA. In control liver from aged rats, three types of PCNA in the cytoplasm and two types in the nucleoplasm were present at decreased levels. In regenerating liver from young rats, the increases in L type in the cytoplasm and H type in the nucleoplasm were remarkable. However, none of the three PCNA types increased significantly during liver regeneration in aged rats. Treatment with DNase resulted in the disappearance of the H type with a concomitant increase in the P and L types. These results suggest that the H type is a complex form consisting of the P and L types of PCNA and DNA. These results suggest that the increase in the L type in the cytoplasm reflects newly synthesized PCNA production for cellular proliferation and that the increase in the H type in the nucleoplasm is a reflection of binding to DNA and the fundamental role of PCNA itself in liver regeneration in young rats. On the other hand, there was little increase in any of the three types in regenerating liver from 24-month-old rats. Thus, PCNA content may be closely related to the decrease in the rate of cellular proliferation in aged animals.

Cell cycle analysis of amount and distribution of nuclear DNA topoisomerase I as determined by fluorescence digital imaging microscopy.

Fluorescence digital imaging microscopy (FDIM) has been used to perform a cell cycle analysis of both the amount and the distribution of nuclear DNA topoisomerase I in individual CEM human leukemia cells. Cells were stained by indirect immunofluorescence methods using a polyclonal antiserum generated with a 21-amino-acid peptide representing amino acids 219-239 of human topoisomerase I. Immunohistochemical staining was followed by staining with Hoechst dye 33342, allowing DNA content to be determined in each cell. Cell cycle analysis showed that nuclear topoisomerase I content doubled (2.2-fold increase) as the cells progressed from G1 to G2/M phases of the cell cycle. However, when normalized for nuclear size, topoisomerase I content per nuclear area remained almost constant (1.3-fold increase). For comparison, we measured the amount of proliferating cell nuclear antigen (PCNA), a protein whose expression fluctuates during the cell cycle. Nuclear PCNA content increased 2.7-fold from G1 to S phase, then declined in G2/M- phases, whereas PCNA content per nuclear area increased 1.7-fold from G1 to S phase. We also measured topoisomerase I content in leucine-deprived cells to determine if altered growth conditions affect topoisomerase I protein expression. Compared to CEM cells in logarithmic growth, leucine-deprived CEM cells had 1.8-fold less topoisomerase I content per nuclear area. Subnuclear distribution studies of proliferating CEM cells showed topoisomerase I to be localized predominantly in the nucleoli throughout the cell cycle. In contrast, leucine-deprived cells exhibited a perinuclear distribution of topoisomerase I. Our results show that FDIM is a useful technique in determining the cell cycle position and both the content and the distribution of topoisomerase I as well as other nuclear proteins in individual cells.

Image analysis of in situ cell cycle related changes of PCNA and Ki‐67 proliferating antigen expression

Abstract. This study reports on the proliferating cell nuclear antigen (PCNA) and Ki‐67 cell cycle related expression and distribution pattern analysed in the same cells. MCF‐7 cells were synchronized by mitotic detachment and triple stained for DNA, PCNA and Ki‐67. The major cell type was identified on each time sample as a function of the PCNA/Ki‐67 pattern, and both antigens as well as DNA were quantified. During G1 phase, the expression of PCNA greatly increased whereas Ki‐67 content decreased. During S phase, nuclear Ki‐67 content continuously increased especially in the second half of this phase, mainly due to the accumulation of the antigen in the nucleoli. During G2 phase, the antigen significantly passed into the nucleoplasm, its content continued to increase and reached its maximum in mitotic cells. Nuclear PCNA content mostly increased in the first part of S phase and sharply declined in mitotic cells as the antigen shifted to the cytoplasm. Cells showing PCNA positive Ki‐67 negative labelling were observed in all time samples from the beginning of the experiment. Their nuclear size, DNA content (of G1 cells), PCNA content (equivalent to the content of some late G, cells) and time occurrence (their percentage increased after the last late G1 cells had disappeared) tend to indicate that these cells have left the cycle by the end of G1 phase to enter a quiescent state. Cells coming out of mitosis split into two groups according to their Ki‐67/PCNA content. The biggest fraction was PCNA negative and Ki‐67 positive while the smallest showed positive staining for both antibodies. Cells of this second cohort slowly lost their 1–67 while their PCNA content increased as they moved through G1. Concurrently, most of the cells of the first cohort (here called Q2 and Q3 cell types) lost their Ki‐67 without increasing their PCNA content; then they joined cells of the second cohort by increasing their PCNA content at the end of G, phase. Some cells of this first cohort can also increase their PCNA and thus reach cells of the first cohort before the end of G1 phase. The existence of these two main cell cohorts suggests that cells after mitosis differ in some way that make them progress dlfferently through G1. Some cells seem to go through early G1 (G1a and late G1 (Glb) while others may come out of mitosis committed to go through the following cycle by directly entering late G1 compartment.

Proliferating cell nuclear antigen in endometrial adenocarcinomas of endometrioid type correlated with histologic grade, stage, previous hormonal treatment, and survival

Only a small number of endometrial carcinomas have been examined for proliferating cell nuclear antigen. The results indicate that a high proliferating cell nuclear antigen content correlates with a poor prognosis. One hundred eight endometrial carcinomas of endometrioid type were examined with the monoclonal antibody PC10 (48 tumors from postmenopausal estrogen users and 60 tumors from nonusers). The PC10 content was weakly but significantly correlated with mitotic count and architectural grade, but not with nuclear grade, stage, or survival. PC10 values in estrogen users were much lower (median, 14%) than in nonusers (median, 26%); the difference was independent of histologic grade and stage. After a median follow-up of 30 months (range, 12 to 66 months) 17 patients had died. The cause of death was established as cancer in only nine cases. No overall difference in PC10 values existed between survivors and nonsurvivors. However, if only the estrogen nonusers were examined the survivors showed a mean PC10 value of 27%, while the nonsurvivors showed a mean PC10 value of 45%. The present study indicates that carcinomas from patients with and without previous hormonal treatment are different with regard to their PC10 content. The quantitative and qualitative estimates of PC10 correlated well.

PCNA and total nuclear protein content as markers of cell proliferation in pea tissue

ABSTRACT The identification of cell proliferation markers has been shown to be a useful tool with which to study basic mechanisms of cell cycle progression. The use of immunofluorescence techniques revealed the presence of the proliferating cell nuclear antigen (PCNA) in pea tissue, where we observed a high PCNA expression in proliferating cells of the root meristem compared to noncycling cells of the differentiated leaf. The presence of PCNA was monitored also during the time-course of seed germination, before, during and after the cell cycle resumption of the embryo cells. PCNA is present in embryo cells not only during and after resumption of the cell cycle but also before, when cells have not yet begun replicating their genome. A bivariate flow cytometric analysis of DNA and nuclear protein content was used to localize precisely the cells of the examined pea tissues in different cell cycle phase subcompartments. A high correlation was found between the degree of cell proliferation and the protein content of G1 nuclei, on the one hand, and the percentage of PCNA positive cells on the other.

Expression of Proliferating Cell Nuclear Antigen (PCNA) and Ki-67 Antigen in Human Malignant Hematopoietic Cells

Human hematopoietic cell lines and fresh lymphoma cells were investigated for their expression of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen using flow cytometry. Ki-67 was detected with a monoclonal antibody and the PCNA content was determined using a human auto-antiserum and in some cases a monoclonal antibody (PC10). The autoantibody to PCNA was found to recognize S-phase cells and the number of positively stained cells was significantly correlated with the S-phase fraction determined from DNA-histograms. There was also a strong association between Ki-67 positivity and the fraction of S-phase cells. By dual parameter analysis of Ki-67 and PCNA detected by the autoantibody a detailed cell cycle analysis could be performed of the lymphoma samples. The recently described PC10 monoclonal antibody was found by FCM to be a good proliferation marker recognizing cycling cells after methanol and detergent treatment, however without any specific cell cycle phase preference.

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.