Changes in G1-phase populations in human glioblastoma and neuroblastoma cell lines influence p66/Be neutron-induced micronucleus yield

Some photon resistant tumours are sensitive to neutrons but no predictive methods exist which could identify such tumours. In a recent study addressing this clinically important issue, we demonstrated that relative biologic effectiveness (RBE) values for p(66)/Be neutrons estimated from micronucleus (MN) data correlate positively with RBE values obtained from conventional clonogenic survival data. However, not all photon-resistant cell lines showed high RBE values when the MN endpoint was used. Now, we examine how the functional status of the p53 tumour suppressor gene and radiation-induced changes in cell cycle phase populations may contribute to this discrepancy. No significant association was established between p53 status and MN yield for both photon and neutron irradiation. The data demonstrated that neutron-, but not photon-, induced MN yield is dependent on the intrinsic ability of cells to activate a G1-phase arrest. In cell lines of comparable photon sensitivity, those showing more extensive depletion of the G1 population express significantly more micronuclei per unit dose of neutrons. These results suggest that differences in cell cycle kinetics, and not the p53 status, may constitute an important factor in damage induction by high linear energy transfer (LET) irradiation and need to be considered when radiation toxicity in clinical radiobiology or radiation protection is assessed using damage endpoints.     

Use of the cytokinesis-block micronucleus assay to measure radiation-induced chromosome damage in lymphoblastoid cell lines

To establish the optimal experimental conditions for the use of the micronuclei (MN) test to determine the level of chromosomal damage induced by ionising radiation (IR) exposure in lymphoblastoid cell lines, a time-course study was performed comparing a normal and an ataxia telangiectasia (AT) cell line, the latter being characterised by an extreme radiation sensitivity. Several parameters were analysed: the use of cytochalasin-B (Cyt-B) to quantify MN, the optimum fixation time to measure radiation-induced MN, the most appropriate treatment dose of IR to distinguish between the normal and the radiosensitive cells and the cell-cycle distribution after irradiation. The results obtained showed that the spontaneous as well as the radiation-induced levels of MN were significantly higher in the AT cell compared to the normal cells (P < 0.001 and P = 0.005, respectively). In both cell types the number of radiation-induced MN were lower in the cultures without Cyt-B than those with Cyt-B (P < 0.001), with the AT cells being distinguished in terms of IR-induced MN from the normal cells only with the addition of Cyt-B. The level of MN formation was independent of the dose of Cyt-B used (3 or 6 microg/ml). The optimum time for radiation-induced MN measured was found to be between 48 and 72 h post-irradiation, with 2 and 4 Gy exposures inducing similar levels of MN. However, as the higher dose caused a greater delay in the cell-cycle, treatment with 2 Gy with MN measurement at 48 h in the presence of 3 microg/ml Cyt-B were chosen as the optimum experimental conditions. This choice was validated using two additional normal and AT cell lines. In conclusion, our results show that the use of Cyt-B increases the sensitivity of the MN test for comparing differences in radiosensitivity between lymphoblastoid cell lines.     

Sanguinarine causes DNA damage and p53-independent cell death in human colon cancer cell lines

The benzophenanthridine alkaloid sanguinarine has antimicrobial and possibly anticancer properties but it is not clear to what extent these activities involve DNA damage. Thus, we studied its ability to cause DNA single and double strand breaks, as well as increased levels of 8-oxodeoxyguanosine, in human colon cancer cells and found DNA damage consistent with oxidation. Since the tumor suppressor p53 is frequently involved in inducing apoptosis following DNA damage we investigated the effect of sanguinarine in wild type, p53-mutant and p53-null colon cancer cell lines. We found them to be equally sensitive to this plant compound, indicating that cell death is not mediated by p53 in this case. In addition, our observation that apoptosis induced by sanguinarine is initiated very rapidly raised the question whether there is enough time for cellular signaling in response to DNA damage. Moreover, the abundance of double strand breaks is not consistent with only oxidative damage to DNA. We conclude that the majority of DNA double strand breaks in sanguinarine-treated cells are likely the result, rather than the cause, of apoptotic cell death and that apoptosis induced by sanguinarine is independent of p53 and most likely independent of DNA damage.     

Maintenance of G1 Checkpoint Controls in Telomerase-Immortalized Endothelial Cells

Here we report the characterization of a series of telomerase-immortalized human umbilical vein endothelial cell lines (i-HUVEC). These cells maintain endothelial characteristics such as marker expression, dependence on basic fibroblast growth factor for proliferation, and the ability to form tube structures on Matrigel. In addition, these cells do not show signs of tumorigenic transformation because their growth is contact-inhibited, serum-dependent, and anchorage-dependent. In addition, i-HUVEC do not grow or survive when implanted subcutaneously in immunocompromised mice. Notably, the i-HUVEC lines maintain normal p53-dependent checkpoint control, inducing expression of p21Cip1/Waf1 in response to DNA damage. These cells subsequently decrease phosphorylation of pRb and arrest in G1. Furthermore, the i-HUVEC lines maintain normal p53-independent checkpoint control, inducing expression of p27Kip1 in response to lovastatin treatment, with a subsequent decrease in pRb phosphorylation. Lovastatin-treated i-HUVEC lines undergo a G1 arrest that can be reversed with comparable kinetics to that of low passage HUVEC. Together these data demonstrate that telomerase-immortalized endothelial cells can retain normal phenotypes and cell cycle regulation. This result could have significant implications in the study of angiogenic processes such as tumor growth, wound healing, and the vascularization of engineered tissue.     

Aberrant regulation and function of wild-type p53 in radioresistant melanoma cells.

Sporadic human tumors and the hereditary cancer predisposition syndrome Li-Fraumeni are frequently associated with mutations in the p53 tumor suppressor gene that compromise its ability to function as a DNA damage checkpoint. A subset of Li-Fraumeni patients with wild-type p53 alleles have mutations in chk2/hcds1, one of the genes signaling the presence of DNA damage to the p53 protein. This suggests that p53 may be kept inactive in human cancer by mutations targeting DNA damage signaling pathways. Melanoma cells are highly radioresistant, yet they express wild-type p53 protein, raising the possibility of defects in the pathways that activate p53 in response to DNA damage. We have described a chk2/hcds1-independent DNA damage signaling pathway that targets Ser-376 within the COOH terminus of p53 for dephosphorylation and leads to increased p53 functional activity. We now report that in several human melanoma cell lines that express wild-type p53, the phosphorylation state of Ser-376 was not regulated by DNA damage. In these cell lines, neither the endogenous wild-type p53 protein nor high levels of ectopic wild-type p53 led to cell cycle arrest or apoptosis. Thus, defective activation of p53 in response to DNA damage may underlie the radioresistance of human melanoma cells.     

Proteasome-dependent degradation of cyclin D1 in 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell cycle arrest

1-Methyl-4-phenylpyridinium ion (MPP(+)), an active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, induces cell death and inhibition of cell proliferation in various cells. However, the mechanism whereby MPP(+) inhibits cell proliferation is still unclear. In this study, we found that MPP(+) suppressed the proliferation with accumulation in G(1) phase without inducing cell death in p53-deficient MG63 osteosarcoma cells. MPP(+) induced hypophosphorylation of retinoblastoma protein and rapidly down-regulated the protein but not mRNA levels of cyclin D1 in MG63 cells. The down-regulation of cyclin D1 protein was suppressed by a proteasome inhibitor, MG132. The cyclin D1 down-regulation by MPP(+) was also observed in p53-positive PC12, HeLa S3, and HeLa rho(0) cells, which are a subclone of HeLa S3 lacking mitochondrial DNA. Moreover, MPP(+) dephosphorylated Akt in PC12 cells, which was rescued by the pretreatment with nerve growth factor. In addition, the pretreatment with nerve growth factor or lithium chloride, a glycogen synthase kinase-3beta inhibitor, suppressed the cyclin D1 down-regulation caused by MPP(+). Our results demonstrate that MPP(+) induces cell cycle arrest independently of its mitochondrial toxicity or the p53 status of the target cells, but rather through the proteasome- and phosphatidylinositol 3-Akt-glycogen synthase kinase-3beta-dependent cyclin D1 degradation.     

Estimation of genetic effects of chronic exposure to low-dose rate gamma-radiation by cytogenetic methods and DNA-comet assay

The study of genetic effects in CBA/lac mice exposed for 1 year to constant low dose-rate gamma-radiation at a dose-rate 63 cGy/year has been carried out. We have shown the significant increase in the DNA breaks' level in spleen lymphocytes by comet-assay beginning from the total absorbed dose of 20 cGy. It is possible that the DNA breaks' level increase resulted from the structural rearrangement of chromatin or induction of lymphocyte proliferation. The results obtained by micronucleus test have proved that the mutagenic effect of chronic low dose-rate gamma-radiation depends on cell type and respectively on cell proliferation rate, cell differentiation, etc. So, by the end of experiment the significant increase in the frequency of PCE with micronuclei (MN) was observed. However, in contrast, the frequency of NCE with MN was not increased. No significant increase in the percent of lung cells with MN was registered.     

Effect of G-rich oligonucleotides on the proliferation of leukemia cells and its relationship with p53 expression

G-rich oligonucleotides (GROs) can inhibit cell proliferation by inducing cell cycle arrest at S phase in tumor cell lines. GROs bind specific cellular proteins, such as nucleolin, a crucial protein interacting with P53; however, little is known about the relationship between GROs and P53. In this study, we have shown that GROs inhibited the proliferation of U937 cells (a human monocytic leukemia cell line without P53 expression) by inducing S-phase arrest. We also showed that GRO colocalized with nucleolin in U937 cells. GRO treatment induced alteration of a series of cell cycle regulatory proteins in U937 cells. Increased Cdk2 expression might promote the cells to enter S phase and subsequent decrease of Cdk2 might induce cell cycle arrest in S phase. Transfection of U937 cells with a wild-type p53 gene caused the formation of nucleolin-P53 complex, which alleviated the effect of GRO on leukemia cells. This alleviated effect is probably due to the decreased uptake of GRO.     

The induction of micronuclei in human lymphocytes by low doses of radiation

The appearance of micronuclei (MN) is delayed with respect to cell division in populations of irradiated human lymphocytes, so that the length of time in culture, as well as the number of divisions, is a factor in MN assays. Using two assays that control for cell kinetics, we measured the yield of cells with MN exposed to graded doses of 60Co gamma rays and 90KVP X-rays. The yields showed a non-linear increase with dose. They can be represented by two straight lines: the one in the range below 0.15 Gy has a slight slope, the other in the range above 0.15 Gy has a significantly greater slope. The radical scavengers cysteamine and glycerol, which reduced the MN yields sharply at 3 Gy, were less effective at 0.3 Gy, indicating that terminal deletions arising from the direct ionization of DNA are a major source of the MN induced by low radiation doses. It is likely that the non-linear dose response is due to the saturation of a DNA repair process.     

Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.     

Cytotoxicity of azadirachtin A in human glioblastoma cell lines

The neem toxin azadirachtin A exhibits selective toxicity on insects. Despite its well-proven efficacy, the mode of action of this toxin remains obscure. The toxicity on vertebrate cells compared to insect cells is also not well characterized. We have cultivated six human glioblastoma cell lines G-28, G-112, G-60 (TP53 mutant) and G-44, G-62, G-120 (TP53 wild-type) in the presence of 28 microM of azadirachtin. This toxin concentration was chosen because it represents the 25 to 50% lethal dose in the glioma cells. Toxicity was measured in terms of cell proliferation (binucleation index), formation of micronuclei and cell survival. In the TP53 mutant cell lines, azadirachtin reduced the proportion of dividing cells and induced formation of micronuclei. Except for G-44 which showed a decrease in binucleation index, proliferation in the TP53 wild-type cell lines was unaffected by azadirachtin. In the TP53 wild-type cell lines, the decrease in micronuclei frequency is attributed to fewer cells entering mitosis to produce micronuclei. This is also apparent from the low surviving fractions. Cell survival was suppressed by 25-69% in all cell lines. The reduction of cell survival is a clear indication that azadirachtin affects reproductive integrity and cell division. The induction of micronuclei reflects DNA damage. Similar studies on damage induction in insect cell lines could elucidate the processes which precede the antifeedant and antimoulting effects of azadirachtin and other neem toxins in insects.     

p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction.

The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events. Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA. In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity. In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination. Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene.     

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII−/− transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.     

siRNA-mediated type 1 insulin-like growth factor receptor silencing induces chemosensitization of a human liver cancer cell line with mutant P53

Overexpression of type 1 insulin-like growth factor receptor (IGF1R) contributes to the progression and metastasis of liver cancer, implying that IGF1R gene is a suitable target of RNA interference (RNAi) for liver cancer therapy. To investigate the possible regulation of IGF1R by P53, we examined the level of IGF1R expression in liver cancer cell lines in response to adriamycin. Levels of IGF1R mRNA and protein in cell lines with wild-type P53 decreased dramatically after P53 induction, but no such reduction of IGF1R was observed in cell lines with mutated P53. Inhibition of wild-type P53 in HEPG2 cells by small interfering RNA (siRNA) significantly upregulated the expression of IGF1R. IGF1R inhibition by siRNA in Huh7 cells with mutated P53 significantly depressed cell proliferation. To investigate the sensitivity of cancer cells to adriamycin after inhibition of IGF1R, we depressed IGF1R expression using siRNA, and then added adriamycin at an IC50 dose. After a further 48 h incubation with adriamycin, proliferation was significantly depressed in the cells treated with siRNA targeting IGF1R, in comparison with siRNA targeting scramble. Furthermore, both TUNEL and pro-caspase-3 expression assay showed a significant increase in apoptosis after combined treatment with adriamycin and siRNA targeting IGF1R. Our results demonstrate that IGF1R is downregulated by P53, and that siRNA targeting of IGF1R increases liver cancer cells sensitivity to adriamycin and promotes apoptosis. siRNA targeting of IGF1R could be potentially useful for increasing sensitivity to anti-cancer drugs, especially in drug-resistant cells with mutated P53.