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PubMed2004

Reconstitution of active telomerase in primary human foreskin fibroblasts: effects on proliferative characteristics and response to ionizing radiation.

PURPOSE: Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear whether cells immortalized by telomerase reactivation behave in a similar manner as their parental primary cells. MATERIALS AND METHODS: Primary human foreskin fibroblasts were transfected with the human catalytic subunit of telomerase, the reverse transcriptase (hTERT), and their growth characteristics and response to DNA damage were characterized. RESULTS: The sole expression of the human hTERT was sufficient to immortalize the human foreskin fibroblasts. With time in culture, the immortalized cells almost doubled their average telomeric length and the clonal population contained almost no post-mitotic fibroblasts anymore. Up to 300 population doublings, no alterations compared with the parental primary cells were seen in terms of clonogenic radiosensitivity, DNA double-strand break repair, radiation-induced increases in p53 and p21(WAF-1,CIP-1) expression, and the G1/S and G2/M cell cycle checkpoints. Moreover, mitogen-induced mitotic arrest of fibroblasts was still possible in the hTERT-immortalized clones. CONCLUSIONS: Immortalizing fibroblasts by reconstitution of active telomerase seems a good, reliable manner to generate a large source of cells with a radiation damage response similar to the primary cells.

Adaptation, Physiological↗
PubMed2003

Borna disease virus nucleoprotein interacts with the CDC2-cyclin B1 complex.

Transition from G(2) to M phase, a cell cycle checkpoint, is regulated by the Cdc2-cyclin B1 complex. Here, we report that persistent infection with Borna disease virus (BDV), a noncytolytic RNA virus infecting the central nervous system, results in decelerated proliferation of infected host cells due to a delayed G(2)-to-M transition. Persistent BDV-infected rat fibroblast cells showed reduced proliferation compared to uninfected cells. In pull-down assays we observed an interaction of the viral nucleoprotein with the Cdc2-cyclin B1 complex. Transfection of the viral nucleoprotein but not of the phosphoprotein also results in decelerated proliferation. This phenomenon was found in BDV-susceptible primary rat fibroblast cells and also in primary mouse cells, which are not susceptible to BDV infection. This is the first evidence that the noncytolytic Borna disease virus can manipulate host cell functions via interaction of the viral nucleoprotein with mitotic entry regulators. BDV preferentially infects and persists in nondividing neurons. The present report could give an explanation for this selective choice of host cell by BDV.

Animals↗
PubMed2005

Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells.

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polzeta, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy.

Animals↗
PubMed2003

ATM's leucine-rich domain and adjacent sequences are essential for ATM to regulate the DNA damage response.

The ATM protein kinase regulates the DNA damage response by phosphorylating proteins involved in cell cycle checkpoints and DNA repair. We report here on the function of the predicted leucine zipper (LZ) motif, and sequences adjacent to this, in regulating ATM activity. The predicted LZ sequence was deleted from ATM, generating ATMDeltaLZ, and expressed in an ATM-negative AT cell line. ATM increased cell survival following exposure to ionizing radiation, whereas expression of ATMDeltaLZ failed to increase cell survival. ATMDeltaLZ retained in vitro kinase activity, but was unable to phosphorylate p53 in vivo. Leucine zippers mediate homo- and heterodimerization of proteins. However, the predicted LZ of ATM did not mediate the formation of ATM dimers. We examined if the predicted LZ of ATM was a dominant-negative inhibitor of ATM function in SW480 cells. Expression of amino acids 769-1436 of ATM, including the predicted LZ, sensitized SW480 cells to ionizing radiation, but did not inhibit ATM's kinase activity or its ability to phosphorylate Brca1. Further, this dominant-negative activity was not dependent on the predicted LZ domain. The central region of the ATM protein therefore contains multiple sequences which regulate cell survival following DNA damage.

Amino Acid Motifs↗
PubMed2006

Control of cell migration: a tumour suppressor function for p53?

Much remains to be learned about how cancer cells acquire the property of migration, a prerequisite for invasiveness and metastasis. Loss of p53 functions is assumed to be a crucial step in the development of many types of cancers, leading to dysregulation of cell cycle checkpoint controls and apoptosis. However, emerging evidence shows that the contribution of the tumour suppressor p53 to the control of tumorigenesis is not restricted to its well-known anti-proliferative activities, but is extended to other stages of cancer development, i.e. the modulation of cell migration. This interesting alternative function has been proposed in light of the effect of p53 on specific features of migrating cells, including cell spreading, establishment of cell polarization and the production of protrusions. The effects of p53 on cell motility are largely mediated through the regulation of Rho signalling, thereby controlling actin cytoskeletal organization. These recent studies connect the regulation of proliferation to the control of cell migration and define a new concept of p53 function as a tumour suppressor gene, suggesting that p53 might be involved in tumour invasion and metastasis. This review focuses on emerging data concerning the properties of p53 that contribute to its atypical role in the regulation of cell migration.

Animals↗
PubMed2001

umuDC-mediated cold sensitivity is a manifestation of functions of the UmuD(2)C complex involved in a DNA damage checkpoint control.

The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD'(2)C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show that umuDC-mediated cold sensitivity can be genetically separated from the role of UmuD'(2)C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that umuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD(2)C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD' in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the umuDC gene products.

Bacterial Proteins↗
PubMed2000

The role of p53 in the response to mitotic spindle damage.

The p53 tumour suppressor protein has defined roles in G1/S and G2/M cell cycle checkpoints in response to a range of cellular stresses including DNA damage, dominant oncogene expression, hypoxia, metabolic changes and viral infection. In addition to these responses, p53 can also be activated when damage occurs to the mitotic spindle. Initially, spindle damage activates a p53-independent checkpoint which functions at the metaphase-anaphase transition and prevents cells from progressing through mitosis until the completion of spindle formation. Cells eventually escape from this block (a process termed 'mitotic slippage'), and an aberrant mitosis ensues in which sister chromatids fail to segregate properly. After a delay period, p53 responds to this mitotic failure by instituting a G1-like growth arrest, with an intact nucleus containing 4N DNA, but without the cells undergoing division. Cells lacking wild-type p53 are still able to arrest transiently at mitosis, and also fail to undergo division, underscoring that the delay in mitosis is p53-independent. However, these cells are not prevented from re-entering the cell cycle and can reduplicate their DNA unchecked, leading to polyploidy. Additionally, p53-null cells which experience spindle failure often show the appearance of micronuclei arising from poorly segregated chromosomes which have decondensed and been enclosed in a nuclear envelope. The ability of p53 to prevent their formation suggests an additional G2 involvement which prevents nuclear breakdown prior to mitosis. The molecular mechanism by which p53 is able to sense mitotic failure is still unknown, but may be linked to the ability of p53 to regulate duplication of the centrosome, the organelle which nucleates spindle formation.

Animals↗
PubMed1998

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein.

We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G1 phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p16(INK4a) was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclin-dependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p16(INK4a) would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions.

Aerobiosis↗
PubMed2004

Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response.

Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres. We made all 63 mutant sequence combinations at template positions 474-476 of the yeast telomerase RNA, TLC1. Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants. Although growth rates and telomere profiles varied widely among the tlc1 mutants, chromosome separation and segregation were always aberrant. The mutants showed defects in sister chromatid separation at centromeres as well as telomeres, suggesting activation of a cell cycle checkpoint. Deletion of the DNA damage response genes DDC1, MEC3, or DDC2/SML1 failed to restore chromosome separation in the tlc1 template mutants. These results suggest that mutant telomere sequences elicit a checkpoint that is genetically distinct from those activated by deletion of telomerase or DNA damage.

Base Sequence↗
PubMed2000

Antisense oligonucleotides targeted to the p53 gene modulate liver regeneration in vivo.

The rapidly proliferating cells of the regenerating liver after partial hepatectomy (PH) present a reproducible in vivo model to study the functional role of the tumor suppressor gene p53. The present study uses the rat 70% PH model along with systemic administration of three different structural types of antisense oligonucleotides (ODNs) designed to suppress p53 expression. We tested the hypothesis that antisense ODNs can inhibit the expression of p53, resulting in the loss of the G(1)-S cell cycle checkpoint and an altered pattern of liver regeneration. Intraperitoneal administration of 5 mg/kg/day antisense phosphorothioate ODN after 70% PH resulted in reduced expression of the p53 protein in the regenerating liver. There were concomitant increases in weight gain of remnant-regenerating liver and expression of proliferating cell nuclear antigen and p21(waf-1) compared with either saline or 5 mg/kg/day mispaired phosphorothioate ODN treatment. Flow cytometric analysis of DNA content of isolated hepatocytes revealed a reduction in the G(0)/G(1) cell population and accumulation of cells with more than 4n DNA in antisense-treated rats. The regenerating livers had significantly diminished cytochrome P-450 (CYP) enzyme activities. Rats treated with p53 antisense ODNs, but not saline or mispair ODN controls, had significantly elevated CYP activities. These observations functionally link the expression of p53 with diminished expression of several CYP isoforms in the liver regeneration model.

Animals↗
PubMed2006

p53-Dependent but ATM-independent inhibition of DNA synthesis and G2 arrest in cadmium-treated human fibroblasts.

This study focused on the activation of cell cycle checkpoint responses in diploid human fibroblasts that were treated with cadmium chloride and the potential roles of ATM and p53 signaling pathways in cadmium-induced responses. The alkaline comet assay indicated that cadmium caused a dose-dependent increase in DNA damage. Cells that were rendered p53-defective by expression of a dominant-negative p53 allele or knockdown of p53 mRNA were more resistant to cadmium-induced inactivation of colony formation than normal and ataxia telangiectasia (AT) cells. Synchronized fibroblasts in S were more sensitive to cadmium toxicity than cells in G1, suggesting that cadmium may target some element of DNA replication. Cadmium produced a dose- and time-dependent inhibition of DNA synthesis. An immediate inhibition was associated with severe delay in progression through S phase and a delayed inhibition seen 24 h after treatment was associated with accumulation of cells in G2. AT and normal cells displayed similar patterns of inhibition of DNA synthesis and G2 delay after treatment with cadmium, while p53-defective cells displayed significantly less of the delayed inhibition of DNA synthesis and accumulation in G2 post-treatment. Total p53 protein and ser15-phosphorylated p53 were induced by cadmium in normal and AT cells. The p53 transactivation target Gadd45alpha was induced in both p53-effective and p53-defective cells after 4 h cadmium treatment, and this was associated with an acute inhibition of mitosis. Cadmium produced a very unusual pattern of toxicity in human fibroblasts, inhibiting DNA replication and inducing p53-dependent growth arrest but without induction of p21(Cip1/Waf1) or activation of Chk1.

Ataxia Telangiectasia↗
PubMed2003

Epigenetic inactivation of CHFR in human tumors.

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin-ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.

Cell Cycle Proteins↗
PubMed1993

Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily.

The RCC1 gene of mammals encodes a guanine nucleotide release protein (GNRP). RCC1 and a homolog in Saccharomyces cerevisiae (MTR1/PRP20/SRM1) have previously been implicated in control of mRNA metabolism and export from the nucleus. We here demonstrate that a temperature-sensitive fission yeast mutant which has a mutation in a homologous gene, and two of three additional (mtr1/prp20/srm1) mutants accumulate nuclear poly(A)+ RNA at 37 degrees C. In S.cerevisiae, maturation of rRNA and tRNA is also inhibited at 37 degrees C. Nevertheless, studies with the corresponding BHK-21 cell mutant indicate that protein import into the nucleus continues. MTR1 homologs regulate RNA processing at a point which is distinct from their regulation of chromosome condensation since: (i) poly(A)+ RNA accumulation in the fission yeast mutant precedes chromosome condensation, and (ii) unlike chromosome condensation, accumulation of nuclear poly(A)+ RNA does not require p34cdc28 kinase activation or protein synthesis. Moreover, experiments involving inhibition of DNA synthesis indicate that the S.cerevisiae homolog does not govern cell cycle checkpoint control. Since RCC1p acts as GNRP for Ran, a small nuclear GTPase of the ras superfamily, we have identified two homologs of Ran in S.cerevisiae (CNR1 and CNR2). Only CNR1 is essential, but both code for proteins extremely similar to Ran and can suppress mtr1 mutations in allele-specific fashion. Thus, MTR1 and its homologs appear to act as GNRPs for a family of conserved GTPases in controlling RNA metabolism and transport. Their role in governing checkpoint control appears to be restricted to higher eukaryotes.

Amino Acid Sequence↗
PubMed2006

TRB3 interacts with CtIP and is overexpressed in certain cancers.

TRB3, a human homolog of Drosophila Tribbles, has been recently shown as a critical negative regulator of Akt and S6 kinase activation in a number of cellular processes. Here we found that TRB3 interacted with an important cell cycle regulator CtIP (CtBP-interacting protein) and the interaction involved the C-terminus of both proteins. Interestingly, TRB3 and CtIP co-localized to the nucleus in HeLa cells and exhibited a unique dot-like pattern. Finally, we demonstrated that TRB3 was overexpressed in multiple tumor tissues. Since CtIP plays important roles in cell cycle checkpoint control and it has been implicated in tumorigenesis, our data suggest that TRB3 may be involved in these biological processes through interacting with CtIP.

B-Lymphocytes↗
PubMed1993

Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15.

Recent evidence has suggested that human cyclin-dependent kinase 2 (CDK2) is an essential regulator of cell cycle progression through S phase. CDK2 is known to complex with at least two distinct human cyclins, E and A. The kinase activity of these complexes peaks in G1 and S phase, respectively. The vertebrate CDC2/cyclin B1 complex is an essential regulator of the onset of mitosis and is inhibited by phosphorylation of CDC2 on Thr-14 and Tyr-15. In vitro, CDC2/cyclin B1 is activated by treatment with the members of the Cdc25 family of phosphatases. We found that, like CDC2, CDK2 is also phosphorylated on Thr-14 and Tyr-15 and that treatment of cyclin A or cyclin E immunoprecipitates with bacterially expressed Cdc25M2 (the mouse homolog of human CDC25B) increased the histone H1 kinase activity of these immune complexes 5- to 10-fold. Tryptic peptide mapping demonstrated that Cdc25M2 treatment of cyclin A or cyclin B1 immune complexes resulted in the specific dephosphorylation of Thr-14 and Tyr-15 on CDK2 or CDC2, respectively. Thus, we have confirmed that Cdc25 family members comprise a class of dual-specificity phosphatases. Furthermore, our data suggest that the phosphorylation and dephosphorylation of CDKs on Thr-14 and Tyr-15 may regulate not only the G2/M transition but also other transitions in the cell cycle and that individual cdc25 family members may regulate distinct cell cycle checkpoints.

Amino Acid Sequence↗
PubMed2002

Analysis of heat-shock related gene expression in head-and-neck cancer using cDNA arrays.

PURPOSE: The aim of the present study was to identify genes regulated in the early response to heat-shock in human head-and-neck cancer cells using a cDNA array. METHODS AND MATERIALS: IMC-3 cells were heat-shocked at 44 degrees C for 30 min, then incubated for 6 h. After 6 h incubation, mRNAs were extracted. Early gene expressions in IMC-3 cells were analyzed using a cDNA array after heat-shock. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify the expressions of mRNA for confirming features by cDNA array analysis in several other cell lines (KB, T3M-1, SCC-TF) as well as in IMC-3 cells. RESULTS: Twenty-eight genes were found to be induced or upregulated by heat-shock in IMC-3 cells. These included genes involved in the apoptosis (e.g., CC3, caspase10), tumor invasion (e.g., CC3, TIMP-3), cell cycle checkpoint control (e.g., DP-1, CDC25A), signal transduction (e.g., MEK1) as well as genes associated with heat stress (e.g., Hsp70B', Hsp40). Gene expressions of CC3 and MEK1 were recognized to be induced by heat-shock in pharyngeal cancer cells (KB, T3M-1) and lingual cancer cells (SCC-TF). CONCLUSION: The observed alteration in gene expression may play a role in various biochemical pathways of cancer cells exposed to heat-shock.

Acetyltransferases↗
PubMed2000

Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants.

The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

Animals↗
PubMed1999

Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes.

UNLABELLED: Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation. DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism. KEYWORDS: camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation

Alkaloids↗