Proliferating Cell Nuclear Antigen Gene Expression Research Articles

UV inducibility of rat proliferating cell nuclear antigen gene promoter

Proliferating cell nuclear antigen (PCNA), also known as a cofactor of DNA polymerase δ, 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. J. Cell. Biochem. 73:423–432, 1999. © 1999 Wiley-Liss, Inc.

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.

Involvement of RFX1 Protein in the Regulation of the Human Proliferating Cell Nuclear Antigen Promoter

The proliferating cell nuclear antigen (PCNA) is an essential eukaryotic DNA replication factor that is transcriptionally regulated by the adenovirus oncoprotein E1A 243R. Inducibility of the human PCNA promoter by E1A 243R is conferred by the cis-acting PCNA E1A-responsive element (PERE), which associates with the ATF-1, cAMP response element-binding protein (CREB), and RFX1 transcription factors and is modulated by cellular proteins such as the coactivator CREB-binding protein (CBP) and tumor suppressor p107 (Labrie, C., Lee, B. H., and Mathews, M. B. (1995) Nucleic Acids Res. 23, 3732-3741; Lee, B. H., and Mathews, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4481-4486; Lee, B. H., Liu, M., and Mathews, M. B. (1998) J. Virol. 72, 1138-1145). RFX1 also forms a complex with sequences in the PCNA promoter of mouse and rat that share homology with the RFX1 consensus site. To explore the role of RFX1 in regulating the PCNA promoter, we examined the effects of mutations in the human PERE on RFX1 binding and gene expression. Mutations within the RFX1 consensus binding site reduced RFX1 binding, whereas mutations upstream of the site, or on its border, increased RFX1 binding. These mutations also affected the transcriptional activity of PCNA-chloramphenicol acetyltransferase reporter constructs in transient expression assays. The relative transcriptional activity of mutant PCNA promoters, both in the presence and absence of E1A 243R, was inversely related to their ability to complex with RFX1. These findings suggest that the binding of RFX1 is influenced by sequences outside its consensus binding site and that this transcription factor plays an inhibitory role in the regulation of PCNA gene expression.

Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional transactivator and viral oncogene. Since cellular transformation has been frequently linked to alterations in genome stability, we investigated the effect of Tax on nucleotide excision repair (NER), a prominent cellular DNA repair pathway. Cells expressing Tax exhibited a reduced capacity for NER as measured by unscheduled DNA synthesis and host cell reactivation assays. The cellular proliferating cell nuclear antigen (PCNA) gene product regulates DNA replication and repair pathways, including NER. Since Tax activates transcription of the PCNA promoter, we investigated whether this activity contributes to the reduction of NER. Tax increased endogenous PCNA protein expression, and analysis of Tax mutant proteins demonstrated that the reduction in NER correlated with Tax transactivation of PCNA gene expression. Direct overexpression of PCNA also reduced NER. We propose that overexpression of PCNA, and disruption of NER induced by Tax, predisposes cells to accumulate DNA damage and contributes to HTLV-1 transformation.

An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro.

The transcription factor E2F controls expression of several genes involved in cell proliferation including c-myc, c-myb, proliferating cell nuclear antigen (PCNA), and cdk2 kinase. Having established that both PCNA and cdk2 kinase are induced in rat mesangial cells (MC) by serum stimulation, we attempted to inhibit MC proliferation in vitro by transfecting these cells with cationic liposomes containing a synthetic double-stranded oligodeoxynucleotide (ODN) with high affinity for E2F. Using a gel mobility shift assay, we detected increased specific binding of E2F in MC following serum stimulation. This binding was completely inhibited by preincubation of MC nuclear extracts with the double-stranded ODN with high affinity for E2F but not by preincubation with a missense ODN containing two point mutations. MC were also transfected with a luciferase reporter gene construct containing three E2F binding sites. Luciferase activity was enhanced by serum stimulation of MC, and this effect was specifically abolished by cotransfection of MC with E2F decoy ODN. Furthermore, RT-PCR analysis revealed that serum-induced upregulation of PCNA and cdk2 kinase gene expression was inhibited by E2F decoy ODN transfection but not by transfection of missense ODN. These changes in gene expression were paralleled by a reduction in PCNA and cdk2 kinase protein expression in E2F decoy ODN transfected cells. MC number increased following serum stimulation. This effect was blunted by transfection with E2F decoy ODN but not by transfection of missense ODN. These data suggest that the transcription factor E2F plays a crucial role in the regulation of MC proliferation and that this factor can be successfully targeted to inhibit MC cell cycle progression.

PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene.

We have previously defined the promoter elements, sites IIa and IIb, in the rice proliferating cell nuclear antigen (PCNA) gene that are essential for meristematic tissue-specific expression. In this study, we isolated and characterized cDNAs encoding proteins that specifically bind to sites IIa and IIb. The two DNA binding proteins, designated PCF1 and PCF2, share > 70% homology in common conserved sequences at the N-terminal regions. The conserved regions are responsible for DNA binding and homodimer and heterodimer formation, and they contain a putative basic helix-loop-helix (bHLH) motif. The structure and DNA binding specificity of the bHLH motif are distinguishable from those of other known bHLH proteins that bind to the E-box. The motif is > 70% homologous to several expressed sequence tags from Arabidopsis and rice, suggesting that PCF1 and PCF2 are members of a novel family of proteins that are conserved in monocotyledons and dicotyledons. A supershift assay using an anti-PCF2 antibody showed the involvement of PCF2 in site IIa (site IIb) binding activities in rice nuclear extracts, particularly in meristematic tissues. PCF1 and PCF2 were also more likely to form heterodimers than homodimers. Our results suggest that PCF1 and PCF2 are involved in meristematic tissue-specific expression of the rice PCNA gene through binding to sites IIa and IIb and formation of homodimers or heterodimers.

Two of three promoter elements identified in a rice gene for proliferating cell nuclear antigen are essential for meristematic tissue‐specific expression

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta, and it is highly conserved among eukaryotes. The rice PCNA promoter, truncated to position -263, was previously shown to confer meristematic tissue-specific expression in transgenic plants. By DNasel footprinting and gel retardation analysis, three cis-acting elements were defined in this truncated promoter, which designated site I (-201 to -194, CCAGGTGG), site IIa (-197 to -188, TGGGCCCGT) and site IIb (-178 to -169, TGGTCCCAC). Site I resembles the G-box, and sites IIa and IIb resemble the conserved motif (T/GGTCCCAT) that is found in promoter regions of auxin-regulated genes. Functional analysis of expression of a PCNA-GUS gene fusion in transgenic tobacco plants revealed that a mutation in site I in the full-length 2.0 kb promoter had no significant effect on the activity. However, the mutation in site I in the truncated -263 promoter, which had 14% of the activity of the full-length promoter, caused a considerable decrease in the activity, suggesting that site I contributes in part to transcriptional activation. Simultaneous disruption of sites IIa and IIb in the full-length promoter caused about 80-85% loss of promoter activity, while separate disruption of site IIa or site IIb resulted in no marked change on the activity. These observations suggest that site IIa and site IIb play an important role in the meristematic tissue-specific expression of the rice PCNA gene, presumably by mediating putative enhancer activities dependent on the far-upstream region.

Rapamycin Selectively Blocks Interleukin-2-induced Proliferating Cell Nuclear Antigen Gene Expression in T Lymphocyte: EVIDENCE FOR INHIBITION OF CREB/ATF BINDING ACTIVITIES

The macrolide rapamycin arrests T lymphocytes stimulated by interleukin-2 (IL-2) at G1/S. We have recently found that IL-2 induced an increase in the binding of discrete transcription factors of the ATF/cAMP-responsive element binding factor (CREB) family at G1/S, and that this effect was inhibited by rapamycin (Feuerstein, N., Huang, D., Hinrichs, S. H., Orten, D. J., Aiyar, N., and Prystowsky, M. B. (1995) J. Immunol. 154, 68-79). We now show, by using high resolution two-dimensional gel electrophoresis, that rapamycin inhibited selectively the synthesis of three discrete IL-2-induced soluble proteins (35 kDa/pI approximately 5, 68 kDa/pI approximately 4, 110 kDa/pI approximately 4.3). Analysis of nuclear proteins demonstrated that rapamycin selectively blocked the expression of proliferating cell nuclear antigen (PCNA), an obligate cofactor of DNA polymerase-delta, an important component for DNA replication. Rapamycin inhibited the IL-2-induced PCNA mRNA, and the murine PCNA promoter activity in IL-2-stimulated cells. Inducible CRE-binding proteins were shown previously to be required for PCNA promoter activity in IL-2-stimulated T lymphocytes. Using DNA binding gel mobility shift assay we demonstrated that rapamycin potently inhibited the binding of CREB/ATF transcription factors to CRE elements in the murine proximal PCNA promoter. These results suggest that PCNA is a preferred target in a rapamycin-sensitive transduction pathway, and that the mechanism by which rampamycin inhibits PCNA gene expression may involve the inhibition of the interaction of CREB/ATF transcription factors with CRE elements in the proximal PCNA promoter.

The anti-proliferative effect of proliferating cell nuclear antigen-specific antisense oligonucleotides on human gastric cancer cell lines.

The proliferating cell nuclear antigen (PCNA) is a nuclear protein that leads DNA synthesis by the DNA polymerase delta. As the PCNA gene is strongly expressed in invasive gastric cancer cells with high proliferative activity, PCNA is suspected of playing an important role in the proliferation and advancement of gastric cancer. Thus, the effects of antisense oligonucleotides specific for PCNA mRNA were examined in seven gastric cancer cell lines. It was found that treatment with antisense oligonucleotides at concentrations of 10-40 microM dose-dependently inhibited the growth of all cell lines; however, random sequence oligonucleotides did not modify the proliferation of any type of cells. These results indicate that PCNA is essential for cell proliferation in gastric cancer cells, and that the growth inhibitory effect results from the inhibition of PCNA gene expression. Therefore, PCNA-specific antisense oligonucleotides may be effective in the treatment of gastric cancer.

Inhibition of c-Fos expression in the UV-irradiated epidermis by topical application of antisense oligodeoxynucleotides suppresses activation of proliferating cell nuclear antigen.

Induction of c-fos protooncogene expression following exposure of mammalian skin to UV irradiation suggests an involvement in UV-induced alterations of epidermal cell proliferation and viability. In the present study we have investigated whether topically administered c-fos antisense oligodeoxynucleotides (ODNs) inhibit c-fos activation in the UV-exposed rat skin and thereby modulate the delayed increase in cellular proliferative activity. The accumulation of c-Fos immunolabeled nuclei in the epidermis was almost completely blocked 18 h post-irradiation by topical treatment with the c-fos antisense ODN. The co-expression of c-Jun was not affected and a random sequence control ODN was ineffective. Epicutaneous application of fluorescein-labeled ODNs revealed penetration into the underlying epidermis. The appearance of nuclear immunoreactivity for proliferating cell nuclear antigen (PCNA) 18 h after UV exposure was significantly suppressed in the epidermis treated with c-fos antisense ODNs. In vitro PCNA is involved in both DNA repair synthesis and DNA replication, and the expression of PCNA mRNA is increased after UV irradiation. Thus, it may be speculated that UV-induced c-Fos transcription factor may be linked to repair of photodamaged DNA and/or cell cycle progression by trans-activating PCNA gene expression.

Expression patterns of DNA replication enzymes and the regulatory factor DREF during Drosophila development analyzed with specific antibodies

Specific antibodies were prepared against Drosophila DNA polymerase epsilon and DREF, a regulatory factor for DNA replication-related genes. Using these antibodies together with those for DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), we examined expression patterns and sub-cellular distributions of these proteins during Drosophila development. DNA polymerase alpha, epsilon and PCNA proteins were maternally stored in unfertilized eggs and maintained at high levels during embryogenesis. With distinct nuclear localization, proteins were observed in embryos at interphase stages throughout the 13 nuclear division cycles, suggesting that they all participate in rapid nuclear DNA replication during these cycles. In contrast, maternal storage of a DREF protein was relatively low and its level increased throughout embryogenesis. Strong nuclear staining with the anti-DREF antibody was not observed until the nuclear division cycle 8. Immunostaining of various larval tissues from transgenic flies carrying the PCNA gene promoter-lacZ fusion gene revealed co-expression of DREF, PCNA and lacZ, suggesting that DREF regulates the expression of PCNA gene in these tissues. In addition, we detected a relatively high level of DREF in adult males as well as females. Since DNA polymerase alpha, epsilon and PCNA are hardly detectable in adult males, DREF very likely regulates genes other than those closely linked to DNA replication in adult males.

DNA Methylation Is Not Involved in Growth Regulation of Gene Expression of Proliferating Cell Nuclear Antigen

Expression of proliferating cell nuclear antigen (PCNA) gene is growth regulated. By Southern blot analysis with restriction enzymes such as Hpa II and MspI, no change of DNA methylation state in this gene was detected during rat liver regeneration. There is only one/ HpaII site located in the promoter region of rat PCNA gene and this HpaII site was found to be demethylated in both normal and regenerating livers. Human hepatoma cell lines Hep G2 and Hep 3B were used to study the relation of PCNA gene expression and the DNA methylation. There are 16 HpaII sites in human PCNA gene, and according to the HpaII and MspI restriction patterns, many of the HpaII sites were methylated in vivo. Upon serum stimulation of quiescent cells, no change of DNA methylation state in the Hpa II and HhaI sites was found. Demethylation by the methylation inhibitor, 5-azacytidine, did not affect the expression of PCNA gene in the hepatoma cells. With the human primary fibroblasts, Y2, the demethylation by 5-azacytidine did not seem to change the growth dependence of PCNA gene expression. This is consistent with the observation that the expression of PCNA gene of some cultured cell lines such as CHO.K1, in which the PCNA gene was unmethylated, showed growth dependence. Also, no variation in methylation pattern of PCNA gene was found among the different rat tissues in which the expression of PCNA varies. Therefore, we conclude that DNA methylation is not involved in growth regulation of the PCNA gene expression.

DNA hypomethylation of proliferating cell nuclear antigen gene in human hepatocellular carcinoma is not due to cell proliferation.

Proliferating cell nuclear antigen (PCNA) gene expresses preferentially in proliferative cells or tissues. The levels of PCNA mRNA are very low in livers of adult mammals. Expression of PCNA gene in hepatocellular carcinoma tissues was, however, elevated; and 5'-CCGG-3' sequences of the gene in neoplastic tissue were less methylated. Such DNA hypomethylation was concluded, on the basis of two observations, not to be due to the cell proliferation in hepatoma tissues. First, while the expression of PCNA was increased during serum-stimulation of quiescent Hep G2 cells, the DNA methylation pattern of PCNA gene remained unchanged. Second, in rat liver regeneration, the PCNA mRNA level rose and declined, but the DNA methylation status of PCNA gene was unaltered. Therefore, the DNA hypomethylation of the PCNA gene found in hepatocellular carcinoma was not due to cell proliferation, but a possible consequence of cell transformation.

A conserved region in intron 1 negatively regulates the expression of the PCNA gene.

The Proliferating Cell Nuclear Antigen (PCNA) gene is a growth-regulated gene, whose expression is under the control of both transcriptional and posttranscriptional mechanisms. In previous work, it was shown that the 73 bp immediately upstream of the CAP site and intron 4 are major regulatory elements. We show here that intron 1 also plays a role in determining the levels of PCNA mRNA. Specifically, we show: 1) deletion of intron 1 increases the expression of PCNA mRNA in serum-deprived cells; 2) a 35 bp sequence in intron 1, containing a reverse CCAAT element specifically binds proteins from nuclear extracts; 3) this intron 1 sequence inhibits the expression of a co-tranfected human PCNA gene in transient expression assays suggesting that it competes for positive transcription factors; 4) mutations in the CCAAT region of the 35bp intron 1 probe abrogate both its protein-binding capacity and its ability to inhibit the expression of a co-transfected wt PCNA gene; and 5) the CCAAT region of human intron 1 is highly conserved in the mouse gene. We conclude that the reverse CCAAT region of intron 1 is a negative regulatory element of PCNA gene expression, and hypothesize that its inhibitory effect is abolished when certain protein(s) bind to it and that inhibition is restored if these proteins are competed out by an homologous sequence.

Proliferating cell nuclear antigen in developing and adult rat cardiac muscle cells.

During early development, rat cardiac muscle cells actively proliferate. Shortly after birth, division of cardiac muscle cells ceases, whereas DNA synthesis continues for approximately 2 weeks at a progressively diminishing rate. Little DNA synthesis or cell division occurs in adult cardiocytes. Thus, developing cardiac muscle cells are an ideal system in which to examine the expression of cell cycle-regulated genes during development. We chose to examine proliferating cell nuclear antigen (PCNA), a gene expressed at the G1/S phase boundary of the cell cycle. Northern blots of RNA from cardiac muscle cells from 18-day-old rat fetuses and from day 0, 5, and 14 neonatal as well as adult rat hearts revealed that the PCNA mRNA was found in cardiac muscle cells from all ages. However, because it was possible that this was a result of fibroblast PCNA gene expression, we used reverse transcription followed by polymerase chain reaction to see if it was possible to detect the message for PCNA in cardiac muscle cells from all ages. Because of the great sensitivity of this technique, RNA was recovered from 25 isolated adult cardiac muscle cells. Polymerase chain reaction amplification products for PCNA produced from the RNA isolated from these cells conclusively demonstrated that mRNA for this gene, which normally is associated with proliferating cells, is expressed in adult cardiac muscle cells that no longer divide. Furthermore, Western blot analysis demonstrated that the PCNA protein was found only in embryonic and neonatal cells and not in adult rat cardiac muscle cells. Therefore, it might be inferred from these data that PCNA might be regulated at the posttranscriptional level in adult cardiac muscle cells.

Repression of the Drosophila Proliferating-Cell Nuclear Antigen Gene Promoter by zerknüllt Protein

A 631-bp fragment containing the 5'-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5'-deletion derivatives revealed that the promoter function resided within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknüllt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even-skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region (-119 to -86) of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region (-607 to +137 or -168 to +137) fused with lacZ were established. When these flies were crossed with the zen mutant, ectopic expression of lacZ was observed in the dorsal region of gastrulating embryos carrying the transgene with either construct. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

Repression of the Drosophila proliferating-cell nuclear antigen gene promoter by zerknüllt protein.

A 631-bp fragment containing the 5'-flanking region of the Drosophila melanogaster proliferating-cell nuclear antigen (PCNA) gene was placed upstream of the chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in Drosophila Kc cells transfected with this plasmid and a set of 5'-deletion derivatives revealed that the promoter function resided within a 192-bp region (-168 to +24 with respect to the transcription initiation site). Cotransfection with a zerknüllt (zen)-expressing plasmid specifically repressed CAT expression. However, cotransfection with expression plasmids for a nonfunctional zen mutation, even-skipped, or bicoid showed no significant effect on CAT expression. RNase protection analysis revealed that the repression by zen was at the transcription step. The target sequence of zen was mapped within the 34-bp region (-119 to -86) of the PCNA gene promoter, even though it lacked zen protein-binding sites. Transgenic flies carrying the PCNA gene regulatory region (-607 to +137 or -168 to +137) fused with lacZ were established. When these flies were crossed with the zen mutant, ectopic expression of lacZ was observed in the dorsal region of gastrulating embryos carrying the transgene with either construct. These results indicate that zen indirectly represses PCNA gene expression, probably by regulating the expression of some transcription factor(s) that binds to the PCNA gene promoter.

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.