Adaptive response and the influence of ageing: effects of low-dose irradiation on cell growth of cultured glial cells

PURPOSE: To examine the molecular mechanism of radiation adaptive response (RAR) for the growth of cultured glial cells and to investigate the influence of ageing on the response. MATERIALS AND METHODS: Glial cells were cultured from young and older rats (1 and 24 months). RAR for the growth of glial cells conditioned with a low dose of X-rays and subsequently exposed to a high dose of X-rays was examined for cell number and BrdU incorporation. Involvement of the subcellular signalling pathway factors in RAR was investigated using their inhibitors, activators, and mutated and knockout glial cells. RESULTS: RAR was observed in cells cultured from young rats but was not in cells from older animals. The inhibitors of protein kinase C (PKC) and DNA-dependent protein kinase (DNA-PK) or phosphatidylinositol 3-kinase (PI3K) suppressed RAR. The activators of PKC instead of low-dose irradiation also caused RAR. Moreover, glial cells cultured from severe combined immunodeficiency (scid) mice (CB-17 scid) and ataxia-telangiectasia mutated (Atm) knockout mice showed no RAR. CONCLUSION: The results indicated that PKC, ATM, DNAPK and/or PI3K were involved in RAR for growth and BrdU incorporation of cultured glial cells and RAR decreased with ageing.     

Role of stress responses in human cell survival following exposure to ultraviolet C radiation.

Purpose : To investigate in human skin and other cells the role of tyrosine kinase and protein kinase-C (PKC) in eliciting cell-signalling responses to UV radiation (UVR) that affect the survival of irradiated cells. Materials and methods : The survival of HeLa S3 cells, NCTC 2544 human keratinocytes and A431 human epidermal carcinoma cells was measured following incubation with various tyrosine kinase or PKC inhibitors and exposure to UVC (254nm) radiation. In addition, Western blotting measured PKC isozyme expression in human keratinocytes following UVC exposure. Results : It was confirmed that inhibition of tyrosine kinase activation reduces the survival of UV-irradiated HeLa S3 cells. However, no effect was seen on the survival of either NCTC 2544 human keratinocytes or A431 human epidermal carcinoma cells. In contrast, specific inhibition of PKC reduced the survival of UV-irradiated keratinocytes but had no effect on HeLa cells. Comparison of the effects of different inhibitors in keratinocytes suggested that this effect was mediated mostly through PKC μ and PKC λ/ ?. In addition, keratinocyte exposure to UVC induced large and temporally distinct increases in PKC μ and PKC λ/ ?. Conclusions : The survival of NCTC 2544 keratinocytes, but not HeLa S3 cells, following UVC exposure is mediated by signalling through PKC, mostly PKC μ and PKC λ/ ?. Further study is required to confirm these results in normal human keratinocytes.     

Role of stress responses in human cell survival following exposure to ultraviolet C radiation

PURPOSE: To investigate in human skin and other cells the role of tyrosine kinase and protein kinase-C (PKC) in eliciting cell-signalling responses to UV radiation (UVR) that affect the survival of irradiated cells. MATERIALS AND METHODS: The survival of HeLa S3 cells, NCTC 2544 human keratinocytes and A431 human epidermal carcinoma cells was measured following incubation with various tyrosine kinase or PKC inhibitors and exposure to UVC (254nm) radiation. In addition, Western blotting measured PKC isozyme expression in human keratinocytes following UVC exposure. RESULTS: It was confirmed that inhibition of tyrosine kinase activation reduces the survival of UV-irradiated HeLa S3 cells. However, no effect was seen on the survival of either NCTC 2544 human keratinocytes or A431 human epidermal carcinoma cells. In contrast, specific inhibition of PKC reduced the survival of UV-irradiated keratinocytes but had no effect on HeLa cells. Comparison of the effects of different inhibitors in keratinocytes suggested that this effect was mediated mostly through PKCmu and PKClambda/iota. In addition, keratinocyte exposure to UVC induced large and temporally distinct increases in PKCmu and PKClambda/iota. CONCLUSIONS: The survival of NCTC 2544 keratinocytes, but not HeLa S3 cells, following UVC exposure is mediated by signalling through PKC, mostly PKCmu and PKClambda/iota. Further study is required to confirm these results in normal human keratinocytes.     

Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells

Purpose : To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. Materials and methods : HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm -2) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. Results : The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. Conclusion : The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.     

Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells

PURPOSE: To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. MATERIALS AND METHODS: HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm(-2)) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. RESULTS: The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. CONCLUSION: The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.     

Correlation of chromosomal aberrations and sister chromatid exchanges in individual CHO cells pre-labelled with BrdU and treated with DNaseI or X-rays

PURPOSE: To analyse the correlation between chromosomal aberrations and sister-chromatid exchanges (SCE) in cells treated in G1 phase with X-rays or DNaseI. MATERIALS AND METHODS: Chinese hamster ovary (CHO) cells were labelled with 5'-bromodeoxyuridine (BrdU) for one round of replication and irradiated in G1 phase with 1.2, 2.4, 3.6 and 4.8 Gy X-rays or treated by electroporation with 1000, 2000, 3000 and 4000 units DNaseI per 800 microl electroporation buffer. Using a computer-aided metaphase relocation system first post-treatment mitoses were analysed for SCE and chromosomal aberrations allowing a precise investigation of correlation between both phenomena. RESULTS: A better correlation between aberrations and SCE was observed for DNaseI than for X-rays. X-rays induced more SCE than expected on the basis of aberrations, whereas the frequencies of SCE induced by DNaseI can mainly be accounted for by chromosomal aberrations. DISCUSSION: The results obtained with X-rays support an earlier observation that radiation induces both "true" SCE that are not related to chromosomal aberrations and result from radiation damage to BrdU, and "false" SCE that result from exchange-type aberrations. The high frequency of aberrations observed in cells treated with DNaseI and the good correlation between aberrations and SCE suggests that the endonuclease induces mainly "false" SCE.     

Cell kinetics of normal and perturbed populations measured by incorporation of bromodeoxyuridine and flow cytometry

Bromodeoxyuridine (BrdU) incorporation and flow cytometry have been used to measure the kinetics of V79 cells growing at different temperatures in vitro and cells of the murine Sa F tumour growing in vivo. By simultaneously measuring total DNA content and BrdU incorporation in individual cells at different times after pulse labelling with BrdU, it is a simple procedure to quantify the movement of cells through the cell cycle. The method has the advantage of speed (2 X 10(4) or more cells are analysed within a day or so of the experiment) and the ability to analyse the results in different ways. A G2 block in tumour cell progression in vivo is readily detected after a dose as low as 2 Gy. The neutron relative biological effectiveness for this G2 block is probably larger than that for tumour growth delay.     

Correlation of chromosomal aberrations and sister chromatid exchanges in individual CHO cells pre-labelled with BrdU and treated with DNaseI or X-rays

Purpose : To analyse the correlation between chromosomal aberrations and sister-chromatid exchanges (SCE) in cells treated in G1 phase with X-rays or DNaseI. Materials and methods : Chinese hamster ovary (CHO) cells were labelled with 5'-bromodeoxyuridine (BrdU) for one round of replication and irradiated in G1 phase with 1.2, 2.4, 3.6 and 4.8 Gy X-rays or treated by electroporation with 1000, 2000, 3000 and 4000 units DNaseI per 800 μ l electroporation buffer. Using a computer-aided metaphase relocation system first post-treatment mitoses were analysed for SCE and chromosomal aberrations allowing a precise investigation of correlation between both phenomena. Results : A better correlation between aberrations and SCE was observed for DNaseI than for X-rays. X-rays induced more SCE than expected on the basis of aberrations, whereas the frequencies of SCE induced by DNaseI can mainly be accounted for by chromosomal aberrations. Discussion : The results obtained with X-rays support an earlier observation that radiation induces both 'true' SCE that are not related to chromosomal aberrations and result from radiation damage to BrdU, and 'false' SCE that result from exchange-type aberrations. The high frequency of aberrations observed in cells treated with DNaseI and the good correlation between aberrations and SCE suggests that the endonuclease induces mainly 'false' SCE.     

低氧时蛋白激酶C对内皮细胞表达血管内皮生长因子的调节作用

目的探讨低氧培养大鼠肺动脉血管内皮细胞生长因子 (VEGF)表达变化与蛋白激酶C(PKC)活性的关系。方法培养大鼠肺动脉血管内皮细胞 ,观察低氧 ( 1 %O2 )培养 0、1、3、6、1 2h大鼠肺动脉血管内皮细胞PKC活性和VEGFmRNA水平变化 ;同时对培养液中VEGF蛋白水平进行测定。培养基中加入PKC抑制剂 (staurosporine)后 ,立即进行低氧培养 ,测定低氧培养不同时间点上述指标的变化。 结果低氧培养 1hPKC活性首先明显升高 (P >0 .0 5 ) ,至 3hVEGFmRNA表达明显升高 (P <0 .0 1 ) ,6h培养液中VEGF蛋白水平显著升高 (P <0 .0 1 )。而加入PKC抑制剂后 ,低氧培养的内皮细胞PKC活性与 0h比较明显下降 (P <0 .0 1 ) ,相应各时间点的VEGFmRNA及蛋白水平与 0h比较无明显变化 (P >0 .0 5 )。结论低氧能够刺激大鼠肺动脉血管内皮细胞VEGF表达升高 ,低氧时PKC活性升高是调节VEGF表达升高的重要因素之一     

Poly(ADP-ribose) polymerase,a major determinant of early cell response to ionizing radiation

Purpose : To determine whether DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP-1) are involved in eliciting the rapid fluctuations of radiosensitivity that have been observed when cells are exposed to short pulses of ionizing radiation. Materials and methods : The effect of DNA-PK and PARP-1 inhibitors on the survival of cells to split-dose irradiation was investigated using Chinese hamster V79 fibroblasts and human carcinoma SQ-20B cells. The responses of PARP-1 proficient and PARP-1 knockout mouse 3T3 fibroblasts were compared in a similar split-dose assay. Results : Inactivation of DNA-PK by wortmannin potentiated radiation-induced cell kill but it did not alter the oscillatory, W-shaped pattern of early radiation response. In contrast, oscillatory radiation response was abolished by 3-aminobenzamide, a reversible inhibitor of enzymes containing a PARP catalytic domain. The oscillatory response was also lacking in PARP-1 knockout mouse 3T3 fibroblasts. Conclusion : The results show that PARP-1 plays a key role in the earliest steps of cell response to ionizing radiation with clonogenic ability or growth as endpoint. It is hypothesized that rapid poly(ADP-ribosylation) of target proteins, or recruitment of repair proteins by activated PARP-1 at the sites of DNA damage, bring about rapid chromatin remodelling that may affect the incidence of chromosomal damage upon re-irradiation.     

Poly(ADP-ribose) polymerase, a major determinant of early cell response to ionizing radiation

PURPOSE: To determine whether DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP-1) are involved in eliciting the rapid fluctuations of radiosensitivity that have been observed when cells are exposed to short pulses of ionizing radiation. MATERIALS AND METHODS: The effect of DNA-PK and PARP-1 inhibitors on the survival of cells to split-dose irradiation was investigated using Chinese hamster V79 fibroblasts and human carcinoma SQ-20B cells. The responses of PARP-1 proficient and PARP-1 knockout mouse 3T3 fibroblasts were compared in a similar split-dose assay. RESULTS: Inactivation of DNA-PK by wortmannin potentiated radiation-induced cell kill but it did not alter the oscillatory, W-shaped pattern of early radiation response. In contrast, oscillatory radiation response was abolished by 3-aminobenzamide, a reversible inhibitor of enzymes containing a PARP catalytic domain. The oscillatory response was also lacking in PARP-1 knockout mouse 3T3 fibroblasts. CONCLUSION: The results show that PARP-1 plays a key role in the earliest steps of cell response to ionizing radiation with clonogenic ability or growth as endpoint. It is hypothesized that rapid poly(ADP-ribosylation) of target proteins, or recruitment of repair proteins by activated PARP-1 at the sites of DNA damage, bring about rapid chromatin remodelling that may affect the incidence of chromosomal damage upon re-irradiation.     

Radiation-induced apoptosis in the scid mouse spleen after low dose-rate irradiation

Purpose : To elucidate the process of radioadaptation, the role of DNA-PK activity was examined using the scid mouse defective in DNA-PKcs. Materials and methods : The induction of apoptosis in the spleens of the C.B-17 Icr scid mouse and the parental mouse was studied after chronic irradiation with γ-rays at 1.5 Gy (0.001Gy min -1 for 25 h) followed by challenge irradiation with X-rays at 3.0 Gy (1.0 Gy min -1 for 3 min). Results : When the wild-type mouse was previously exposed to chronic irradiation (1.5Gy) at a low dose-rate (0.001 Gy min -1) , apoptosis induced by acute irradiation (3.0 Gy, 1.0Gy min -1) was significantly suppressed, especially in the splenic white pulp. There was no change by acute irradiation after chronic irradiation in the scid mouse, although an effect was detected in the spleen after acute irradiation alone. Conclusions : These data suggest that DNA-PK activity might play a major role in the radioadaptive response following pre-irradiation at a low dose-rate.     

Radiation-induced apoptosis in the scid mouse spleen after low dose-rate irradiation

PURPOSE: To elucidate the process of radioadaptation, the role of DNA-PK activity was examined using the scid mouse defective in DNA-PKcs. MATERIALS AND METHODS: The induction of apoptosis in the spleens of the C.B-17 Icr scid mouse and the parental mouse was studied after chronic irradiation with gamma-rays at 1.5 Gy (0.001 Gy min(-1) for 25 h) followed by challenge irradiation with X-rays at 3.0 Gy (1.0 Gy min(-1) for 3 min). RESULTS: When the wild-type mouse was previously exposed to chronic irradiation (1.5 Gy) at a low dose-rate (0.001 Gy min(-1)), apoptosis induced by acute irradiation (3.0 Gy, 1.0 Gy min(-1)) was significantly suppressed, especially in the splenic white pulp. There was no change by acute irradiation after chronic irradiation in the scid mouse, although an effect was detected in the spleen after acute irradiation alone. CONCLUSIONS: These data suggest that DNA-PK activity might play a major role in the radioadaptive response following pre-irradiation at a low dose-rate.     

Induction and detection of bystander effects after combined treatment of cells with 5-bromo-2'-deoxyurine, Hoechst 33 258 and ultraviolet A light

PURPOSE: A combined treatment of cells with 5-bromo-2'-deoxyurine (BrdU), Hoechst 33 258 and ultraviolet A (UVA) light was used to introduce chromosomal aberrations in cells for the study of bystander effects in non-labelled cells. MATERIALS AND METHODS: Mixtures of BrdU-labelled and non-labelled Chinese hamster cells (V79) in S phase were exposed to Hoechst 33 258 and/or UVA light. Metaphase cells were collected and analysed for chromosomal aberrations by Giemsa staining. BrdU immunostaining was performed to verify BrdU incorporation in the cells. RESULTS: Combined treatment with BrdU, Hoechst dye and UVA light induced reduced cell survival and increased chromosomal aberrations, whereas treatment with Hoechst 33 258 and/or UVA light had no effect on cells. Elevated frequencies of chromosomal aberrations were found in non-labelled cells mixed with BrdU-labelled cells and exposed to Hoechst dye and UVA light, suggesting the induction of bystander effects by damaged BrdU-labelled cells. These bystander clastogenic effects were also observed in non-labelled cells mixed with dying cells, indicating a contribution of dying cells in the induction of the bystander effects. CONCLUSIONS: The combined treatment with BrdU, Hoechst 33 258 and UVA light is a valid method for the study of bystander effects as it enables both induction of DNA damage and discrimination of targeted cells and bystander cells.     

Ageing and local growth factors in muscle

Muscle responds to mechanical overload by increasing its size. In contrast, as a muscle gets older it atrophies. The mechanisms regulating these differing responses are not fully understood. Animal studies have shown that older muscles are less well able to repair following contraction-induced injury than young muscles. It is becoming clear that local growth factors produced within the muscle may play important roles in both repair, adaptation and ageing. The growth hormone/insulin like growth factor 1 (GH/IGF-I) axis is important during growth and development, but circulating levels of these hormones decline in later life. However, many tissues including muscle, produce IGF-I for autocrine and paracrine actions. Genetic manipulation of IGF-I in muscle has shown that it has considerable anabolic affects on muscle both in young and old animals. Insulin like growth factor 1 exists in multiple isoforms and one isoform, which differs from the systemic or liver type (IGF-IEa), appears to be particularly sensitive to mechanical signals and to muscle damage. This isoform (IGF-IEc) has been termed mechano growth factor (MGF). The anabolic actions of IGF-I and MGF are through stimulating protein synthesis and by playing a role in the activation, proliferation and differentiation of satellite cells. These effects are discussed in relation to human studies of muscle adaptation to strength training in older people who seem to retain an ability to increase muscle mass and strength through this type of exercise.     

脑生素促海马细胞生长的扫描电镜研究

 为观察脑生素(BB)对培养神经细胞的促生长和促分裂作用,将生后2～3 d的大鼠海马细胞分离并培养在DMEM培养基上,实验分为加BB和未加BB对照组,采用扫描电镜技术观察了BB促海马培养细胞(神经细胞和胶质细胞)发育过程中的形态特征.结果表明:经25 d培养,加BB的海马培养神经细胞和胶质细胞较对照组更为活跃,证实BB对神经细胞的生长有明显的促进作用.     

Mutagenic effect of low energy neutrons on human chromosome 11

PURPOSE: The shape of the dose-effect curve for neutrons, i.e. the question as to whether the curve is linear or supralinear in the low-dose region, is still not clear. Therefore, the mutagenic effect of very low doses of low-energy neutrons was determined. MATERIALS AND METHODS: Human-hamster hybrid A(L) cells contain human chromosome 11, which expresses the membrane protein CD59. This membrane protein can be detected immunologically and quantified by flow cytometry. The A(L) cells were irradiated with neutrons of 0.565, 2.5 or 14.8 MeV and the results were compared with those after 200 kVp X-rays. Before irradiation, cells spontaneously mutated in the CD59 gene were removed by magnetic cell sorting (MACS). RESULTS: The relative biological effectiveness (RBE) for CD59 mutation induction was 19.8 (+/-2.7) for 0.565 MeV, 10.2 (+/-1.9) for 2.5 MeV, and 10.2 (+/-1.6) for 14.8 MeV neutrons. Linear mutation responses were obtained with all radiations except for 14.8 MeV neutrons where a supralinear curve may be a better fit. The deletion spectrum of mutated cell clones showed 29 Mbp deletions on average after irradiation with 0.069 Gy of 0.565 MeV neutrons. This scale of deletions is similar to that after 3 Gy 100 kV X-rays (=34 Mbp). For 50% cell survival, the RBE of the neutrons was 11 compared with 200 kV X-rays. CONCLUSIONS: Neutrons of low energies (0.565 or 2.5 MeV) produce a linear dose-response for mutation in the tested dose range of 0.015-0.15 Gy. The neutron curve of 14.8 MeV can be approximated by a curvilinear or linear function.     

醛糖还原酶抑制剂对糖尿病大鼠肾小球蛋白激酶C活性的影响

目的 探讨醛糖还原酶抑制剂Sorbinil对糖尿病大鼠肾小球蛋白激酶C活性的影响。方法 采用腹腔内注射链脲菌素法制作糖尿病大鼠模型，将大鼠分为模型组、Sorbinil组、格列喹酮 苯那普利组和正常对照组。8周后处死大鼠，分离肾小球，分别测定各组肾小球细胞浆和细胞膜蛋白激酶C活性。结果 糠尿病模型组细胞浆蛋白激酶C活性显著降低，细胞膜蛋白激酶C活性显著升高。用两组西药治疗后，苯那普利 格列奎酮组无明显改善，而Sorbinil组可显著提高细胞浆蛋白激酶C活性、降低细胞膜蛋白激酶C活性。结论 Sorbinil可通过改善多元醇代谢而降低蛋白激酶C活性，同时，亦或存在其他途径如直接对蛋白激酶C的抑制作用等等。Sorbinil通过调节多元醇代谢等途径间接或直接降低蛋白激酶C活性，是其治疗糖尿病肾病的作用机理之一。