Chelation therapy with 3,4,3-Li(1,2-HOPO) after pulmonary exposure to plutonium in rats.

Internal exposure to plutonium can occur through inhalation for the nuclear worker, but also for the public if the radionuclide was released into the atmosphere in the context of a nuclear accident or terrorist attack. DieThylenetriaminePentaAcetic acid (DTPA) is currently still the only authorized chelator that can be used to decorporate internalized plutonium. The Linear HydrOxyPyridinOne-based ligand named 3,4,3-Li(1,2-HOPO) remains the most promising drug candidate to replace it in the hopes of improving chelating treatment. This study aimed to assess the efficacy of 3,4,3-Li(1,2-HOPO) in removing plutonium from rats exposed to the lungs, depending on the timing and route of treatment, and almost always compared to DTPA at a ten-fold higher dose used as a reference chelator. First, early intravenous injection or inhalation of 3,4,3-Li(1,2-HOPO) demonstrated superior efficacy over DTPA in preventing plutonium accumulation in liver and bone in rats exposed by injection or lung intubation. However, this superiority of 3,4,3-Li(1,2-HOPO) was much less pronounced with delayed treatment. In rats given plutonium in the lungs, the experiments also showed that 3,4,3-Li-HOPO reduced pulmonary retention of plutonium more effectively than DTPA only when the chelators were injected early but not at delayed times, while it was always the better of the two chelators when they were inhaled. Under our experimental conditions, the rapid oral administration of 3,4,3-Li(1,2-HOPO) was successful in preventing systemic accumulation of plutonium, but not in decreasing lung retention. Thus, after exposure to plutonium by inhalation, the best emergency treatment would be the rapid inhalation of a 3,4,3-Li(1,2-HOPO) aerosol to limit pulmonary retention of plutonium and prevent extrapulmonary deposition of plutonium in target systemic tissues.

Excretion of Pu-238 during Long-term Chelation Therapy by Repeated DTPA Inhalation.

ABSTRACT: An individual underwent an extensive diethylenetriaminepentaacetate (DTPA) chelation therapy that started several months after plutonium incorporation, most likely by inhalation of a soluble compound. After receiving multiple intravenous infusions of DTPA, the patient continued the treatment by pulmonary delivery of aerosolized DTPA. The purpose of the present work is to provide and discuss the bioassay data obtained during the DTPA aerosol therapy and compare them with those under the DTPA infusion therapy that have been largely interpreted elsewhere. As with DTPA given intravenously, each delayed DTPA inhalation increased the clearance of plutonium not only in urine but also in feces, thus demonstrating the ability to remove plutonium retained by extrapulmonary tissues. Also, the slow decline of increased plutonium urinary elimination together with enhanced fecal excretion are two features coherent with the contribution of intracellular chelation to overall decorporation. The therapeutic benefit of DTPA inhalation appeared lower than with DTPA infusion, most likely due to a lower amount of DTPA reaching the systemic compartments where plutonium chelation predominates. The results suggest that DTPA administration through aerosol could be an alternative to the invasive procedure using a needle, i.e., intravenous injection/infusion, when protracted decorporation therapy is needed following transuranic internalization. Indeed, the patient may be more inclined to undergo a chelation treatment for a longer period because taking DTPA by inhalation may make it less cumbersome and painful.

Interpretation of Enhanced Fecal and Urinary Plutonium Excretion Data under a 2-y Regular DTPA Treatment Started Months after Intake.

ABSTRACT: In a worker who had internalized plutonium, most likely through inhalation of a somewhat soluble compound, an extensive diethylenetriaminepentaacetate (DTPA) treatment regimen was initiated several months after contamination. Numerous radiotoxicological analyses were performed in both fecal and urinary specimens collected, sometimes for three consecutive days after DTPA administration. Activity measurements showed the continued effectiveness of DTPA intravenous infusions in removing plutonium from tissues of retention even if the treatment regimen started very belatedly after contamination. In the present case, the activity excreted through urine within the first 24-h after a DTPA infusion contributed only about half of that activity excreted within the first three days (i.e., the cumulative activity of the first three 24-h urine collections). In addition, the careful study of the data revealed that DTPA-induced excretion of plutonium via fecal pathway significantly contributed to the overall decorporation. The intracellular chelation of plutonium may be responsible for this enhanced excretion of activity in feces as well as for the delayed and sustained increased clearance of activity in urine. The authors would suggest that the occupational physicians offer to individuals who internalized moderately soluble or soluble plutonium compounds undergo a long-term DTPA treatment, especially when it is not initiated promptly after intake. Under this scenario, measurements of plutonium in successive urine and fecal collections after treatment should be required to get a better estimate of the therapeutic benefit. Also, intracellular chelation and fecal route should be taken into account for better interpretation of radiotoxicological data and modeling of plutonium kinetics under delayed DTPA treatment.

DTPA-Coated Liposomes as a New Delivery Vehicle for Plutonium Decorporation.

Administration of diethylenetriaminepentaacetic acid (DTPA) is the treatment approach used to promote the decorporation of internalized plutonium. Here we evaluated the efficacy of PEGylated liposomes coated with DTPA, primarily designed to prevent enhanced plutonium accumulation in bones, compared to marketed nonliposomal DTPA and liposomes encapsulating DTPA. The comparative effects were examined in terms of reduction of activity in tissues of plutonium-injected rats. The prompt treatment with DTPA-coated liposomes elicited an even greater efficacy than that with liposome-encapsulated DTPA in limiting skeletal plutonium. This advantage, undoubtedly due to the anchorage of DTPA to the outer layer of liposomes, is discussed, as well as the reason for the loss of this superiority at delayed times after contamination. Plutonium complexed with DTPA-coated liposomes in extracellular compartments was partly diverted into the liver and the spleen. These complexes and those directly formed inside hepatic and splenic cells appeared to be degraded, then released from cells at extremely slow rates. This transitory accumulation of activity, which could not be counteracted by combining both liposomal forms, entailed an underestimation of the efficacy of DTPA-coated liposomes on soft tissue plutonium until total elimination probably more than one month after treatment. DTPA-coated liposomes may provide the best delivery vehicle of DTPA for preventing plutonium deposition in tissues, especially in bone where nuclides become nearly impossible to remove once fixed. Additional development efforts are needed to limit the diversion or to accelerate cell release of plutonium bound to DTPA-coated liposomes, using a labile bond for DTPA attachment.

Fetuin exhibits a strong affinity for plutonium and may facilitate its accumulation in the skeleton.

After entering the blood, plutonium accumulates mainly in the liver and the bones. The mechanisms leading to its accumulation in bone are, however, completely unknown. We already know that another uptake pathway not involving the transferrin-mediated pathways is suspected to intervene in the case of the liver. Fetuin, a protein playing an important role in bone metabolism, is proposed as a potential transporter of Pu from serum to bone. For the first time, the binding constants of these two proteins (transferrin and fetuin) with tetravalent plutonium at physiological pH (pH 7.0) were determined by using capillary electrophoresis (CE) coupled with inductively coupled plasma mass spectrometry (ICP-MS). Their very close values (log10 KPuTf = 26.44 ± 0.28 and log10 KPuFet = 26.20 ± 0.24, respectively) suggest that transferrin and fetuin could compete to chelate plutonium, either in the blood or directly at bone surfaces in the case of Pu deposits. We performed competition reaction studies demonstrating that the relative distribution of Pu-protein complexes is fully explained by thermodynamics. Furthermore, considering the average concentrations of transferrin and fetuin in the blood, our calculation is consistent with the bio-distribution of Pu observed in humans.

Chelation Treatment by Early Inhalation of Liquid Aerosol DTPA for Removing Plutonium after Rat Lung Contamination.

Occupational contamination is a potential health risk associated with plutonium inhalation. DTPA remains the chelating drug of choice to decorporate plutonium. In this study, plutonium was found to be more effectively removed from lungs by a single inhalation of nebulized DTPA solution at only 1.1 µmol.kg-1 than by a single intravenous (i.v.) dose of DTPA at 15 µmol.kg-1. When DTPA was inhaled promptly after contamination, it removed the transportable fraction of plutonium prior blood absorption, thereby preventing both liver and bone depositions. Conversely, DTPA injection was better than inhalation at reducing the extrapulmonary burden, probably due to the much greater circulating dose, favoring the mobilization of plutonium already translocated. Thus, prompt inhalation, concomitantly supplemented with i.v. injection, of DTPA induced an important decrease in extrapulmonary deposits. Repeated DTPA inhalations over several weeks were more efficient than a single inhalation in limiting both pulmonary and extrapulmonary plutonium retention, due at least in part to the chelation of the transportable fraction of lung plutonium. Furthermore, repeated DTPA injections remained better at reducing liver and bone plutonium retentions. Taken together, our results suggest that multiple DTPA inhalations may be considered an effective treatment after inhalation of plutonium, particularly given the ease of this needle-free delivery, for the two following conditions: 1. A treatment combining i.v. injection and inhalation should be given in an emergency scenario to efficiently chelate the activity already absorbed; 2. Inhalations should be administered daily to effectively trap the early transferable fraction.

Americium biodistribution in rats after wound contamination with different physicochemical forms in the presence or absence of plutonium: analyses using STATBIODIS.

Americium (Am) biodistribution data obtained after wound contamination in rats were analysed to evaluate and quantify the influence of different physicochemical forms of Am in the presence or absence of plutonium (Pu). The biodistribution data were individual Am daily urinary excretion and tissue retention. The data were analysed with STATBIODIS, a statistical tool developed in the laboratory and based on the R language. Non-parametric methods were selected to comply with the data characteristics. Am systemic tissue retention and urinary excretion data were much greater for contamination with soluble physicochemical forms than insoluble forms. Meanwhile, Am relative biodistribution between the main retention tissues (skeleton, liver and kidney) remained the same. Hence, after absorption into blood the radionuclide behaviour was independent of the physicochemical form. The presence of Pu did not change the Am biodistribution. Comparisons of the biodistribution data from the laboratory with mean values published by other laboratories showed that soluble to moderately soluble forms of Am resulted in similar urine excretion after contamination, whether it was intravenous, intramuscular, subcutaneous injection or incision. Findings from this work will contribute to improve the understanding and interpretation of wound contamination cases with different physicochemical forms and mixtures of actinides including Am.

Delivery of DTPA through Liposomes as a Good Strategy for Enhancing Plutonium Decorporation Regardless of Treatment Regimen.

In this study, we assessed the efficacy of unilamellar 110-nm liposomes encapsulating the chelating agent diethylenetriaminepentaacetic acid (DTPA) in plutonium-exposed rats. Rats were contaminated by intravenous administration of the soluble citrate form of plutonium. The comparative effects of liposomal and free DTPA at similar doses were examined in terms of limitation of alpha activity burden in rats receiving various treatment regimens. Liposomal DTPA given at 1 h after contamination more significantly prevented the accumulation of plutonium in tissues than did free DTPA. Also, when compared to free DTPA, liposome-entrapped DTPA was more efficient when given at late times for mobilization of deposited plutonium. In addition, repeated injections of liposomal DTPA further improved the removal of plutonium compared to single injection. Various possible mechanisms of action for DTPA delivered through liposomes are discussed. The advantage of liposomal DTPA over free DTPA was undoubtedly directly and indirectly due to the better cell penetration of DTPA when loaded within liposomes, mainly in the tissues of the mononuclear phagocytic system. The decorporation induced by liposomal DTPA may result first from intracellular chelation of plutonium deposited in soft tissues, predominantly in the liver. Afterwards, the slow release of free DTPA molecules from these same tissues may enable a sustained action of DTPA, probably mainly by extracellular chelation of plutonium available on bone surfaces. In conclusion, decorporation of plutonium can be significantly improved by liposomal encapsulation of DTPA regardless of the treatment regimen applied.

Decorporation Approach after Rat Lung Contamination with Plutonium: Evaluation of the Key Parameters Influencing the Efficacy of a Protracted Chelation Treatment.

While the efficacy of a protracted zinc (Zn)- or calcium (Ca)-diethylenetriaminepentaacetic acid (DTPA) treatment in reducing transuranic body burden has already been demonstrated, questions about therapeutic variables remain. In response to this, we designed animal experiments primarily to assess both the effect of fractionation of a given dose and the effect of the frequency of dose fraction, with the same total dose. In our study, rats were contaminated intravenously with plutonium (Pu) then treated several days later with Ca-DTPA given at once or in various split-dose regimens cumulating to the same total dose and spread over several days. Similar efficacies were induced by the injection of the total dose or by splitting the dose in several smaller doses, independent of the number of doses and the dose level per injection. In a second study, rats were pulmonary contaminated, and three weeks later they received a Ca-DTPA dose 11-fold higher than the maximal daily recommended dose, administered either as a single bolus or as numerous multiple injections cumulating to the same dose, based on different injection frequency schedules. Independent of frequency schedule, the various split-dose regimens spread over weeks/months were as efficient as single delivery of the total dose in mobilizing lung plutonium, and had a therapeutic advantage for removal of retained hepatic and bone plutonium burdens. We concluded that cumulative dose level was a therapeutic variable of greater importance than the distribution of split doses for the success of a repeated treatment regimen on retained tissue plutonium. In addition, pulmonary administration of clodronate, which aims at killing alveolar macrophages and subsequently releasing their plutonium content, and which is associated with a continuous Ca-DTPA infusion regimen, suggested that the efficacy of injected Ca-DTPA in decorporating lung deposit is limited, due to its restricted penetration into alveolar macrophages and not because plutonium, as a physicochemical form, is unavailable for chelation.

Decorporation of Pu/Am Actinides by Chelation Therapy: New Arguments in Favor of an Intracellular Component of DTPA Action.

Diethylenetriaminepentaacetic acid (DTPA) is currently still the only known chelating drug that can be used for decorporation of internalized plutonium (Pu) and americium (Am). It is generally assumed that chelation occurs only in biological fluids, thus preventing Pu/Am deposition in target tissues. We postulate that actinide chelation may also occur inside cells by a mechanism called "intracellular chelation". To test this hypothesis, rats were given DTPA either prior to (termed "prophylactic" treatment) or belatedly after (termed "delayed" treatment) Pu/Am injection. DTPA decorporation efficacy was systematically tested for both plutonium and americium. Both prophylactic and delayed DTPA elicited marked decreases in liver Pu/Am. These results can be explained by chelation within subcellular compartments where DTPA efficacy increased as a function of a favorable intracellular DTPA-to-actinide molar ratio. The efficacy of intracellular chelation of liver actinides decreased with the delay of treatment. This is probably explained by progressive actinide binding to the high-affinity ligand ferritin followed by migration to lysosomes. Intracellular chelation was reduced as the gap between prophylactic treatment and contamination increased. This may be explained by the reduction of the intracellular DTPA pool, which declined exponentially with time. Skeletal Pu/Am was also reduced by prophylactic and delayed DTPA treatments. This decorporation of bone actinides may mainly result from extracellular chelation on bone surfaces. This work provides converging evidence for the involvement of an intracellular component of DTPA action in the decorporation process. These results may help to improve the interpretation of biological data from DTPA-treated contamination cases and could be useful to model DTPA therapy regimens.

Differential contribution of XPC, RAD23A, RAD23B and CENTRIN 2 to the UV-response in human cells.

Several genes in human cells are activated by physical genotoxic agents in order to regenerate cell homeostasis. Among the pathways contributing to this response, nucleotide excision repair (NER) is unique in restoring the nucleotide sequence of the DNA molecule without generating mutations. The first step of NER is mediated by a protein complex composed of XPC, RAD23B, an ubiquitin receptor and CENTRIN 2, an EF-hand calcium binding protein. These three proteins are multifunctional and participate in other important biochemical pathways. We silenced the XPC, RAD23A or RAD23B genes in HeLa cells for a long period of time by using Epstein Barr Virus-derived plasmids carrying sequences coding for small interfering RNA. XPC silencing confirms an essential role for XPC in DNA repair and cell survival after ultraviolet light irradiation. RAD23A and RAD23B participate in DNA repair and cell survival with diverging functions. Our data also indicate that CENTRIN 2 is recruited onto nuclear damaged areas quickly after irradiation and that XPC plays an important role during its internalization into the nucleus of human cells. Furthermore, the inhibition of XPC expression correlates with a decreased amount of CENTRIN 2 transcript and protein, indicating that XPC is required for the fine tuning of CENTRIN 2 gene expression. Moreover, XPC-silenced cells present a reduced concentration of CENTRIN 2 that affects both its centrosomal and nuclear localization suggesting that XPC deficiency may indirectly slow down cell division.

The combined effects of xeroderma pigmentosum C deficiency and mutagens on mutation rates in the mouse germ line.

Spontaneous and induced mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germ line of xeroderma pigmentosum group C (Xpc) knockout mice defective in global genome nucleotide excision repair. Spontaneous and radiation-induced mutation rates in homozygous Xpc(-/-) males were significantly higher than those in isogenic wild-type (Xpc(+/+)) and heterozygous (Xpc(+/-)) mice. In contrast, exposure to the monofunctional alkylating agent ethylnitrosourea resulted in similar increases in ESTR mutation rates across all genotypes. ESTR mutation spectra in the germ line of Xpc(-/-), Xpc(+/-) and Xpc(+/+) did not differ. Considering these data and the results of other publications, we propose that the Xpc-deficient mice possess a mutator phenotype in their germ line and somatic tissues that may significantly enhance carcinogenesis across multiple tissues.

Poly(ethylene oxide) facilitates the characterization of an affinity between strongly basic proteins with DNA by affinity capillary electrophoresis.

In order to study kin17 protein-DNA affinity, we have developed a fast and reproducible capillary electrophoresis (CE) analysis of a strongly basic protein: kin17 protein, using a nonpermanent coating based on poly(ethylene oxide) (PEO) to avoid adsorption of kin17. The coating procedure was optimized to provide a residual and stable electroosmotic flow (EOF = 5 x 10(-5) cm(2)/V x s), exhibiting RSD of 0.3% and excellent long-term stability. Good intraday and interday reproducibility of kin17 migration times (0.8 and 0.3% relative standard deviation (RSD), respectively) enabled us to consider that the recovery percentage obtained for kin17 protein was satisfactory (79%). The potential of this PEO-based coating procedure was evaluated for affinity CE method in order to study the affinity of kin17 protein for two single-stranded DNA (ssDNA) models: polydeoxyadenylic acid and polydeoxycytidilic acid (pdA and pdC). Binding constants (1.5 x 10(7) +/- 17% and 1.7 x 10(7) + 25%M(-1)) were evaluated assuming a 1:1 affinity between kin17 and pdA or pdC, respectively.

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins.

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

Homophilic anchorage of brain-hexokinase to mitochondria-porins revealed by specific-peptide antibody cross recognition.

In brain tumors the main source of energy is from glycolysis, which is initiated by hexokinase 1 (HK1), an enzyme bound to the mitochondrial porin. Disruption of HK binding greatly affects tumor cell survival. Little is known about the acceptor site of HK1. Therefore, a polyclonal antibody (Pab) directed to MIAAQLLAYYFTELK (MK) peptide, corresponding to the 15-amino acids of the N-terminal sequence of brain HK1 was obtained. Anti MK antibody (aMK-Pab)bound specifically to HK as shown by ELISA. The aMK-Pab binding to MK peptide was antibody-concentration dependent and was completely abolished by its preincubation with the peptide at 6 x 10-8 M. The aMK-Pab recognized cytosolic HK (cHK) and HK solubilized (sHK)from rat-brain mitochondrial preparations, but not the yeast HK which does not have the MK sequence. An anti-brain HK Pab (aHK-Pab) directed to purified HK recognized the MK peptide; aHK-Pab bound to MK and this binding was inhibited by preincubation of the antibody with the MK peptide. It was previously demonstrated that brain HK anchors to mitochondria porins, also designated as voltage dependent-anion channels (VDAC) via the MK sequence. A specific anti-VDAC antibody (aVDAC-Pab) which specifically bound the N and C-terminal sequences of VDACwas found to bind to c-HK, sHK and MK-coated wells and this binding was abolished by aVDACPabpreincubation with MK peptide. These data suggest that the three Pabs cross-react with an epitope present in HK and VDAC, and which was presented in the MK peptide. Comparison of alignment of HK or VDAC sequences, available in the protein data bank (PDB), did not allow putative homologues responsible for the cross-reaction to be identified, suggesting that the epitope is conformational. This, added to inhibition of mitochondria-isolated HK binding by the MK peptide,suggests that there is an homophilic-type interaction between HK and porin, through a peptidic structure represented at least in part in the MK peptide.

Germline mutation rates at tandem repeat loci in DNA-repair deficient mice.

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of non-exposed and irradiated severe combined immunodeficient (scid) and poly(ADP-ribose) polymerase (PARP-1-/-) deficient male mice. Non-exposed scid and PARP-/- male mice showed considerably elevated ESTR mutation rates, far higher than those in wild-type isogenic mice and other inbred strains. The irradiated scid and PARP-1-/- male mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated wild-type isogenic males. ESTR mutation spectra in the scid and PARP-1-/- strains did not differ from those in the isogenic wild-type strains. Considering these data and the results of previous studies, we propose that a delay in repair of DNA damage in scid and PARP-1-/- mice could result in replication fork pausing which, in turn, may affect ESTR mutation rate in the non-irradiated males. The lack of mutation induction in irradiated scid and PARP-1-/- can be explained by the high cell killing effects of irradiation on the germline of deficient mice.

Selective interactions of human kin17 and RPA proteins with chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner.

Several proteins involved in DNA synthesis are part of the so-called 'replication factories' that are anchored on non-chromatin nuclear structures. We report here that human kin17, a nuclear stress-activated protein, associates with both chromatin and non-chromatin nuclear structures in a cell cycle- and DNA damage-dependent manner. After L-mimosine block and withdrawal we observed that kin17 protein was recruited in the nucleus during re-entry and progression through S phase. These results are consistent with a role of kin17 protein in DNA replication. About 50% of the total amount of kin17 protein was detected on nuclear structures and could not be released by detergents. Furthermore, the amount of kin17 protein greatly increased in both G(1)/S and S phase-arrested cells in fractions containing proteins anchored to nuclear structures. The detection of kin17 protein showed for the first time its preferential assembly within non-chromatin nuclear structures in G(1)/S and S phase-arrested cells, while the association with these structures was found to be less stable in the G(2)/M phase, as judged by fractionation of human cells and immunostaining. In asynchronous growing cells, kin17 protein interacted with both chromatin DNA and non-chromatin nuclear structures, while in S phase-arrested cells it interacted mostly with non-chromatin nuclear structures, as judged by DNase I treatment and in vivo UV cross-linking. In the presence of DNA damage in S phase cells, the distribution of kin17 protein became mainly associated with chromosomal DNA, as judged by limited formaldehyde cross-linking of living cells. The physical interaction of kin17 protein with components of the nuclear matrix was confirmed and visualized by indirect immunofluorescence and immunoelectron microscopy. Our results indicate that, during S phase, a fraction of the human kin17 protein preferentially associates with the nuclear matrix, a fundamentally non-chromatin higher order nuclear structure, and to chromatin DNA in the presence of DNA damage.

Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes.

The Homo sapiens kin17 ((HSA)kin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that (HSA)kin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. These results are consistent with a major role of (HSA)kin17 protein in DNA replication factories. Other treatments hampering replication fork progression and/or inducing double-strand breaks also triggered an accumulation and a concentration of the chromatin-bound (HSA)kin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced (HSA)kin17 foci were retained in the nucleus after detergent extraction, suggesting a strong association with nuclear structures. Gel filtration analyses of cellular extracts showed that endogenous (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000. Interestingly, HU-induced G(1)-S arrest triggered an increase in the molecular weight of complexes containing (HSA)kin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound (HSA)kin17 protein. We hypothesize that in the presence of unrepaired DNA damage, (HSA)kin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair.

Human kin17 protein directly interacts with the simian virus 40 large T antigen and inhibits DNA replication.

Kin17 is an evolutionarily conserved DNA-binding protein, which forms intranuclear foci in proliferating cells. Recent data have suggested that human kin17 protein is associated with cell proliferation and unrepaired DNA lesions. Herein, we show that human fibroblasts (MRC5-V2 and CHSV4) immortalized with SV40 overexpress endogenous kin17 protein, as compared with normal diploid human fibroblasts. We observed that certain carcinoma cell lines also up-regulated kin17 protein, suggesting that increased kin17 protein levels may be a consequence of the immortalized phenotype. We report here that the endogenous kin17 protein is located in nucleoplasmic foci and colocalizes with SV40 large T antigen. Purification of human kin17 protein allowed analysis of the physical interaction with T antigen by several in vitro and in vivo assays. Large T antigen and human kin17 protein are part of the same high molecular weight multiprotein complex in human cells. Furthermore, human kin17 protein interacts with T antigen bound to the SV40 DNA origin of replication. Strikingly, the overexpression of human kin17 protein in vivo and the introduction of increased amounts of human kin17 protein in an in vitro assay reduced T-antigen-dependent DNA replication, suggesting that kin17 protein may be involved in the DNA replication process in human cells.