Bone morphogenetic protein 4 is involved in cadmium-associated bone damage.

Cadmium (Cd) is a well-characterized bone toxic agent and can induce bone damage via inhibiting osteogenic differentiation. Bone morphogenetic protein (BMP)/SMAD signaling pathway can mediate osteogenic differentiation, but the association between Cd and BMP/SMAD signaling pathway is yet to be illuminated. To understand what elements of BMPs and SMADs are affected by Cd to influence osteogenic differentiation and if BMPs can be the biomarkers of which Cd-induced osteoporosis, human bone marrow mesenchymal stem cells (hBMSCs) were treated with cadmium chloride (CdCl2) in vitro to detect the expression of BMPs and SMADs, and 134 subjects were enrolled to explore if the BMPs can be potential biomarkers of Cd-associated bone damage. Our results showed that Cd exposure significantly promoted the adipogenic differentiation of hBMSCs and inhibited its osteogenic differentiation by inhibiting the expression of BMP-2/4, SMAD4, and p-SMAD1/5/9 complex. And mediation analyses yielded that BMP-4 mediated 39.32% (95% confidence interval 7.47, 85.00) of the total association between the Cd and the risk of Cd-associated bone damage. Moreover, during differentiation, BMP-4 had the potential to enhance mineralization compared with CdCl2 only group. These results reveal that BMP-4 can be a diagnostic biomarker and therapeutic target for Cd-associated bone damage.

Genetic variants of the XRCC7 gene involved in DNA repair and risk of human bladder cancer.

OBJECTIVE: To investigate the association between the polymorphisms of the KU70 and X-ray repair cross complementing group 7 (XRCC7) genes and the risk of bladder cancer. METHODS: This hospital-based case-control study included 213 patients with newly diagnosed bladder transitional cell carcinoma and 235 cancer-free controls frequency-matched by age and sex. Two polymorphisms, KU70 and XRCC7, using a method involving polymerase chain reaction-restriction fragment length polymorphism were genotyped. RESULTS: The risk of bladder cancer decreased in a dose-response manner as the number of XRCC76721G alleles increased (adjusted odds ratio [OR] = 0.70, 95% confident interval [CI] = 0.47-1.03 for 6721GT and OR = 0.31, 95% CI = 0.10-0.99 for 6721GG; P(trend) = 0.013). However, when we used 6721 (GT + GG) as the reference, we found a statistically significant increased risk of bladder cancer associated with the 6721TT genotype (OR = 1.53, 95% CI = 1.04-2.25). In the stratification analysis, this increased risk was more pronounced among subgroups of patients aged >65 years (OR = 2.27; 95% CI = 1.25-4.10) and ever smokers (OR = 2.06, 95% CI = 1.15-3.68). Furthermore, we observed a 3.24-fold increased risk (95% CI = 1.35-7.78) for smokers aged >65 years carrying 6721TT genotype compared with those carrying the 6721 (GG + GT) genotype. However, the KU70-61C > G polymorphism was not associated with a significantly increased risk of bladder cancer. CONCLUSIONS: The XRCC7 but not the KU70 polymorphism appears to be involved in the etiology of human bladder cancer. Larger studies with more detailed data on environmental exposure are needed to verify these initial findings.

[Construction and identification of antisense c-Jun N-terminal kinase 1 eukaryotic fluorescent expressing plasmids and JNK1-/- human embryo lung fibroblasts cell line].

OBJECTIVE: To construct antisense c-Jun N-terminal kinase 1 (JNK1) eukaryotic fluorescent expressing vector and JNK1-/- human embryo lung fibroblasts cell line. METHODS: Trizol reagent was used to extract total RNA in HELF. The proper primers of JNK1 were chosen and synthesized. RT-PCR and gene recombinant techniques were used to construct the fragment of JNK1. After purification, the PCR products were cut, and JNK1 were inserted reversely into eukaryotic fluorescent expressing vector pEGFP-C1. Enzyme-cutting and DNA auto-sequencing were used to prove the successful construction of JNK1 eukaryotic expressing vector. Then plasmids were extracted and transfected into HELF cells and screen by G418 24 h later. Monoclone was chosen and cultured. Fluorescent imaging and Western blot were used to identify the JNK1-/- HELF cell line. RESULTS: Sequence analysis of pEGFP-C1-as JNK1 plasmids was same as expected. The expression level of JNK1 was inhibited markedly. CONCLUSION: Construction of antisense JNK1 eukaryotic fluorescent expressing vectors and JNK1-/- HELF cell line is successful.

Functional polymorphisms of JWA gene are associated with risk of bladder cancer.

The JWA gene is a novel cell differentiation-related gene thought to be a responsive gene in response to DNA damage and repair induced by environmental stressors. Recently, a novel single nucleotide polymorphism (SNP) was identified in the promoter of the JWA gene (-76GC) that may alter the transcription activity and thus play a role in increased risk of bladder cancer. Further, studies were conducted to screen for more novel variants in the JWA exons by using PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) followed by PCR-RFLP (PCR restriction fragment length polymorphism) methods. Finally, the functional relevance of the newly identified genetic variants in a hospital-based case-control study of 215 bladder cancer patients and 250 cancer-free controls was evaluated. In addition to the -76GC polymorphism, another novel SNP (454CA in exon2 and 723TG in exon 3) of JWA was identified. The -76GC allele and genotype frequencies were found to vary in different ethnic groups. The -76C allele and 454A allele were both associated with significantly increased risk of bladder cancer. In contrast, the 723GG genotype was associated with a decreased risk of bladder cancer. Furthermore, -76C and 454A together increased the risk of bladder caner using haplotype and stratification analysis. In conclusion, the three novel functional genetic polymorphisms of JWA gene, -76GC, 454CA, and 723TG, appear to contribute to the etiology of bladder cancer.

Single nucleotide polymorphism of the JWA gene is associated with risk of leukemia: a case-control study in a Chinese population.

The JWA gene was initially cloned as a novel cell differentiation-associated gene and was subsequently found to be an environmental responsive gene. The JWA gene also produced a marked effect during chemical-induced multidirectional differentiations of primary and human myeloid leukemia cells. Recently, a novel single nucleotide polymorphism (SNP) in exon2 of the JWA gene (454CA) was identified that may play a role in risk of bladder cancer. The aim of this study was to investigate the association between the 454CA (NM_006407.2) in JWA exon2 variants and risk of leukemia in a hospital-based case-control study of 202 leukemia patients and 289 cancer-free controls. Results indicated that 454A allele was found to associate with significantly increased risk of leukemia, although the 454CA is a synonymous polymorphism in coding region of the JWA gene. In conclusion, the potentially functional genetic polymorphism 454CA of the JWA gene appears to contribute to the risk of multiple kinds of leukemia in a south Chinese population.

Arsenite-induced alterations in Ku70-deficient cells: a model to study genotoxic effects.

As one of three subunits of DNA-dependent protein kinase (DNA-PK), Ku70 protein plays an important role in repair of DNA double-strand breaks (DNA DSB). To further understand the functions of Ku70 protein and the mechanisms underlying arsenite-induced genotoxic effects, the effects of Ku70 deficiency were examined. The Ku70-deficient cell line HLFK and null vector cell line HLFC were established after recombinant plasmid of Ku70 gene antisense RNA and null pEGFP-C1 vector were transferred into human embryo lung fibroblasts (HLF) cells. Experiments were undertaken to detect DNA DSB damage by neutral single-cell gel electrophoresis assay (SCGE), chromosomal alterations by micronucleus test, and cell cycle progression by flow cytometry in HLFC and HLFK cells treated with control, 1, 2.5, 5, or 10 microM sodium arsenite for 2, 4, or 24 h, respectively. Western blot analysis results showed that Ku70 protein content in HLFK cells decreased to 38% of those in HLFC cells. The median lethal concentrations (LC50) of sodium arsenite to HLFC and HLFK cells for 24 h were 27.38 microM and 21.80 microM, respectively. Results of neutral SCGE assay showed that there were concentration-dependent increases in tail length of DNA DSB, in percent of cells with DNA DSB tails, and in severity of DNA DSB damage in HLFK and HLFC cells. The increases in these indices in HLFK cells were significantly higher than those found in HLFC cells exposed to similar amounts of metal. The ability of DNA DSB to repair in HLFK cells was less than that seen in HLFC cells. Sodium arsenite produced concentration-dependent elevation in micronuclei and abnormal nuclei formation. The Ku70-deficiency enhanced the susceptibility to chromosomal alterations induced by sodium arsenite. Low concentrations of sodium arsenite induced cell arrest at G1; however, at high concentrations of metal this G1 arrest effect disappeared. These results suggested that Ku70 protein plays an important role in repair of DNA DSB damage and for maintainance of genome stability.

The role of oxidative stress in hormesis induced by sodium arsenite in human embryo lung fibroblast (HELF) cellular proliferation model.

Hormetic dose-response relationships induced by environmental agents are often characterized by a low-dose stimulation and a high-dose inhibition. The mechanisms underlying hormesis induced by environmental agents still remain an enigma; however, hormetic consequences may have significant implications for health risk assessments. To investigate the role of oxidative stress in hormetic phenomena associated with cell proliferation induced by sodium arsenite, the levels of reactive oxygen species (ROS), lipid peroxidation (LPO), and heat-shock proteins (HSP) and the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were measured in human embryo lung fibroblast (HELF) cells after treatment with sodium arsenite at various concentrations for differing times. Results showed that sodium arsenite induced significant cell proliferation at low concentrations (0.5 microM for 12, 24, and 48 h), but inhibited cell growth at high amounts (5 and 10 microM for 24 and 48 h), reflected as a beta concentration-response curve. Data indicated that the relationship between ROS levels and sodium arsenite exposure concentration displayed a positive correlation. It was found out that sodium arsenite at high concentrations induced LPO damage. The activities of SOD were enhanced at low metal concentrations but inhibited with high amounts in a concentration-dependent manner. Similarly, heat-shock protein 27 (HSP27) levels were increased by sodium arsenite of low concentrations with early exposure time (3, 6, and 12 h), but decreased with high metal concentrations with greater exposure time (24 and 48 h). Sodium arsenite decreased HSP70 expression at lower concentrations, but increased HSP70 expression at higher concentration. The results indicated that this cellular hormetic model of cell proliferation induced by sodium arsenite occurred in HELF cells, which may explain contradictory effects seen with this metal. Sodium arsenite at low concentrations induced enhanced ROS generation without cytotoxicity and a cellular protective effect. In contrast, sodium arsenite at high concentrations produced marked ROS formation, marked oxidative stress, and cellular damage, as evidenced by LPO.

Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells.

Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high-dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. In the present study, we aimed at investigating the relationship between biphasic effect of arsenite on cell proliferation and apoptosis and activation of JNK and ERK1/2 in human embryo lung fibroblast (HELF) cells. Our results demonstrated that cell proliferation may be stimulated at lower concentrations (0.1 and 0.5 microM) arsenite but inhibited at higher concentrations (5 and 10 microM). When cell apoptosis was used as the endpoint, the concentration-response curves were changed to U-shapes. During stimulation phospho-JNK levels were significantly increased at 3, 6, and 12 h after 0.1 or 0.5 microM arsenite exposure. Phospho-ERK1/2 levels were increased with different concentrations (0.1-10 microM) of arsenite at 6, 12, and 24 h. Blocking of JNK pathway with 20 microM SP600125 or ERK1/2 by 100 microM PD98059 significantly inhibited biphasic effect of arsenite in cells. Data in the present study suggest that activation of JNK and ERK1/2 may be involved in biphasic effect of arsenite when measuring cell proliferation and apoptosis in HELF cells. JNK activation seems to play a more critical role than ERK1/2 activation in the biphasic process.

[The relationship between hormesis of proliferation and oxidative stress induced by sodium arsenite in human embryo lung fibroblasts].

OBJECTIVE: To investigate the relationship between the hormesis of proliferation and oxidative stress induced by sodium arsenite (Na(2)AsO(2)) in human embryo lung fibroblasts (HELF). METHODS: HELF were treated with Na(2)AsO(2) of 0.0, 0.1, 0.5, 1.0, 5.0 and 10.0 micromol/L for 4 hours or 24 hours, respectively. The cell proliferation, the reactive oxygen species (ROS) level, the malondialdehyde (MDA) content and the activity of glutathione peroxide (GSH-Px) and the superoxide dismutase (SOD) in HELF were detected respectively. RESULTS: The HELF proliferation induced by 0.1 and 0.5 micromol/L Na(2)AsO(2) was significantly higher than that in the control group (P < 0.01). The HELF proliferation induced by 5.0 and 10.0 micromol/L Na(2)AsO(2) was significantly lower than that in the control group (P < 0.01) with the dose-effect relation of an inverted U curve. The ROS level induced by Na(2)AsO(2) of between 0.5 and 10.0 micromol/L was significantly increased (P < 0.05, P < 0.01). The positive correlation was found between the ROS level and the exposure dose of Na(2)AsO(2) (r = 0.934, P < 0.01). The 5.0 and 10.0 micromol/L Na(2)AsO(2) induced the significant increase of the MDA contents (P < 0.01) and the significant decrease of the GSH-Px activity compared to those in the control group (P < 0.01). The SOD activity in 0.5 micromol/L Na(2)AsO(2) group was significantly higher than that in the control group (P < 0.01) while the SOD activity induced by 5.0 and 10.0 micromol/L Na(2)AsO(2) was significantly decreased (P < 0.01) if compared with the control group with the dose-effect relation of an inverted U curve. CONCLUSION: The sodium arsenite can induce the hormesis of proliferation in HELF with the dose-effect relation of an inverted U curve. The mechanisms probably relates to different levels of oxidative stress induced by sodium arsenite of different concentrations.

[Expression deficiency of JWA enhanced DNA damage and delayed DNA repair in HeLa cells induced by benzo (a) pyrene exposure].

OBJECTIVE: To investigate the role of JWA gene in benzo (a) pyrene [B (a) P] induced DNA damage and repair effects in HeLa cells. METHODS: The antisense JWA express vector (pEGFP-C1-asJWA) was constructed and stably transfected into HeLa cells. JWA deficient HeLa cells (asJWA-HeLa) was then screened and established. The general characteristics of asJWA-HeLa cells were investigated. DNA damage and repair cell culture model was conducted by treating the cells with 50 micromol/L B (a) P plus S9 for 3 hours and then the cells were maintained further 0, 1, 3, and 24 hours for DNA repairing. The damaged DNA was detected by single cell gel electrophoresis assay (comet assay). RESULTS: JWA deficient HeLa cells (with a 31% of JWA protein expression as compared with the control) were obtained successfully. Compared with the empty vector transfected cells (C1-HeLa) and the untransfected HeLa cells, asJWA-HeLa cells were more sensitive to B (a) P exposure and with a delayed DNA repair process. CONCLUSION: The JWA determined might function as a potential effective environmental responsive gene and actively participate the process of B (a) P exposure associated with intracellular signal pathways of DNA damage and repair.

JWA, a novel signaling molecule, involved in all-trans retinoic acid induced differentiation of HL-60 cells.

JWA (AF070523) was originally identified as a novel all-trans retinoic acid (ATRA) responsible gene in primary human tracheal bronchial epithelial cells. For the notable performance achieved by ATRA in the differentiation induction therapy, we investigated the role of JWA in ATRA-mediated differentiation of the human myeloid leukemia HL-60 cells. We found that concomitant with the progressive cell differentiation, JWA expression was up-regulated by ATRA in a dose- and time-dependent manner. Inhibition of JWA expression by RNA interference partially blocked ATRA-induced differentiation and growth inhibition of HL-60 cells. Pre-treatment of phorbol-12-myristate-13-acetate (TPA), a PKC activator, decreased ATRA-mediated differentiation, companied with the down-regulation of JWA expression. Arsenic trioxide (As(2)O(3), 0.5 microM) enhanced the cellular differentiation induced by 0.01 microM ATRA, but had no noticeable effect on the differentiation induced by 0.1 microM ATRA. Concurrent with the enhancement, JWA expression was up-regulated. All the data suggest that up-regulation of JWA expression is essential for ATRA-induced differentiation of HL-60 cells. And JWA, associated with PKC, is involved in its signal pathways. Ideal therapeutic efficacy with low toxicity may be obtained if low doses of ATRA (0.01 microM) and As(2)O(3) (0.5 microM) are combined. These findings may present a novel mechanism that cellular differentiation and growth inhibition induced by ATRA are mediated at least in part through regulation of JWA expression. JWA may be a novel molecular marker for ATRA-induced HL-60 cell differentiation. ATRA up-regulates JWA expression by stimulating the transcriptional activity of JWA gene promoter.

JWA, a novel signaling molecule, involved in the induction of differentiation of human myeloid leukemia cells.

Dysregulation of hematopoietic cellular differentiation contributes to leukemogenesis. Unfortunately, relatively little is known about how cell differentiation is regulated. JWA (AF070523) is a novel all-trans retinoic acid (ATRA) responsible gene that initially isolated from ATRA-treated primary human tracheal bronchial epithelial cells. For the notable performance achieved by ATRA in the differentiation induction therapy, we investigated the role of JWA in the induction of differentiation of human myeloid leukemia cells. Our results showed that JWA was not only regulated by ATRA but also by several other differentiation inducers such as phorbol-12-myristate-13-acetate (TPA), arabinoside (Ara-C), and hemin, involved in the mechanisms of differentiation along different lineages of myeloid leukemia cells arrested at different stages of development. Generally, JWA was up-regulated by these inducers in a time-dependent manner. Inhibition of JWA by RNA interference decreased the induced cellular differentiation. However, in NB4 cells treated with ATRA, dissimilar with others, the expression of JWA was down-regulated, and the induced cellular differentiation could be enhanced by silencing of JWA. Collectively, JWA works as a potential critical molecule, associated with multi-directional differentiation of human myeloid leukemia cells. In NB4 cells, JWA may function as a lineage-restricted gene during differentiation along the monocyte/macrophage-like or granulocytic pathway.

[A case-control study on JWA promoter -76G-->C polymorphism and the susceptibility of bladder cancer].

OBJECTIVE: This case-control study was aimed to detect the single nucleotide polymorphisms (SNPs) in JWA promoter region, to assess the effect of SNP on transcriptional activity, and to probe the relationship between SNP and the risk of bladder cancer. METHODS: The design of one control per case was adopted. The JWA gene promoter region in 155 patients with bladder cancer and in 155 cancer-free controls was amplified by PCR-SSCP technique, and the SNP were confirmed by direct DNA sequencing. The recombinant plasmids of JWA promoter fragment which contain the SNP were constructed as CAT reporter gene and were transfected transiently into NIH 3T3 cells for disclosing whether SNP changes the transcriptional activity of the promoter. RESULTS: A novel SNP -76 G-->C at promoter region of JWA gene was found. The frequencies of the C allele and GC genotype at JWA promoter -76G-->C in bladder cancer group (10.00% and 20.00% respectively) were significantly higher than those in control group (5.16% and 10.32% respectively) (P < 0.05). The transcriptional activity of -76GC allele genotype was significantly down-regulated as compared with that of -76GG allele genotype (P < 0.01). Multivariate logistic regression analysis revealed that JWA polymorphism at promoter -76G-->C is an independent novel risk factor for bladder cancer. CONCLUSION: The JWA -76G-->C variant genotype may play an important role in transcription regulation of JWA gene and in the susceptibility to bladder cancer.

[Expression of novel environmental responsive protein JWA involved in the oxidative stress responsiveness in MCF-7 cells].

OBJECTIVE: To study the expression and the possible role of JWA protein in oxidative stress-induced damage of MCF-7 cells, especially the relationship between JWA and heat shock proteins (HSPs). METHODS: MCF-7 cells were exposed to different concentration of H(2)O(2) (0.01,0.10, 1.00 mmol/L) for different time (10, 30, 60 and 180 min) respectively. DNA damage was detected by using DNA gel electrophoresis. The MTT assay was used to analyze the effect of H(2)O(2) on the cytotoxicity and relative cell proliferation ratio of the cells. The expressions of JWA, HSP70, HSP27 and HSF1 were determined by Western-blot. RESULTS: The inhibitory effect on MCF-7 cells viability induced by H(2)O(2) was shown a dose-and time-dependent manner and MCF-7 cells proliferation, and was almost completely inhibited by the exposure of H(2)O(2) at 1.00 mmol/L for 180 min. Hydrogen peroxide treatment of MCF-7 cells caused oxidative stress which up-regulated the expressions of JWA, HSP70 and heat shock factor 1 (HSF1) in a dose-dependent manner, and the expression pattern of JWA was very similar to those of HSP70 and HSF1 but not to HSP27. CONCLUSION: JWA might enhance intracellular defenses against H(2)O(2)-induced oxidative damage in human breast carcinoma cells. JWA is determined functioning as an effective environmental responsive protein and as a parallel molecule of HSP70 actively participates in the signal pathways of oxidative damage which might be regulated by HSF1.

[JWA gene acts as sensitive molecule responsive to oxidative stress and apoptosis in K562 cells].

OBJECTIVE: To investigate the expression of JWA gene, heat shock proteins (hsp70 and hsp27) and p53, and to explore the role and the possible mechanism of JWA gene involved in H2O2-induced oxidative stress of K562 cells. METHODS: 0.01, 0.1, 1 mmol/L H2O2 treated K562 cells at 10, 30, 60 and 180 min to established the models of DNA damage. Furthermore, K562 cells were induced apoptosis by 0.1 mmol/L H2O2 at different time (6-48 h) and different concentration (0.5-1,000 micromol/L) of H2O2 at 48 h. DNA damage and cell apoptosis were detected by DNA gel electrophoresis. And the immunoblotting assay was used for detecting expressions of JWA protein and correlated genes (hsp27, hsp70 and p53). RESULTS: During the DNA damage, JWA was much more sensitive to H2O2 than those heat shock proteins, and its expression pattern was very similar to that of hsp70. And at low concentration of H2O2-exposure (0.01 mmol/L), the expressions of JWA and heat shock proteins were all increased greatly. In addition, JWA, hsp70, hsp27 and p53 overexpressed and their expression pattern were similar during cell apoptosis. CONCLUSION: JWA should be functioning as an effective environmental responsive gene and should actively participate the signal pathways of oxidative stress which might be associated with hsp70 and p53.

Regulation of a novel cell differentiation-associated gene, JWA during oxidative damage in K562 and MCF-7 cells.

Oxidative stress, or the production of oxygen-centered free radicals, has been hypothesized as the major source of DNA damage that can lead to a variety of diseases including cancer. It is known that 8-hydroxy-deoxyguanosine (8-oxo-dG) is a useful biomarker of oxidative DNA damage. Our recent data showed that JWA, initially being cloned as a novel cell differentiation-associated gene, was also actively responsive to environmental stressors, such as heat-shock, oxidative stress and so on. In the present study, we have applied a modified comet assay and bacterial repair endonucleases system (endonuclease III and formamidopyrimidine glycosylase) to investigate if JWA is involved in hydrogen peroxide (H2O2)-induced DNA damage and repair in K562 and MCF-7 cells, and to demonstrate if the damage is associated with 8-oxo-dG. The results from the comet assay have shown that the average tail length and the percentage of the cells with DNA tails are greatly induced by H2O2 treatment and further significantly enhanced by the post-treatment of repair endonucleases. The H2O2-induced 8-oxo-dG formation in K562 and MCF-7 cells is dose-dependent. In addition, the data have clearly demonstrated that JWA gene expression is actively induced by H2O2 treatment in K562 and MCF-7 cells. The results suggest that JWA can be regulated by oxidative stress and is actively involved in the signal pathways of oxidative stress in the cells.

[Genetic polymorphisms of nucleotide repair gene hMTH1 in southern Chinese Han population].

In order to study the genetic polymorphisms of nucleotide repair gene hMTH1 in southern Chinese Han population, the polymorphisms of the gene's promoter and its five exons among peripheral blood lymphocytes of 172 Chinese Han people were analyzed with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. The sequences of the promoter and exon 1 of hMTH1 gene were conserved. A T to C polymorphism was detected at the 73th base in exon 2. The genotype frequencies of TT and TC were 93.02% and 6.98%, respectively. The allelic frequencies of T and C were 96.51% and 3.49%, respectively. A T to C polymorphism was detected at codon 45 in exon 3, which was first reported. The genotype frequencies of TT and TC were 95.35% and 4.65%, respectively. The allelic frequencies of T and C were 97.67% and 2.33%, respectively. A G to A polymorphism was detected at codon 83 in exon 4. The genotype frequencies of GG and GA were 89.53% and 10.47%, respectively. The allelic frequencies of G and A were 94.77% and 5.23%, respectively. A C to T polymorphism was detected at codon 119 in exon 5. The genotype frequencies of CC and CT were 95.93% and 4.07%, respectively. The allelic frequencies of C and T were 97.97% and 2.03%, respectively.

[Effects of hydroquinone on DNA and nucleus damage in human embryo lung fibroblasts].

OBJECTIVE: To study DNA and nucleus damage in human embryo lung fibroblast (HLF) exposed to hydroquinone (HQ) and its genotoxicity. METHODS: HLF were treated with HQ (0, 10, 20, 40, 80 micro mol/L, respectively) for 3 h and DNA damage was detected by comet assay. HLF was also treated with the same concentrations of HQ for 1 h and micronucleus test was performed after they were cultured for 24 h. RESULTS: Comet assay showed that percentage of cells with tails in each groups treated with varied doses of HQ was 12%, 19%, 42%, 79% and 95%, respectively, with mean tail length of 7.87, 9.35, 11.03, 19.28 and 23.32 micro m, respectively, in an obvious dose-dependent manner (P < 0.05). Very significant increase in percentage of cells with tails and length of their comet tail were observed in those groups treated with HQ of 20, 40 and 80 micro mol/L (P < 0.01). And, proportion of high and severe DNA damage increased with dose of HQ. HQ could also induce formation of micronucleus and abnormal nucleus in all groups treated by varied doses of HQ, with rates of micronucleus and abnormal nucleus of 2%, 3%, 10%, 9% and 15%, and 6%, 7%, 16%, 27% and 28%, respectively, in a significant dose-dependent manner. There was significant increase in rates of micronuclei and abnormal nuclei in cells treated with HQ at doses of 20, 40 and 80 micro mol/L (P < 0.05). CONCLUSIONS: Exposure to HQ could cause DNA and nucleus damage inducing genotoxic effects on HLF.

[Construction of double-strand break repair protein hKu70 deficient cell strain and its biologic characters].

OBJECTIVE: To construct DNA double-strand break (DSB) repair protein hKu70 deficient cell strain and to observe its biological characters for studying the functions of hKu70 gene and the effects of occupational harmfulness factors on DSB repair. METHODS: Human lung fibroblasts (HLF) were transfected with the eukaryotic expression plasmids of hKu70 gene antisense RNA (pEGFP-C1-K) to construct hKu70 protein deficient cells (named as "HLFK"). The protein expression levels of hKu70 gene in HLFC and HLFK were detected by the Western blotting to estimate the effects of antisense inhibition. Morphology, growth character and growth status in soft agar of transfected HLFK were observed. RESULTS: pEGFP-C1-K vector was successfully expressed in HLF. The protein expression level of hKu70 gene in HLFK was decreased by 42% as compared with that in HLFC. No obvious changes of the biologic characters were observed in HLFK. CONCLUSION: The hKu70 protein deficient cell strain was successfully constructed. The hKu70 protein deficiency alone didn't induce obvious changes of the biological characters in HLFK.