By fractionating whole cell extracts into cytoplasmic, soluble nuclear, and chromatin-bound fractions, we observed that RecQ4 protein bound more tightly to chromatin upon UV irradiation. Genome instability is thought to play an important role in the development and progression of cancer and has also been implicated in the aging process. RTS patients display clinical profiles that include growth deficiency, photosensitivity with poikiloderma, signs of premature aging including the graying and loss of hair and cataracts, and an increased cancer predisposition especially to osteosarcoma 2.
The cellular function of RecQ4 is obscure, although some recent studies indicate that RecQ4 may be implicated in different DNA metabolic processes. Secondly, Petkovic et al. Thirdly, Woo and Werner 11 , 12 have reported that RecQ4 plays a role in oxidative stress. Fourthly, cells defective in RecQ4 escaped from the S-phase arrest following UV or hydroxyurea treatment The NER pathway consists of at least 30 proteins involved in sequential damage recognition, dual incision, repair synthesis, and ligation steps Given its affinity for damaged DNA and its ability to interact with many core NER factors, XPA is anticipated to verify NER lesions and to play a central role in positioning the repair machinery correctly around the injury In this study, we have examined the cellular localization of RecQ4 in response to multiple DNA damage agents.
Further, two proteins could interact with each other, and their interaction was increased by UV exposure.
Chemistry And Biological Actions Of 4 Nitroquinoline 1 Oxide
For drug treatments, cells were incubated with 1. The RecQ4 antibody was affinity-purified with Amino-Link Plus immobilization kit Pierce following the manufacturer's instructions. Western blotting of whole cell lysates from T cells and HeLa cells showed that the purified antibody only recognized RecQ4 protein. Immunofluorescence —Cells were grown on mm coverslips overnight prior to treatment. Secondary antibody dilutions are as follows: rhodamine red-X-conjugated AffiniPure goat anti-rabbit 1: and fluorescein isothiocyanate-conjugated AffiniPure goat anti-mouse 1: 50 Jackson ImmunoResearch, Inc.
Subcellular Fractionation —The cellular protein fractionation was performed essentially as described Lysates were cleared by centrifugation, and protein concentration was determined by the Bradford assay Bio-Rad. Chromatin-bound proteins were obtained after centrifugation at 13, rpm for 20 min. For the control, DNA was harvested from nonirradiated cells.
The membranes were then incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibodies Amersham Biosciences , and bound antibodies were visualized using the ECL chemiluminescent method. The immunoprecipitates were separated from supernatant by centrifugation and washed with phosphate-buffered saline containing 0. HeLa cells were treated with different DNA damage agents as indicated. For chemical treatment, cells were incubated with 1. No treat , left untreated. After 4 h, the color formed was quantitated by a spectrophotometric plate reader Berkman, Inc.
Indirect immunofluorescence staining analysis revealed that most RecQ4 was homogeneously localized in nucleus of mock-treated HeLa cells Fig. At 4 h after UV irradiation, cells were immunostained with anti-RecQ4 antibody red.
C , quantitative results of A. The averages of three different experiments are shown. For each experiment, at least cells for each time point were counted, and the percentage of cells containing RecQ4 foci was calculated. The error bar represents S.
Photodynamic Activity of 4-Nitroquinoline-l-oxide and Related Compounds
D , quantitative results of B. For each experiment, at least cells for each dose were counted, and the percentage of cells containing RecQ4 foci was calculated.
As shown in Fig. To test this hypothesis, we generated pBabe-RecQ4 retroviral construct. The stable cell lines infected with control virus or pBabe-RecQ4 virus were generated.
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Since reduced DNA repair is often linked to the hypersensitivity to UV irradiation, we performed the MTT assay to determine the cellular viability in response to UV irradiation with these two cell lines. Cells were seeded onto well plate and irradiated with increasing doses of UV. At 48 h after irradiation, cell viability was determined. RecQ4 has a functional role in NER. Total cell lysates were prepared from stably transfected cells and probed with anti-RecQ4 upper panel and actin lower panel antibodies, respectively. The cells were treated with increasing doses of UV irradiation followed by a h recovery.
Points , mean of four different measurements; bars , S. RecQ4 and XPA interacted with each other. Then co-immunoprecipitation assays were performed with anti-XPA antibody, and immunoprecipitates were detected by anti-RecQ4 antibody upper panel. The immunoprecipitates were analyzed by Western blotting with anti-XPA antibody. Furthermore, DNase I treatment of cell lysates did not abrogate the interaction of XPA and RecQ4, indicating that this interaction is not mediated by the separate binding of two proteins to the chromatin Fig.
Taken together, these data demonstrated that RecQ4 and XPA could interact with each other both in vivo and in vitro. At 4 h after irradiation, whole cell lysates were prepared for co-immunoprecipitation with anti-FLAG resin M2-beads. However, no interaction between RecQ4 and RPA was detected under the same conditions, suggesting that the interaction of RecQ4 with XPA is intrinsic to two proteins and is not mediated by the formation of multiprotein complex in cells.
Immunofluorescence microscopy was performed. XPA null cells from a xeroderma pigmentosum patient GM, Coriell Institute were used for immunofluorescence staining analysis. RecQ4 was normally expressed in these cells data not shown. Remarkably, the RecQ4 foci were no longer formed Fig. To investigate whether RecQ4 could also increasingly associate with chromatin after UV treatment, extracts of HeLa cells were fractionated into three parts: cytoplasmic protein, soluble nuclear protein, and chromatin-bound protein.
As a positive control, we clearly detected that XPA translocated from cytoplasm to nuclei, with most of XPA bound to chromatin at 0. More RecQ4 protein bound to chromatin after UV irradiation. The quantities of RecQ4 protein were estimated by densitometry and then normalized to untreated controls, which were designated as 1. Mutation in the RecQ4 causes Rothmund-Thomson syndromes, which have severe physiological consequences in humans, the most prominent of which are cancer susceptibility and premature aging. At the cellular level, defects in RecQ4 cause genome instability, particularly trisomy, aneuploidy, deletions, translocations, and high frequencies of chromosomal rearrangements However, there are limited and conflicting data regarding the potential role of RecQ4 in the DNA damage response 8 , 10 — There are two studies about RecQ4 foci published recently.
Petkovic et al. Werner et al. The discrepancy in these reports is hard to interpret, but the different cell fixation approaches and the different sources of antibodies used for immunostaining might be the reason, as discussed previously 8. The RecQ4 nuclear foci greatly decreased 21 h after UV irradiation, and at this time point, either DNA lesions are removed or cells die from apoptosis.
The following day, the medium was replaced with medium containing 4NQO or Taxol, followed by incubation for 48 h. The total cell lysates of treated cells were separated by electrophoresis on The membrane was subsequently stained with Ponceau S and cut into several pieces according to the molecular weight of each designed protein. The blots were subsequently incubated with individual primary antibodies POLD4, ,; cat.
The statistical software SPSS There was a positive correlation between the decrease and the 4NQO concentration Fig. Exposure to tobacco smoke and UV radiation can result in various types of DNA damage and subsequently lead to cancer formation. Therefore, 4NQO-induced mutagen sensitivity assays have been used to study susceptibility in 4NQO-treated cells in the present study. Low POLD4 expression weakens the nucleotide excision repair capability. These are two reasons for the cause of genomic instability and ultimately the increase in the risk Fig.
NER, nucleotide excision repair. In previous studies, POLD4 expression in small cell lung cancer and a small section of non-small cell lung cancers is lower when compared to normal samples 9. Clinical data showed that almost all small cell lung cancers have a smoking history, indicating the possibility of a correlation between smoking-induced lung cancer and POLD4. The most studied polycyclic aromatic hydrocarbon is benzopyrene.
In the present study, the benzopyrene analogue 4NQO was used. These insults appear similar to the damage induced by other carcinogens that are present in tobacco In our previous study 9 , the low expression of POLD4 in small cell and non-small cell lung cancer suggested that there is a possible association between smoking and POLD4 genes.
In the present study, another type of cytotoxic drug, Taxol, was selected as a control to cause cytotoxicity, however, the repair of DNA damage did not depend on the NER capacity. In conclusion, the present study reported that a significant tobacco carcinogen, benzopyrene analogue 4NQO, downregulated the POLD4 expression level, and thereby caused the decreased NER, further resulting in genomic instability, and ultimately an increased risk of lung cancer formation.
Combined with the results of our previous study, we have proposed the mechanisms of action for POLD4 in smoking-induced lung cancer. The present study was supported partly by grants from the National Natural Science Foundation of China no. Hecht SS: Tobacco carcinogens, their biomarkers and tobacco-induced cancer. Nat Rev Cancer. Osada H and Takahashi T: Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer.
PLoS Comput Biol. Hecht SS: Chemoprevention of cancer by isothiocyanates, modifiers of carcinogen metabolism. J Nutr. Chem Res Toxicol. Hecht SS: Lung carcinogenesis by tobacco smoke.
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