Characteristics of p53 protein stabilization in cells of patients with ataxia-telangiectasia after gamma-irradiation

Stabilization of P53 protein in cells isolated from patients with a grave hereditary disease ataxia-telangiectasia (AT), characterized by strongly enhanced sensitivity to ionizing radiation and impairment of cell cycle control after DNA damage, was studied. The level of expression of these reactions by patients may vary, and it tends to be linked with the severity of the disease. In all AT strains studied, both acquired by the authors and obtained from foreign colleagues, we observed the alteration of timing and character of stabilization of P53 protein, after the action of ionizing radiation in sublethal dosage, as compared to that in cells from healthy donor.     

ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53

In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM-/- fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEF(p53-/-). However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21(CIP1) induction, resulting in partial release of the G(2)/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.     

Discrimination of heterozygote currency of ataxia-telangiectasia by indirect immunofluorescent analysis

The qualitative differences in P53 protein stabilization after ionizing irradiation in different doses were found in cells of members of ataxia-telangiectasia (AT) family--proband AT6SP, her sister AT(S)6SP and father AT(F)6SP. The method of indirect immunofluorescence with confocal microscopy was used.     

A defect in DNA topoisomerase II activity in ataxia-telangiectasia cells

DNA topoisomerase type I and II activities were determined by serial dilution in nuclear extracts from control and ataxia-telangiectasia lymphoblastoid cells. Topoisomerase I activity, assayed by relaxation of supercoiled plasmid DNA, was found to be approximately the same in both cell types. In order to remove interference from topoisomerase I, the activity of topoisomerase II was measured by the unknotting of knotted P4 phage DNA in the presence of ATP. The activity of topoisomerase II was markedly reduced in two ataxia-telangiectasia cell lines, AT2ABR and AT8ABR, compared to controls. This reduction in activity was detected with increasing concentration of protein and in time course experiments at a single protein concentration. A third cell line, AT3ABR, did not have a detectably lower activity of topoisomerase II when assayed under these conditions. The difference in topoisomerase II activity in the ataxia-telangiectasia cell lines examined may reflect to some extent the heterogeneity observed in this syndrome.     

Coupling of histone mRNA levels to radioresistant DNA synthesis in ataxia-telangiectasia cells

Cloned genomic DNA for human histone H1, H3 and H4 genes has been used to determine the effects of -radiation on histone mRNA levels and synthesis in ataxia-telangiectasia cells. Synthesis of histone mRNA was determined in cells synchronized with aphidicolin. Effects of irradiation on DNA synthesis and passage through S phase were also monitored. Irradiation was found to slow the passage of control cells through the cell cycle but had no effect on progression of ataxia-telangiectasia cells. H1 and core histone mRNA synthesis was inhibited by radiation in two control cell lines after release from aphidicolin block. No inhibition was observed in one ataxia-telangiectasia cell line and a small degree of inhibition in a second. An increased level of mRNA was observed in both irradiated control and ataxia-telangiectasia cells at 5–7 h post-irradiation compared to unirradiated cells. Similar results were obtained in log phase cells. These results demonstrate that histone mRNA synthesis is radioresistant in ataxia-telangiectasia cells and is coupled to radioresistant DNA synthesis in these cells.     

A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.

Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells. Finally, IR induction of the human GADD45 gene, an induction that is also defective in AT cells, was dependent on wild-type p53 function. Wild-type but not mutant p53 bound strongly to a conserved element in the GADD45 gene, and a p53-containing nuclear factor, which bound this element, was detected in extracts from irradiated cells. Thus, we identified three participants (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.     

Discrimination of heterozygous carriage of ataxia-telangiectasia by the method of indirect immunofluorescent analysis

Qualitative differences of the stabilization of protein P53 were studied by methods of indirect immunofluorescence with the use of confocal microscopy in cells obtained from members of the family of a patient with ataxia-telangiectasia (AT)—the proband AT6SP and her blood relatives: the sister AT(S)6SP and the father AT(F)6SP—at different time periods after irradiation with various doses of ionizing radiation.     

53BP1, an activator of ATM in response to DNA damage

p53 Binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DNA damage checkpoint proteins with C-terminal BRCT domains and is most likely the human ortholog of the budding yeast Rad9 protein, the first cell cycle checkpoint protein to be described. 53BP1 localizes rapidly to sites of DNA double strand breaks (DSBs) and its initial recruitment to these sites has not been shown to be dependent on any other protein. Initially, 53BP1 was thought to be a mediator of DNA DSB signaling, but now it has been shown to function upstream of ataxia-telangiectasia mutated (ATM), in one of at least two parallel pathways leading to ATM activation in response to DNA damage. Currently, only a single tudor and two BRCT domains are recognized in 53BP1; however, their precise functional role is not understood. Elucidating the function of 53BP1 will be critical to understanding how cells recognize DNA DSBs and how ATM is activated.     

Human cytomegalovirus IE1-72 activates ataxia telangiectasia mutated kinase and a p53/p21-mediated growth arrest response

Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type (p53(+/+)) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19(Arf) and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G(1) to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.     

Enhanced expression of procollagenase in ataxia-telangiectasia and xeroderma pigmentosum fibroblasts

Summary Ataxia-telangiectasia and xeroderma pigmentosum are human hereditary diseases in which patients are cancer prone and demonstrate increased sensitivity to DNA damage by ionizing and ultraviolet radiation, respectively. In culture, both ataxia-telangiectasia and xeroderma pigmentosum skin fibroblasts show increased synthesis and secretion of the extracellular matrix proteins fibronectin and collagen. To determine whether these differences in protein production result from fundamental abnormalities in regulation of genes associated with cellular interactions, we compared the effects of trifluoperazine and 12-O-tetradecanoylphorbol-13-acetate on expression of the extracellular matrix-degrading metalloproteinases, procollagenase and prostromelysin, by normal, ataxia-telangiectasia, and xeroderma pigmentosum fibroblasts. After trifluoperazine treatment the overall levels of these metalloproteinases were much greater in three ataxia-telangiectasia cell strains and in cells from xeroderma pigmentosum complementation groups A and D than in normal cells. In contrast, cells from xeroderma pigmentosum complementation group C produced only slightly more procollagenase than normal cells. 12-O-tetradecanoylphorbol-13-acetate also induced higher than normal levels of procollagenase in some ataxia-telangiectasia and xeroderma pigmentosum strains, but less than that induced by trifluoperazine. Because increased extracellular accumulation of matrix-degrading enzymes has long been implicated in metastatic progression, this altered expression of procollagenase and prostromelysin in ataxia-telangiectasia and xeroderma pigmentosum cells could play an important role in the pathogenesis of various tumors in individuals with these genetic diseases. This work was supported by the Office of Health and Environmental Research, U. S. Department of Energy (contract DE-AC03-76-SF01012) (J. A., J. P. M.) and by a Fellowship in Medical Research from the A. P. Giannini/Bank of America Foundation (J. A.). A summary of these results has appeared previously in abstract form (1).     

Defective DNA topoisomerase II in ataxia-telangiectasia cells

A number of characteristics in the human genetic disorder ataxia-telangiectasia are compatible with an alteration to chromatin structure or the recognition of that structure by an enzyme or DNA binding protein. We describe here reduce activity of DNA topoisomerase type II in a number of Epstein Barr Virus-transformed ataxia-telangiectasia lymphoblastoid cell lines. Enzyme activity was reduced 10-fold or greater in 4 out of 5 cell lines compared to controls. In the remaining cell line approximately a 2-3 fold reduction was evident in partially purified extracts. DNA topoisomerase type I activity was found to be the same as controls in all the cell lines. Northern blot analysis revealed that the same level of DNA topoisomerase II mRNA was expressed in ataxia-telangiectasia and control cell lines. The size and amount of the enzyme did not differ appreciably from that observed in control cells. The reduced activity of DNA topoisomerase II in ataxia-telangiectasis cells might be explained by amino acid substitutions, small deletions in DNA or by a defect in post-translational modification in these cells.     

Altered levels of primary antioxidant enzymes in progeria skin fibroblasts

Free radicals are involved in the aging process. In this study, the profile of primary antioxidant enzymes that scavenge reactive oxygen species (ROS) was examined for the first time in human skin fibroblasts from progeria, a premature aging disease. Altered levels of antioxidant enzymes were found in progeria cells. Basal levels of MnSOD were decreased in progeria cells as well as a blunted induction in response to chronic stress. This change may contribute to the accelerated aging process in progeria cells. In contrast, the levels of CuZnSOD showed no progeria-related change. Two H2O2 removing enzymes demonstrated a significant reduction in progeria cells: only 50% of normal CAT activity and 30% of normal GPX activity can be detected in progeria cells. This diminished H2O2 removing capacity in progeria cells may lead to an imbalance of intracellular ROS and therefore may play an important role in the development of progeria.     

High-voltage electron microscopy of human diploid fibroblasts during ageing in vitro. Morphometric analysis of mitochondria

Since recent studies have suggested a diminished mitochondrial functional capacity in late-passage ('old') compared to early-passage ('young') normal fibroblasts and fibroblasts from the Hutchinson-Gilford (progeria) syndrome of premature ageing, we analysed whole-cell preparations on the high voltage electron microscope to look for mitochondrial and related defects. All strains examined showed considerable heterogeneity in cell size and intracellular morphology. Mitochondria were readily seen in all cells, predominantly as long slender rods with frequent branching, but occasional circular and saccular forms were also evident. Various parameters of mitochondrial mass including mean number, weight, and total length of mitochondria per cell weight tended to increase in old and progeria cells, but only the former attained statistical significance due to the heterogeneity and consequent variance. A significant finding was the decreased width of mitochondria in old and progeria cells. Cystic blebs were evident in mitochondria of some cells with an apparent increase in old and progeria fibroblasts. These blebs appeared to be due to weakening of the inner membrane, allowing dilatation of the outer membrane which otherwise appeared intact. The number of osmiophilic inclusions per cell weight, particularly lipofuscin granules and autophagic vacuoles, was significantly increased in old and progeria cells. In conclusion, despite some morphological changes, mitochondria of old and progeria cells maintain a structurally and bioenergetically adequate mass compatible with continued cellular viability.     

Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells.

Considerable evidence supports a defect at the level of chromatin structure or recognition of that structure in cells from patients with the human genetic disorder ataxia-telangiectasia. Accordingly, we have investigated the activities of enzymes that alter the topology of DNA in Epstein Barr Virus-transformed lymphoblastoid cells from patients with this syndrome. Reduced activity of DNA topoisomerase II, determined by unknotting of P4 phage DNA, was observed in partially purified extracts from 5 ataxia-telangiectasia cell lines. The levels of enzyme activity was reduced substantially in 4 of these cell lines and to a lesser extent in the other cell line compared to controls. DNA topoisomerase I, assayed by relaxation of supercoiled DNA, was found to be present at comparable levels in both cell types. Reduced activity of topoisomerase II in ataxia-telangiectasia is compatible with the molecular, cellular and clinical changes described in this syndrome.     

Stem cell depletion in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS or progeria) is a very rare genetic disorder with clinical features suggestive of premature aging. Here, we show that induced expression of the most common HGPS mutation (LMNA c.1824C>T, p.G608G) results in a decreased epidermal population of adult stem cells and impaired wound healing in mice. Isolation and growth of primary keratinocytes from these mice demonstrated a reduced proliferative potential and ability to form colonies. Downregulation of the epidermal stem cell maintenance protein p63 with accompanying activation of DNA repair and premature senescence was the probable cause of this loss of adult stem cells. Additionally, upregulation of multiple genes in major inflammatory pathways indicated an activated inflammatory response. This response has also been associated with normal aging, emphasizing the importance of studying progeria to increase the understanding of the normal aging process.     

Reduced DNA repair in progeria cells and effects of gamma-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells

A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33-50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by gamma-ray irradiation to the cells before UV irradiation. The dose of gamma-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, gamma-ray irradiation further reduced UDS with a lower gamma-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a 'built-in' defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by gamma-ray irradiation.     

Elevated levels of glycoprotein gp200 in progeria fibroblasts

The glycosylation of proteins in fibroblasts from people with the premature ageing disease Hutchinson-Gilford Progeria Syndrome (progeria) was investigated.Protein was prepared from fibroblast cell lines established from skin biopsy taken from progeria patients and control donors. Glycoproteins were labelled by the covalent attachment of the steroid hapten digoxygenin to the sugar group. After separation of total protein by SDS-PAGE and electroblotting onto Immobilon-P, glycoproteins were detected by enzyme immunoassay.We have observed a glycoprotein of M_r 200 kDa which is consistently present in protein preparations from progeria fibroblasts and which is absent, or markedly reduced, in preparations from control fibroblasts. This suggests that it may be useful as a marker for progeria. Similar analysis of progeria lymphoblast and control lymphoblast cultures did not show this altered pattern of glycosylated proteins, indicating that it may be cell-type specific.Glycoproteins were also detected by labelling fibroblastsin vitro with D-[6-³H] glucosamine hydrochloride followed by SDS-PAGE of isolated protein and subsequent fluorography. Profiles of glycoproteins from progeria and control fibroblasts were consistent with those obtained from labelling of carbohydrate groups with digoxygenin. Protease digestion of cell protein verified that the band at M_r 200 kDa contains a protein core.Characteristic features of progeria primarily involve the connective tissue and include wrinkled and loose skin, loss of soft tissue, thin limbs and stiff joints. Death of progeria patients is usually a result of cardiovascular abnormalities. The most consistent manifestations thus involve the connective tissue.The glycoprotein of M_r 200 kDa which we have observed in progeria fibroblastsin vitro could reflect a perturbation in glycosylation which may underly the connective tissue defects seen in progeria.Abbreviations EMEM Eagle's Minimum Essential Medium with Earle's Salts - FCS Fetal Calf Serum - PBS Ca²⁺- and Mg²⁺-free Phosphate-Buffered Saline - PDL Population Doubling Level - SDS Sodium Dodecyl Sulphate - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis     

Cleavage and phosphorylation of XRCC4 protein induced by X-irradiation

We report the p35 and p60 forms of XRCC4 protein, appearing in human leukemia MOLT-4 or U937 cells following X-irradiation or hyperthermia. p35 appeared in conjunction with the cleavage of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the fragmentation of internucleosomal DNA, and was suppressed by Ac-DEVD-CHO. p35 was also produced in vitro by treating MOLT-4 cell lysate with recombinant caspases, suggesting that p35 was a caspase-cleaved fragment of XRCC4 in apoptotic cell death. p60 was sensitive to treatment with phosphatase or wortmannin and was undetectable in M059J cells deficient in DNA-PKcs. However, p60 was found in ataxia-telangiectasia cells after irradiation. These results indicated p60 as a phosphorylated form of XRCC4, requiring DNA-PKcs but not ataxia-telangiectasia mutated (ATM).     

9R穿膜肽可增强P53抗肿瘤效果

为解决P53蛋白难以进入细胞内部发挥治疗作用的瓶颈难题.将p53基因融合插入带有9个精氨酸作为穿膜肽的表达载体中表达融合蛋白CPPs-P53,并与没有穿膜肽的P53蛋白进行比较,利用Western blotting方法检测蛋白的表达情况,MTT及Annexin V/PI双染法检测细胞生长抑制率及细胞凋亡率.Western blotting检测表明已成功在原核表达系统中表达融合蛋白CPPs-P53和P53蛋白,且蛋白纯度均已达到90％以上;MTT检测表明,P53蛋白对肿瘤细胞的生长虽有一定的抑制作用,但融合蛋白CPPs-P53与之相比,对肿瘤细胞生长的抑制效果显著增强,细胞生长抑制率有明显的提升,并且细胞生长抑制率呈现剂量依赖性;Annexin V/PI双染检测细胞凋亡情况也表明P53虽可以在一定程度上诱导肿瘤细胞的凋亡,但与P53蛋白相比较,融合蛋白CPPs-P53诱导的凋亡细胞明显增加,凋亡率是P53蛋白的2～3倍.由此说明在抑制肿瘤细胞的生长和诱导细胞凋亡方面,CPPs-P53比没有穿膜肽的P53蛋白的效果更显著.