Proliferating Cell Nuclear Antigen Transcription Research Articles

The Proliferating Cell Nuclear Antigen (PCNA) Transcript Variants as Potential Relapse Markers in B-Cell Acute Lymphoblastic Leukemia.

Leukemia is the most common childhood malignancy in Mexico, representing more than 50% of all childhood cancers. Although treatment leads to a survival of up to 90% in developing countries, in our country, it is less than 65%. Additionally, ~30% of patients relapse with poor prognosis. Alternative splicing plays an important role in transcriptome diversity and cellular biology. This mechanism promotes an increase in the assortment of proteins with potentially distinct functions from a single gene. The proliferating cell nuclear antigen (PCNA) gene encodes two transcripts for the same protein of 261 amino acids, which is associated with several important cellular processes and with several types of cancer. However, the diversity of the transcript variants expressed in this condition is not clear. Then, we used microarray gene expression to identify changes in the exon expression level of PCNA. The data were validated using RT-PCR and Sanger sequencing, and three additional transcripts (PCNA_V3, PCNA_V4, and PCNA_V5) were identified. Computational analyses were used to determine the potential proteins resulting, their structure, and interactions with PCNA native protein and themselves. Additionally, the PCNA transcript variants were inhibited using specific siRNA, determining that their inhibition contributes to the malignant characteristics in vitro. Finally, we quantified the PCNA transcript variants in acute lymphoblastic leukemia samples and identified their expression in this disease. Based on the clinical characteristics, we determined that PCNA_V2 and PCNA_V4 are expressed at significantly low levels in relapsed B-ALL patients. We conclude that the low expression of PCNA_V2 and PCNA_V4 could be a potential molecular marker of relapse in acute lymphoblastic leukemia patients.

Ring finger protein 6 enhances chemo-resistance by transcriptionally activating proliferating cell nuclear antigen expression and attenuating DNA damage in lung adenocarcinoma

The E3 ubiquitin ligase RING finger protein 6 (RNF6) is elevated in several cancers, including prostate and colorectal cancers. Here, we extended the finding of elevated RNF6 expression levels and its association with poor prognosis in patients with lung adenocarcinoma (LUAD). Genome-wide RNA sequencing in H3255 cells with RNF6 knockdown, followed by analysis of differentially expressed genes using Clusters of Orthologous Groups and gene set enrichment analysis revealed aberrations in genes related to DNA repair, especially double-strand break (DSB) repair. RNF6 knockdown increased γH2AX foci, a biomarker for DSBs in H3255 and A549 LUAD cells, and enhanced DNA damage induced by chemotherapy in cisplatin-resistant A549/CDDP cells. In a series of experiments in cultured cells, as well as in nude mice carrying xenografts, RNF6 knockdown restored the sensitivity of A549/CDDP cells to cisplatin treatment. Mechanistically, RNA sequencing in RNF6-knockdown cells revealed the significant downregulation of proliferating cell nuclear antigen (PCNA), an oncogene that promotes DNA repair. Re-chromatin immunoprecipitation assay results suggested the formation of a RNF6-TCF4 complex that binds to the PCNA promoter to activate its transcription. Downregulation of RNF6 reduced TCF4 recruitment to PCNA promoters in H3255 and A549 cells, indicating that RNF6 regulates PCNA transcription to a certain extent by regulating TCF4 binding to PCNA promoters. The collective results implicate RNF6 overexpression as a molecular target in the management of cisplatin-resistant LUAD.

Periodic Oxaliplatin Administration in Synergy with PER2-Mediated PCNA Transcription Repression Promotes Chronochemotherapeutic Efficacy of OSCC.

Developing chemotherapeutic resistance affects clinical outcomes of oxaliplatin treatment on various types of cancer. Thus, it is imperative to explore alternative therapeutic strategies to improve the efficacy of oxaliplatin. Here, it is shown that circadian regulator period 2 (PER2) can potentiate the cytotoxicity of oxaliplatin and boost cell apoptosis by inhibiting DNA adducts repair in human oral squamous cell carcinoma (OSCC) cells. The circadian timing system is closely involved in controling the activity of DNA adducts repair and gives it a 24 h rhythm. The mechanistic dissection clarifies that PER2 can periodically suppress proliferating cell nuclear antigen (PCNA) transcription by pulling down circadian locomotor output cycles kaput–brain and muscle arnt‐like 1 heterodimer from PCNA promoter in a CRY1/2‐dependent manner, which subsequently impedes oxaliplatin‐induced DNA adducts repair. Similarly, PER2 is capable of improving the efficacy of classical DNA‐damaging chemotherapeutic agents. The tumor‐bearing mouse model displays PER2 can be deployed as an oxaliplatin administration timing biomarker. In summary, it is believed that the chronochemotherapeutic strategy matching PER2 expression rhythm can efficiently improve the oxaliplatin efficacy of OSCC.

D-Aspartate amends reproductive performance of aged roosters by changing gene expression and testicular histology.

Male broiler breeders (n=32) of 55 weeks of age were administered four different doses of capsulated d-aspartate (DA; 0, 100, 200 or 300mgkg-1day-1, p.o. (DA0, DA100, DA200 and DA300 respectively)) for 12 successive weeks to assess reproductive performance, blood testosterone, testicular histology and transcript levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), androgen receptor (AR), LH receptor (LHR), 3β-hydroxysteroid dehydrogenase (3BHSD), proliferating cell nuclear antigen (PCNA), glutamate ionotropic receptor NMDA type subunit 1 (GRIN1) and glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B). Blood samples and ejaculates were collected, and bodyweight was recorded weekly for 10 weeks. AI was performed weekly for the last 2 weeks to determine the number of sperm penetration holes in the perivitelline layer, fertility and hatchability. Testes histology and transcript levels were evaluated in the 12th week. Bodyweight, numbers of Leydig cells and blood vessels, testis index and levels of sperm abnormalities were not affected (P>0.05) by the treatment. However, sperm total and forward motility, plasma membrane integrity and functionality of sperm, ejaculate volume, testosterone concentration and fertility were higher (P<0.05) in both the DA200 and DA300 groups compared with the other groups. In the DA100 and DA200 groups, sperm concentration, number of spermatogonia, thickness of the seminiferous epithelium and the diameter of tubules were significantly higher (P<0.05) than the other DA-treated groups. The number of penetration holes, hatchability and malondialdehyde concentration were higher in the DA200, all DA-treated and DA300 groups respectively compared with the control and other treatment groups. Except for P450scc, AR, LHR and PCNA transcript levels in the DA300 groups, the relative expression of the genes evaluated improved significantly in the other DA-treated groups. Based on these experimental findings, it is concluded that DA improves reproductive performance of aged roosters.

Pretreatment of BMSCs with TZD solution decreases the proliferation rate of MCF‑7 cells by reducing FGF4 protein expression.

The present study aimed to investigate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) that had been pretreated with pioglitazone and/or rosiglitazone on the growth and proliferation rate of MCF-7 cells. The adhesive interaction between the BMSCs and the MCF-7 cancer cells revealed that the pretreatment of BMSCs with a combination of two types of thiazolidinedione drug reduced the growth and proliferation rate of the MCF-7 cells. The proliferation rate of the MCF-7 cells could also be reduced by the non-adhesive interaction of the cancer cells with BMSCs pretreated with pioglitazone and/or rosiglitazone. The growth and proliferation rate reduction effects on the MCF-7 cells may be attributed to the reduction in the protein level of fibroblast growth factor 4 (FGF4) in the conditioned medium of the pretreated BMSCs. The evidence that the low protein level of FGF4 in the conditioned medium of the pretreated BMSCs perturbed the proliferation rate of the MCF-7 cells by reducing the levels of Ki-67 and proliferating cell nuclear antigen transcripts in the cancer cells was also demonstrated in the present study using a FGF4-neutralizing antibody. All the above findings demonstrate that future studies on the correlation between FGF4 and pretreated BMSCs would be beneficial.

Cyclosporine A up-regulates Sonic hedgehog in gingiva: role of the up-regulation on gingival cell proliferation.

Sonic hedgehog protein (SHH) is a mitogen that stimulates cell proliferation. Cyclosporine A enhances the proliferation of gingival cells; however, the relationships of SHH to cyclosporine A or to cyclosporine A-enhanced gingival cell proliferation have not been described. Here, we investigated SHH expression in gingiva in vitro and in vivo after cyclosporine A treatment and tested the effect of SHH inhibition on cyclosporine A-enhanced gingival fibroblast proliferation in vitro. In human gingival fibroblasts, cyclosporine A treatment increased the expression of SHH transcripts and SHH protein, and stimulated cell proliferation; the addition of cyclopamine, an SHH signaling inhibitor, suppressed cyclosporine A-enhanced cell proliferation. Up-regulated expression of SHH and up-regulation of proliferating cell nuclear antigen transcripts and protein were observed in the edentulous gingiva of cyclosporine A-treated rats. Cyclosporine A up-regulates gingival SHH expression in vitro and in vivo, and the inhibition of the SHH pathway counteracts the stimulatory effect of cyclosporine A on gingival fibroblast proliferation. Therefore, we suggest that SHH mediates a novel molecular mechanism for cyclosporine A-induced gingival complications.

Construction and Functional Analysis of Luciferase Reporter Plasmid Containing PCNA Gene Promoter

PCNA (proliferating cell nuclear antigen) is a protein related to tumor development, which has been used extensively in breast cancer diagnosis and prognosis. PCNA has proven to be a useful marker to evaluate cell proliferation and prognosis when combined with other breast cancer markers. Construction of PCNA promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating PCNA transcription. In this study, a human PCNA promoter luciferase reporter construct was generated by PCR amplification of PCNA promoter. The PCR fragment was digested and cloned into pGL3 vector. The promoter sequence was verified by sequencing. The results showed that luciferase reporter plasmids of PCNA promoter were successfully constructed. Then the effects of some key transcription factors, which play important roles in breast cancer cell proliferation, were investigated by luciferase reporter assays in MCF-7 cells. The results showed that ERα can enhance transcriptional activity of PCNA. Furthermore, 17-β-estradiol (E2) also shows an obvious impact in activating PCNA transcription. Our data illuminated that E2 enhances ERα-induced proliferation potential of MCF-7 cells by stimulating the transcriptional activity of PCNA. Our research will provide a model to screen some novel factors in regulating proliferation marker transcription.

Effect of insulin on the cell cycle of germinating maize seeds (Zea maysL.)

AbstractDuring seed germination, metabolism is reactivated, DNA is repaired and cell division is restarted in the meristems. The mechanisms that co-ordinate cell growth and division in maize embryonic axes during germination are not well understood. However, the presence of a factor similar to IGF (insulin-like growth factor) that accelerates germination has been reported. In the present work, the regulation of the cell-cycle restart by bovine insulin [which has been demonstrated to produce similar effects as insulin-like growth factor of maize (ZmIGF) in maize seeds] was studied in germinating embryonic axes. Our results showed that bovine insulin differentially stimulates growth, S6K phosphorylation,S6rptranscript accumulation on the polysomal fraction, as well asde novoDNA synthesis in the radicles and the coleoptiles of the embryonic axis. A stronger and earlier effect was observed in radicles compared to coleoptiles; therefore, the effect of insulin on the cell cycle of the root meristem was studied by flow cytometry. The G1–S transition was stimulated and cell proliferation was induced. Furthermore, it was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) that bovine insulin increased E2F and PCNA (proliferating cell nuclear antigen) transcription after 15 h of germination and PCNAde novosynthesis at 15 h of germination. These results show that bovine insulin preferentially stimulates growth in the radicles of germinating embryonic axes and suggest that its effect on the G1–S transition and the activation of cell proliferation is mediated by the induction of E2F and PCNA transcription.

A proposed conserved role for an avocado fw2.2-like gene as a negative regulator of fruit cell division

Previous studies using 'Hass' avocado and its small fruit (SF) phenotype as a model showed that SF is limited by cell number, not by cell size. In an attempt to explore the molecular mechanisms regulating avocado fruit cell division, we isolated four distinct avocado cell proliferation-related genes and investigated their expression characteristics, comparing normal fruit (NF) and SF developmental patterns. Three cDNAs termed PaCYCA1, PaCYCB1 and PaPCNA, encoding two mitotic cyclins and a proliferating cell nuclear antigen (PCNA), were first isolated from young NF tissues. The accumulation of their transcripts was predominant in mitotically active organs, including young fruitlets, leaves and roots. Furthermore, a fourth full-length cDNA, designated Pafw2.2-like, encoding a FW2.2 (fruit-weight)-like protein, was isolated from SF tissues. FW2.2 is postulated to function as a negative regulator of cell division in tomato fruit. Remarkably, northern analysis revealed that the accumulation of the mitotic cyclins and of PCNA transcripts gradually decreased in NF tissues during growth, whereas in SF, their levels had already decreased at earlier stages of fruit development, concomitant with an earlier arrest of fruit cell division activity. In contrast, parallel sq-RT-PCR analysis showed that Pafw2.2-like mRNA accumulation was considerably higher in SF tissues than in the same NF tissues essentially at all examined stages of fruit growth. Together, our data suggest essential roles for the two mitotic cyclins genes and the PCNA gene in regulating avocado fruit development. Furthermore, the possibility that Pafw2.2-like acts as does fw2.2 in tomato, is discussed.

Cloning of proliferating cell nuclear antigen gene from the dinoflagellate Prorocentrum donghaiense and monitoring its expression profiles by real-time RT-PCR

The growth rate of marine phytoplankton at the species level is an important parameter for estimating a phytoplankton population in the field. Therefore, monitoring this rate may have potential to be useful for providing early warnings for red tide blooms. We investigated the proliferating cell nuclear antigen (PCNA) gene of the red tide species Prorocentrumdonghaiense Lu et Goebel to study the relationship between its expression level and the growth rate. The gene was isolated and the full cDNA sequence encoding PCNA was obtained. It consisted of 1,057 base pairs (bp), comprising 81 bp of the 5′ untranslated region (UTR) and 196 bp of the 3′ UTR, and had an open reading frame (ORF) of 780 bp, encoding 259 amino acid residues. The coding region was highly conserved compared with other organisms PCNA gene. Using real-time PCR, we found that the average of PCNA transcript, normalized to per cell basis, decreased obviously from 26.12 ± 3.04 copies in the exponential and transition phase to 5.95 ± 0.79 copies in the stationary phase. However, in the decline phase, the copies of PCNA transcription per cell is below one copy. Furthermore, the expression level of PCNA changed consistently with the growth rate as assessed by the relative quantitation method. These results indicate that PCNA has a potential as a molecular marker of growth phase and rate for P. donghaiense. This may help in forecasting blooms, allowing for effective control measures to be implemented in a timely fashion.

Co‐immunoprecipitation of putative proteins that interact with mosquito proliferating cell nuclear antigen

We have sequenced cDNAs encoding proliferating cell nuclear antigen (PCNA) from Aedes albopictus cells and from Aedes aegypti mosquitoes. The mosquito cDNAs contained an open reading frame encoding a 260 amino acid protein with a calculated mass of 29.0 kDa and a pI of 4.46. There was a single amino acid difference between PCNA proteins from Ae. albopictus and Ae. aegypti. In An. gambiae, the PCNA homolog contained 260 residues, and the pcna gene was interrupted by a single 67 nucleotide intron in the betaC2 region of the protein. A phylogenetic comparison grouped known Dipteran PCNA sequences into two clusters, representing the Nematocera and the Cyclorrhapha. PCNA transcripts measured 1.1 kb, and were stable, as was PCNA protein. Mosquito PCNA was efficiently recognized by a commercially available mouse anti-PCNA monoclonal antibody, which coprecipitated 29 kDa and 35 kDa proteins from mosquito cells representing different growth states. These results support the feasibility of recovering mosquito cell cycle inhibitory proteins by virtue of their interaction with PCNA.

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.

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.

Proliferating Cell Nuclear Antigen Transcription Is Repressed through an E2F Consensus Element and Activated by Geminivirus Infection in Mature Leaves

Proliferating Cell Nuclear Antigen Transcription Is Repressed through an E2F Consensus Element and Activated by Geminivirus Infection in Mature Leaves

Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves.

The geminivirus tomato golden mosaic virus (TGMV) amplifies its DNA genome in differentiated plant cells that lack detectable levels of DNA replication enzymes. Earlier studies showed that TGMV induces the accumulation of proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerase delta, in mature cells of Nicotiana benthamiana. We sought to determine if PCNA protein accumulation reflects transcriptional activation of the host gene. RNA gel blot analysis detected an approximately 1200-nucleotide PCNA transcript in young leaves. The same RNA was found in mature leaves of infected but not healthy plants. Reporter gene analysis showed that a 633-bp promoter fragment of the N. benthamiana PCNA gene supports high levels of expression in cultured cells and in young but not mature leaves of healthy transgenic plants. In contrast, PCNA promoter activity was detected in both young and mature leaves of TGMV-infected plants. Developmental studies established a strong relationship between symptom severity, viral DNA accumulation, PCNA promoter activity, and endogenous PCNA mRNA levels. Mutation of an E2F consensus element in the PCNA promoter had no effect on its activity in young leaves but increased transcription in healthy mature leaves. Unlike the wild-type PCNA promoter, TGMV infection had no detectable effect on the activity of the mutant E2F promoter. Together, these results demonstrate that geminivirus infection induces the accumulation of a host replication factor by activating transcription of its gene in mature tissues, most likely by overcoming E2F-mediated repression.

Proliferating Cell Nuclear Antigen Transcription Is Repressed through an E2F Consensus Element and Activated by Geminivirus Infection in Mature Leaves

Proliferating Cell Nuclear Antigen Transcription Is Repressed through an E2F Consensus Element and Activated by Geminivirus Infection in Mature Leaves

Isolation and Analysis of a T Cell Clone Variant Exhibiting Constitutively Phosphorylated Ser133 cAMP Response Element-Binding Protein

In driving T cell proliferation, IL-2 stimulates a new program of gene expression that includes proliferating cell nuclear antigen (PCNA), a requisite processivity factor for DNA polymerase delta. PCNA transcription is regulated in part through tandem CRE sequences in the promoter and CRE binding proteins; IL-2 stimulates CREB phosphorylation in the resting cloned T lymphocyte, L2. After culturing L2 cells for greater than 91 days, we consistently isolate a stable variant that exhibits constitutive CREB phosphorylation. L2 and L2 variant cells were tested for IL-2 responsiveness and rapamycin sensitivity with respect to specific kinase activity, PCNA expression and proliferation. In L2 cells, IL-2 stimulated and rapamycin inhibited the following: cAMP-independent CREB kinase activity, PCNA expression and proliferation. In L2 variant cells, CREB kinase activity was constitutively high; IL-2 stimulated and rapamycin blocked PCNA expression and proliferation. These results indicate that IL-2 induces a rapamycin-sensitive, cAMP-independent CREB kinase activity in L2 cells. However, phosphorylation of CREB alone is not sufficient to drive PCNA expression and L2 cell proliferation in the absence of IL-2.

Differential Regulation of p53-dependent and -independent Proliferating Cell Nuclear Antigen Gene Transcription by 12 S E1A Oncoprotein Requires CBP

The tumor suppressor protein p53 and the adenoviral 12 S E1A oncoprotein are both known to elicit their biological effects mainly by regulating the transcription of important cellular genes. The human proliferating cell nuclear antigen (PCNA) gene is a transcriptional target of both p53 and E1A. We have analyzed the effects of p53 and 12 S E1A, separately as well as together, on PCNA gene transcription. Our results showed that whereas both p53 and 12 S E1A separately activated PCNA transcription, 12 S E1A repressed p53-mediated transcriptional activation. Thus, 12 S E1A uses a dual strategy of transcriptional activation and repression to take control of the cellular PCNA gene regulation. The cyclic AMP-response element in the PCNA core promoter, besides being crucial for basal transcription, synergizes with p53 to activate transcription. The cyclic AMP response element-binding protein (CREB)-binding protein (CBP) is an essential component of both the transcriptional activation and repression by E1A. Our data demonstrate for the first time that E1A can modulate CBP function to activate PCNA transcription, while at the same time repressing p53-mediated activation by disrupting CBP interaction with p53, thereby uncoupling PCNA transcription from the regulatory effects of p53.

G0/G1 cell cycle arrest in the brain of Sarcophaga crassipalpis during pupal diapause and the expression pattern of the cell cycle regulator, proliferating cell nuclear antigen

During pupal diapause in the flesh fly, Sarcophaga crassipalpis, the cells of the brain are arrested in the G0/G1 phase of the cell cycle. When diapause is terminated with a topical application of hexane, cell cycling is evident within 12 hours. Four G1 and S phase regulatory genes were examined by Northern blot analysis to evaluate their expression patterns in relation to this cell cycle arrest. A distinction between diapausing and nondiapausing individuals was noted only for Proliferating Cell Nuclear Antigen ( PCNA). PCNA was highly expressed after diapause was terminated but not during diapause. In contrast, cyclin E, p21, and p53 were expressed equally at all times. In situ hybridization using PCNA probes further indicated a correlation between PCNA transcription (expression) in the brain and cell cycling. Our evidence thus suggests a potential role for PCNA as an important regulator of cell cycle arrest during diapause.

Addition of calmodulin antagonists to NRK cells during G1 inhibits proliferating cell nuclear antigen expression

The mRNAs of most proteins involved in DNA synthesis show an S phase correlated expression when mammalian cells are stimulated to proliferate from G0. This is the case for proliferating cell nuclear antigen (PCNA), a cofactor of DNA polymerase δ that is essential for the synthesis of the leading and lagging strands of DNA. Normal rat kidney cells re-entering the cell cycle from quiescence start DNA synthesis at 12 h and reach a maximum at 20 h. The expression of PCNA parallels the synthesis of DNA. Progression through the S phase was inhibited by addition of the anticalmodulin drug W13 to the cells during G1, 5 h after activation. W13 also inhibited the increase in both PCNA protein and mRNA indicating that calmodulin regulates its expression. Using TK −ts13 cells transfected with a plasmid containing the thymidine kinase gene under the control of the human 2.8 kb PCNA promoter, we demonstrated that this promoter is not regulated by calmodulin. The half-life of PCNA mRNA during G1/S transition was not modified by the treatment with W13, indicating that the decrease in the mRNA found when calmodulin was inhibited is not due to changes in its stability. Run-on assays revealed that control cells produced predominantly complete PCNA transcripts during S phase, while short incomplete transcripts were generated in W13-treated cells at the same time. These results indicate that calmodulin participates in a more direct or indirect way during G1 in the activation of PCNA expression. From data presented here it can be suggested that calmodulin activates the release of a transcriptional block leading to an increase in the amount of PCNA during S phase.