Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G2-phase arrest and subsequent G1-phase arrest: TP53-dependent and TP53-independent roles

Through a detailed study of cell cycle progression, protein expression, and kinase activity in gamma-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G2-phase and subsequent G1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G2 phase, normal cells chronically arrest in the subsequent G1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21(WAF1/CIP1)) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.     

Caffeine induces TP53-independent G(1)-phase arrest and apoptosis in human lung tumor cells in a dose-dependent manner

Caffeine is a model radiosensitizing agent that is thought to work by abrogating the radiation-induced G(2)-phase checkpoint. In this study, we examined the effect that various concentrations of caffeine had on cell cycle checkpoints and apoptosis in cells of a human lung carcinoma cell line and found that a concentration of 0.5 mM caffeine could abrogate the G(2)-phase arrest normally seen after exposure to ionizing radiation. Surprisingly, at a concentration of 5 mM, caffeine not only induced apoptosis by itself and acted synergistically to enhance radiation-induced apoptosis, but also induced a TP53-independent G(1)-phase arrest. Examination of the molecular mechanisms by which caffeine produced these effects revealed that caffeine had opposing effects on different cyclin-dependent kinases. CDK2 activity was suppressed by caffeine, whereas activity of CDC2 was enhanced by suppressing phosphorylation on Tyr15 and by interfering with 14-3-3 binding to CDC25C. These data indicate that the effect of caffeine on cell cycle checkpoints and apoptosis is dependent on dose and that caffeine acts through differential regulation of cyclin-dependent kinase activity.     

Association between TP53 codon 72 single-nucleotide polymorphism and radiation sensitivity of human fibroblasts

Inherent radiosensitivity varies widely between individuals. We hypothesized that amino acid substitution variants in two highly radiation-responsive proteins, TP53 (p53) and CDKN1A (p21, Waf1, Cip1), are associated with and could explain individual variations in radiosensitivity. The two non-synonymous single-nucleotide polymorphisms (SNPs) TP53 codon 72 Arg/Pro G>C and CDKN1A codon 31 Ser/Arg C>A were genotyped in 92 normal fibroblast cell strains of different radiosensitivity. The clonogenic surviving fraction at 2 Gy (SF2) ranged between 0.15 and 0.50 (mean = 0.34, SD = 0.08). The mean SF2 was used to divide the cell strains into radiosensitive (45) and normal groups (47). A significant association was observed between SF2 and the TP53 codon 72 haplotype (C compared to G, P = 0.01). No association was observed between CDKN1A codon 31 haplotype and radiosensitivity (P = 0.86). The variant TP53 Arg72 allele was associated with a decrease in radiosensitivity, presumably due to suboptimal function leading to less stringent control of cell division. We conclude that certain SNPs in susceptible genes can influence cellular radiation response. Such risk alleles could ultimately be used as predictive markers for radiosensitivity to help stratifying individuals during assessment of risk of radiation exposure.     

Wild-type TP53 inhibits G(2)-phase checkpoint abrogation and radiosensitization induced by PD0166285, a WEE1 kinase inhibitor

The WEE1 protein kinase carries out the inhibitory phosphorylation of CDC2 on tyrosine 15 (Tyr15), which is required for activation of the G(2)-phase checkpoint in response to DNA damage. PD0166285 is a newly identified WEE1 inhibitor and is a potential selective G(2)-phase checkpoint abrogator. To determine the role of TP53 in PD0166285-induced G(2)-phase checkpoint abrogation, human H1299 lung carcinoma cells expressing a temperature-sensitive TP53 were used. Upon exposure to gamma radiation, cells cultured under nonpermissive conditions (TP53 mutant conformation) underwent G(2)-phase arrest. However, under permissive conditions (TP53 wild-type conformation), PD0166285 greatly inhibited the accumulation of cells in G(2) phase. This abrogation was accompanied by a nearly complete blockage of Tyr15 phosphorylation of CDC2, an increased activity of CDC2 kinase, and an enhanced sensitivity to radiation. However, under permissive conditions (TP53 wild-type conformation), PD0166285 neither disrupted the G(2)-phase arrest nor increased cell death. The compound inhibited Tyr15 phosphorylation only partially and did not activate CDC2 kinase activity. To understand the potential mechanism(s) by which TP53 inhibits PD0166285-induced G(2)-phase checkpoint abrogation, two TP53 target proteins, 14-3-3rho and CDKN1A (also known as p21), that are known to be involved in G(2)-phase checkpoint control in other cell models were examined. It was found that 14-3-3rho was not expressed in H1299 cells, and that although CDKN1A did associate with CDC2 to form a complex, the level of CDKN1A associated with CDC2 was not increased in response to radiation or to PD0166285. The level of cyclin B1, required for CDC2 activity, was decreased in the presence of functional TP53. Thus inhibition of PD0166285-induced G(2)-phase checkpoint abrogation by TP53 was achieved at least in part through partial blockage of CDC2 dephosphorylation of Tyr15 and inhibition of cyclin B1 expression.     

Low-dose hyper-radiosensitivity: a consequence of ineffective cell cycle arrest of radiation-damaged G2-phase cells

This review highlights the phenomenon of low-dose hyper- radiosensitivity (HRS), an effect in which cells die from excessive sensitivity to small single doses of ionizing radiation but become more resistant (per unit dose) to larger single doses. Established and new data pertaining to HRS are discussed with respect to its possible underlying molecular mechanisms. To explain HRS, a three-component model is proposed that consists of damage recognition, signal transduction and damage repair. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell cycle checkpoint that is specific to cells irradiated in the G2phase. This checkpoint exhibits a dose expression profile that is identical to the cell survival pattern that characterizes HRS and is probably the key control element of low-dose radiosensitivity. This premise is strengthened by the recent observation coupling low- dose radiosensitivity of G2-phase cells directly to HRS. The putative role of known damage response factors such as ATM, PARP, H2AX, 53BP1 and HDAC4 is also included within the framework of the HRS model.     

c-fos expression in human skin fibroblasts by reperfusion after oxygen deficiency: a recovery change of human skin fibroblasts after oxygen deficiency stress

A dramatic and specific induction of c-fos mRNA was observed in human skin fibroblasts in vitro culture by oxygen reperfusion after oxygen deficiency treatment. C-fos mRNA reached a maximum about 30-60 min after oxygen reperfusion and declined to basal level after 120 min. And this phenomenon was duration of oxygen deficiency-dependent, and remarkably observed for 0.5-2 hr of oxygen deficiency. More long duration of oxygen deficiency induced a decreasing tendency of c-fos mRNA overexpression due to essential and irreversible cellular damage. Thus increased c-fos gene expression might be an early event in cellular recovery process in particularly human skin fibroblasts with oxygen deficiency.     

DHT formation and degradation in cultured human skin fibroblasts: DHT accumulation in the genital skin

The conversion of testosterone to dihydrotestosterone (DHT) by 5 alpha-reductase and the interconversion between DHT and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) by 3 alpha-hydroxy-steroid oxidoreductase (3 alpha-HSOR) were studied in fibroblasts derived from the genital skin of 22 males and 6 females, and from the nongenital skin of 19 males and 9 females with normal gonadal function. The formation of DHT from testosterone (5 alpha-reduction) was significantly greater in fibroblasts from genital skin than in those from nongenital skin in both males (2.15 +/- 1.43 vs 0.81 +/- 0.46 pmol/mg protein/h, mean +/- SD, P less than 0.001) and females (2.52 +/- 1.99 vs 0.69 +/- 0.18, P less than 0.01). Furthermore, DHT formation from 3 alpha-diol (3 alpha-HSOR oxidation) was also significantly greater in genital skin fibroblasts than in nongenital skin fibroblasts of males (5.47 +/- 3.37 vs 2.52 +/- 1.74 pmol/mg protein/h, P less than 0.01). However, the degradation of DHT to 3 alpha- and/or 3 beta-diol (3 alpha- and/or 3 beta-HSOR reductions) was not different between genital and nongenital skin fibroblasts of either males or females. Respective ratios of DHT formation to DHT degradation (5 alpha-reduction/3 alpha-HSOR reduction, 3 alpha-HSOR oxidation/3 alpha-HSOR reduction) were also significantly greater (P less than 0.002) in genital skin fibroblasts than in nongenital skin fibroblasts of males. On the other hand, both DHT formation and degradation were not different between male and female genital skin fibroblasts. These results suggest that the increased production of DHT in genital compared to nongenital skin results from increased 5 alpha-reduction and 3 alpha-HSOR oxidation.     

The importance of abrogation of G2-phase arrest in combined effect of TRAIL and ionizing radiation

BACKGROUND: In this work we studied the relationship between the enhanced expression of DR5 receptor and the effect of combination of TRAIL and ionizing radiation on cell cycle arrest and apoptosis induction in human leukemia cell line HL-60. MATERIAL AND METHODS: DR5, APO2.7 and cell cycle were analyzed by flow cytometry. Proteins Bid and Mcl-1 were analyzed by Western-blotting. For clonogenic survival, colony assay on methylcellulose was used. RESULTS: Ionizing radiation caused significantly enhanced positivity of DR5 receptors 24 h after irradiation with high doses (6 and 8 Gy). An increase of DR5 receptor positivity after a dose of 2 Gy was not statistically significant and application of TRAIL 48 h after irradiation did not increase the apoptosis induction. However, a decrease of radiation-induced G(2) phase arrest and an increase of apoptosis were observed when TRAIL was applied 16 h before irradiation with the dose of 2 Gy. Incubation with 6 microg/l TRAIL for 16 h reduced D(0) value from 2.9 Gy to 1.5 Gy. The induction of apoptosis by TRAIL was accompanied by Bid cleavage and a decrease of antiapoptotic Mcl-1 16 h after incubation with TRAIL. CONCLUSION: TRAIL in concentration of 6 microg/l applied 16 h before irradiation by the dose of 1.5 Gy caused the death of 63% of clonogenic tumor cells, similarly as the dose of 2.9 Gy alone, which is in good correlation with the enhanced apoptosis induction.     

Ewing Sarcoma: influence of TP53 Arg72Pro and MDM2 T309G SNPs

The Ewing Sarcoma is an important tumor of bone and soft tissue. The SNPs Arg72Pro of TP53 and T309G of MDM2 have been associated with many cancer types and have been differently distributed among populations worldwide. Based on a case–control design, this study aimed to assess the role of these SNPs in 24 Ewing Sarcoma patients, compared to 91 control individuals. DNA samples were extracted from blood and genotyped for both SNPs by PCR–RFLP and confirmed by DNA sequencing. The results showed an association between the G allele of the T309G and Ewing Sarcoma (P = 0.02). Comparing to the TT carriers, the risk of G allele carriers was 3.35 (95 % CI = 1.22–9.21) with P = 0.02. At the genotypic level, an association of the TT genotype with the control group (P = 0.03) was found. Comparing to the TT genotype, the risk of TG and GG was 2.97 (95 % CI = 1.03–8.58) with P = 0.04 and 5.00 (95 % CI = 1.23–20.34) with P = 0.02, respectively. No associations regarding the Arg72Pro SNP were found. Considering that the T309G has been associated with several types of cancer, including sarcomas, our results indicate that this SNP may also be important to Ewing Sarcoma predisposition.     

Lupeol induces p53 and cyclin-B-mediated G2/M arrest and targets apoptosis through activation of caspase in mouse skin

Lupeol, present in fruits and medicinal plants, is a biologically active compound that has been shown to have various pharmacological properties in experimental studies. In the present study, we demonstrated the modulatory effect of lupeol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced alterations on cell proliferation in the skin of Swiss albino mice. Lupeol treatment showed significant (p < 0.05) preventive effects with marked inhibition at 48, 72, and 96 h against DMBA-mediated neoplastic events. Cell-cycle analysis showed that lupeol-induced G2/M-phase arrest (16-37%) until 72 h, and these inhibitory effects were mediated through inhibition of the cyclin-B-regulated signaling pathway involving p53, p21/WAF1, cdc25C, cdc2, and cyclin-B gene expression. Further lupeol-induced apoptosis was observed, as shown by an increased sub-G1 peak (28%) at 96 h, with upregulation of bax and caspase-3 genes and downregulation of anti-apoptotic bcl-2 and survivin genes. Thus, our results indicate that lupeol has novel anti-proliferative and apoptotic potential that may be helpful in designing strategies to fight skin cancer.     

Prolonged cell cycle arrest in irradiated human diploid skin fibroblasts: the role of nutrient deprivation

Ionizing radiation has been reported to cause an irreversible cell cycle arrest in normal human diploid fibroblasts. However, colony survival assays show that even at high doses of gamma radiation, human diploid fibroblasts do not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling. In this study, we resolve the apparent discrepancy between colony survival assays and the observed radiation-induced prolonged arrest. Using flow cytometry analysis, we have confirmed that human diploid fibroblasts do exhibit a prolonged cell cycle arrest in both G(1) and G(2)/M phases of the cell cycle. However, a single replacement of fresh growth medium stimulated a fraction of the arrested population of cells to transiently re-enter the cell cycle. Daily medium changes stimulated these irradiated human diploid fibroblasts to continue cycling until they were contact-inhibited. Thus the fraction of human diploid fibroblasts which survive radiation exposure and are capable of cycling appears to permanently arrest as a result of nutrient insufficiency. Western blot analysis demonstrated a radiation-induced elevation in TP53 (formerly known as p53) protein levels within 2 h postirradiation, followed by a decrease to levels comparable to those in unirradiated controls. The TP53 and CDKN1A (formerly known as p21) protein levels were indistinguishable after 24 h and remained elevated for a 6-day period of observation in both control and irradiated cultures. Our studies indicate that human diploid fibroblasts are capable of re-entering the cell cycle after exposure to ionizing radiation and that this re-entry is dependent on a constant supply of nutrients provided by fresh medium changes. The fraction of cells capable of resuming cell cycling is consistent with the surviving fraction of cells in colony assays.     

p53-independent regulation of cyclin B1 in normal human fibroblasts during UV-induced G2-arrest

Recently we demonstrated, using normal human fibroblasts (NHFs), that UVc radiation induces a G2/M arrest which was even more pronounced when p53 expression was inhibited. So, the aim of this study was to evaluate in NHFs the relationship between UV-induced G2/M arrest and cyclin B1 regulation and to investigate if p53 could contribute to the cyclin B1 regulation in these conditions. Following exposure of asynchronous NHFs to UV light, we showed that the induced G2/M arrest was accompanied by a dose-dependent down-regulation of cyclin B1 mRNA as evaluated by RT-PCR. Concomitantly, using flow cytometric analysis, we observed a strong accumulation of cyclin B1 protein which was correlated to the apparition of the G2/M arrest. In order to study the contribution of p53 to the cyclin B1 accumulation in response to UV exposure, we inhibited p53 induction using p53 antisense oligonucleotides. We found that the inhibition of p53 protein induction after UV exposure had no effect on the level of cyclin B1 mRNA. Moreover, although inhibition of p53 protein induction increased the number of the cells in the G2-M phase, the mean content of cyclin B1 protein was not augmented in these cells. These results indicate clearly that the induction of p53 protein following UV exposure does not regulate the level of cyclin B1 mRNA or protein in normal cells.     

An association between low-dose hyper-radiosensitivity and the early G2-phase checkpoint in normal fibroblasts of cancer patients

In our previous study, low-dose hyper-radiosensitivity (HRS) effect was demonstrated for normal fibroblasts (asynchronous and G2-phase enriched) of 4 of the 25 cancer patients investigated. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, the study indicated that G2-phase enrichment had no influence on HRS identification. The conclusion contradicts that reported for human tumor cells, and suggests different mechanism of HRS in normal human cells. In the present paper we report, for the first time, the activity of early G2-phase checkpoint after low-dose irradiation in normal fibroblasts of these 4 HRS-positive patients and 4 HRS-negative patients and answer the question regarding the role of this checkpoint in normal human cells. The response of the early G2-phase checkpoint was determined by assessment of the progression of irradiated cells into mitosis using the mitotic marker, phosphorylated histone H3. We found evident differences in the activity of the early G2-phase checkpoint between HRS-positive and HRS-negative fibroblasts. In HRS-positive fibroblasts the checkpoint was not triggered and DNA damage was not recognized after doses lower than 0.2 Gy resulting in HRS response. On the contrary, in HRS-negative fibroblasts the early G2-phase checkpoint was activated regardless of the dose in the range 0.1–2 Gy. In conclusion, although cell cycle phase has no effect on the presence of HRS effect in normal human fibroblasts, the data reported here indicate that HRS response in these cells is associated with the functioning of early G2-phase checkpoint in a threshold-dose dependent manner, similarly as it takes place in most of human tumor and other cells.     

Accumulation of the cell cycle regulators TP53 and CDKN1A (p21) in human fibroblasts after exposure to low- and high-LET radiation

The accumulation of the cell cycle regulators TP53 and CDKN1A (p21/CIP1/WAF1) was investigated after exposure to X rays and carbon ions (170 keV microm(-1)) and xenon, bismuth and uranium ions (8900-15,000 keV microm(-1)) in normal human fibroblasts. The influence of the overall dose and the LET of these radiation types was studied systematically and the kinetics of the cell response was followed up to 24 h after exposure. The accumulation of TP53 protein was dependent on the dose and the LET, and TP53 levels declined to lower levels for all radiation types within 24 h after exposure. CDKN1A levels increased and peaked at 3 to 6 h after exposure. The persisting level of this protein at 24 h was strongly dependent on the dose and the LET for X rays and carbon ions. The exposure to very high-LET ions (8900-15,000 keV microm(-1)) did not lead to a further increase in CDKN1A, suggesting a saturation effect for the induction of this protein. The cellular effects of elevated CDKN1A after particle irradiation are discussed.     

Prolongation of duration of G2 arrest delays and finally blocks entry into M phase in contrast to stable and reversible G1 arrest: study of a G1/G2 temperature-sensitive mutant of rat 3Y1 fibroblasts

Proliferation of 3Y1tsF121 cells was arrested in G1 and G2 phases after a shift up to 39.8 degrees C (restrictive temperature). Both arrests were reversible: after a shift down to 33.8 degrees C (permissive temperature), these cells effectively entered the next phases. However, the entry into M phase of the G2-arrested cells was delayed depending on the time in arrest. The G2-arrested cells finally became incapable of entering M phase with a prolonged incubation at 39.8 degrees C. Under the same condition, G1-arrested cells did not lose their ability to proliferate, and their delay of entry into S phase was slight. Therefore, cells in G2 phase are, in a sense, more unstable than the cells in G1 phase. These results also suggest that the time required for entry into M phase may depend on the preparedness for the initiation of M phase and, that it may be prolonged under the condition where the preparedness for entry into M phase is diminished.     

Analysis of genomic integrity and p53-dependent G1 checkpoint in telomerase-induced extended-life-span human fibroblasts

Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.