Involvement of TP53 in apoptosis induced in human lymphoblastoid cells by fast neutrons

We investigated the involvement of TP53 in apoptosis induced by fast neutrons in cells of three human B-lymphoblast cell lines derived from the same donor and differing in TP53 status: TK6 (wild-type TP53), WTK1 (mutant TP53) and NH32 (knockout TP53). Cells were exposed to X rays or to fast neutrons at doses ranging from 0.5 to 8 Gy. Apoptosis was determined by measurements of the sub-G0 /G1-phase DNA content and by the externalization of phosphatidylserine. Fast neutrons induced extensive apoptosis in TK6 cells, as shown by the formation of hypodiploid particles, the externalization of phosphatidylserine, and the activation of caspases. In contrast, cell death was triggered at a significantly lower rate in cells lacking functional TP53. However, TP53-independent cell death also expressed the morphological and biochemical hallmarks of apoptosis. Proliferation tests and clonogenic assays showed that fast neutrons can nevertheless kill WTK1 and NH32 cells efficiently. The absence of functional TP53 only delays radiation-induced cell death, which is also mediated by caspases. These results indicate that fast-neutron irradiation activates two pathways to apoptosis and that the greater relative biological effectiveness of fast neutrons reflects mainly an increase in clonogenic cell death.     

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity.

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.     

Induction of telomerase activity by irradiation in human lymphoblasts

Neuhof, D., Ruess, A., Wenz, F. and Weber, K. J. Induction of Telomerase Activity by Irradiation in Human Lymphoblasts. Radiat. Res. 155, 693-697 (2001). Telomerase activity is a radiation-inducible function, which suggests a role of this enzyme in DNA damage processing. Since the tumor suppressor TP53 plays a central role in the regulation of the cellular response to DNA damage, our study explored the ability of ionizing radiation to change telomerase activity and telomere length in two closely related human lymphoblast cell lines with different TP53 status. TK6 cells (wild-type TP53) and WTK1 cells (mutated TP53) were exposed to different doses of X rays, and telomerase activity was measured by PCR ELISA at different times after irradiation. A dose-dependent increase in telomerase activity was observed. One hour after irradiation with 4 Gy, TK6 and WTK1 cells showed an approximately 2.5-fold increase; for lower doses (0.1 to 1 Gy), telomerase induction was seen only in TK6 cells. Telomerase induction was observed by 0.5 h after irradiation, with a further increase up to 24 h. Irradiated TK6 and WTK1 cells had longer telomeres (+1.3 kb) than unirradiated cells 14 days after exposure. Our data demonstrate a dose-dependent induction of telomerase activity and lengthening of telomeres by ionizing radiation in human lymphoblasts. Induction of telomerase activity by radiation does not generally appear to be controlled by the TP53-dependent DNA damage response pathway. However, for low doses, induction of telomerase requires wild-type TP53.     

Apoptosis induced by MNNG in human TK6 lymphoblastoid cells is p53 and Fas/CD95/Apo-1 related

Agents inducing O(6)-methylguanine (O(6)MeG) in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), are not only highly mutagenic and carcinogenic but also cytotoxic because of the induction of apoptosis. In CHO fibroblasts, apoptosis triggered by O(6)MeG requires cell proliferation and MutSalpha-dependent mismatch repair and is related to the induction of DNA double-strand breaks (DSBs). Furthermore, it is mediated by Bcl-2 degradation and does not require p53 for which the cells were mutated [Cancer Res. 60 (2000) 5815]. Here we studied cytotoxicity and apoptosis induced by MNNG in a pair of human lymphoblastoid cells expressing wild-type p53 (TK6) and mutant p53 (WTK1) and show that TK6 cells are more sensitive than WTK1 cells to cell killing (determined by a metabolic assay) and apoptosis. Apoptosis was a late response observed <24h after treatment and was related to accumulation of p53 and upregulation of Fas/CD95/Apo-1 receptor as well as Bax. The data indicate that MNNG induces apoptosis in lymphoblastoid cells by activating the p53-dependent Fas receptor-driven pathway. This is in contrast to CHO fibroblasts in which, in response to O(6)MeG, the mitochondrial damage pathway becomes activated.     

Deficiencies of double-strand break repair factors and effects on mutagenesis in directly gamma-irradiated and medium-mediated bystander human lymphoblastoid cells

Using RNA interference techniques to knock down key proteins in two major double-strand break (DSB) repair pathways (DNA-PKcs for nonhomologous end joining, NHEJ, and Rad54 for homologous recombination, HR), we investigated the influence of DSB repair factors on radiation mutagenesis at the autosomal thymidine kinase (TK) locus both in directly irradiated cells and in unirradiated bystander cells. We also examined the role of p53 (TP53) in these processes by using cells of three human lymphoblastoid cell lines from the same donor but with differing p53 status (TK6 is p53 wild-type, NH32 is p53 null, and WTK1 is p53 mutant). Our results indicated that p53 status did not affect either the production of radiation bystander mutagenic signals or the response to these signals. In directly irradiated cells, knockdown of DNA-PKcs led to an increased mutant fraction in WTK1 cells and decreased mutant fractions in TK6 and NH32 cells. In contrast, knockdown of DNA-PKcs led to increased mutagenesis in bystander cells regardless of p53 status. In directly irradiated cells, knockdown of Rad54 led to increased induced mutant fractions in WTK1 and NH32 cells, but the knockdown did not affect mutagenesis in p53 wild-type TK6 cells. In all cell lines, Rad54 knockdown had no effect on the magnitude of bystander mutagenesis. Studies with extracellular catalase confirmed the involvement of H2O2 in bystander signaling. Our results demonstrate that DSB repair factors have different roles in mediating mutagenesis in irradiated and bystander cells.     

The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.     

Pifithrin-alpha, an inhibitor of p53, enhances the genetic instability induced by etoposide (VP16) in human lymphoblastoid cells treated in vitro

Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency.The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.     

DNA (cytosine-5)-methyltransferase 1 as a mediator of mutant p53-determined p16<Superscript>ink4A</Superscript> down-regulation

In cancer, gene silencing via hypermethylation is as common as genetic mutations in p53. Understanding the relationship between mutant p53 and hypermethylation of other tumor suppressor genes is essential when elucidate mechanisms of tumor development. In this study, two isogenic human B lymphoblast cell lines with different p53 status include TK6 containing wild-type p53 and WTK1 with mutant p53 were used and contrasted. Lower levels of p16^ink4A protein were detected in WTK1 cells than in TK6 cells, which were accompanied by increased DNA (cytosine-5)-methyltransferase 1 (DNMT1) gene expression as well as hypermethylation of the p16 ^ink4A promoter. siRNA experiments to transiently knock down wild-type p53 in TK6 cells resulted in increase of DNMT1 expression as well as decrease of p16^ink4A protein. Conversely, siRNA knockdown of mutant p53 in WTK1 cells did not alter either DNMT1 or p16^ink4A protein levels. Furthermore, loss of suppression function of mutant p53 to DNMT1 in WTK1 was caused by the attenuation of its binding ability to the DNMT1 promoter. In summary, we provide evidences to elucidate the relationship between mutant p53 and DNMT1. Our results indicate that mutant p53 loses its ability to suppress DNMT1 expression, and thus enhances methylation levels of the p16 ^ink4A promoter and subsequently down-regulates p16^ink4A protein. Z. Guo and M.-H. Tsai contributed equally to this work.     

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.     

The effect of folate deficiency on the cytotoxic and mutagenic responses to ethyl methanesulfonate in human lymphoblastoid cell lines that differ in p53 status

Folic acid deficiency acts synergistically with alkylating agents to increase genetic damage at the HPRT locus in Chinese hamster ovary cells in vitro and in rat splenocytes in vivo. The present studies extend these observations to human cells and, in addition, investigate the role of p53 activity on mutation induction. The human lymphoblastoid cell lines TK6 and WTK1 are derived from the same parental cell line (WI-L2), but WTK1 expresses mutant p53. Treatment of folate-replete or deficient WTK1 and TK6 cells with increasing concentrations (0-50microg/ml) of ethyl methanesulfonate (EMS) resulted in significantly different HPRT mutation dose-response relationships (P<0.01), indicating that folate deficiency increased the EMS-induced mutant frequency in both cell lines, but with a greater effect in TK6 cells. Molecular analyses of 152 mutations showed that the predominant mutation (65%) in both cell types grown in the presence or absence of folic acid was a G>A transition on the non-transcribed strand. These transitions were mainly at non-CpG sites, particularly when these bases were flanked 3' by a purine or on both sides by G:C base pairs. A smaller number of G>A transitions occurred on the transcribed strand (C>T=14%), resulting in 79% total G:C>A:T transitions. There were more genomic deletions in folate-deficient (15%) as compared to replete cells (4%) of both cell types. Mutations that altered RNA splicing were common in both cell types and under both folate conditions, representing 33% of the total mutations. These studies indicate that cells expressing p53 activity exhibit a higher rate of mutation induction but are more sensitive to the toxic effects of alkylating agents than those lacking p53 activity. Folate deficiency tends to reduce toxicity but increase mutation induction after EMS treatment. The p53 gene product did not have a major influence on the molecular spectrum after treatment with EMS, while folate deficiency increased the frequency of deletions in both cell types.     

Role of the mismatch repair system and p53 in the clastogenicity and cytotoxicity induced by bleomycin

The mismatch repair (MMR) system and p53 protein play a pivotal role in maintaining genomic stability and modulate cell chemosensitivity. Aim of this study was to examine the effects of either MMR-deficiency or p53 inactivation, or both, on cellular responses to bleomycin. The MMR-deficient colon carcinoma cell line HCT116 and its MMR-proficient subline HCT116/3-6, both expressing wild-type p53, were transfected with an expression vector encoding a dominant-negative p53 mutant, or with the empty vector. Four transfected clones, having the following phenotypes, MMR-proficient/p53 wild-type, MMR-proficient/p53 mutant, MMR-deficient/p53 wild-type, MMR-deficient/p53 mutant, were subjected to treatment with bleomycin. Loss of MMR function alone was associated with increased resistance to apoptosis, chromosomal damage and inhibition of colony formation caused by bleomycin. Loss of p53 alone resulted in abrogation of G1 arrest and increased sensitivity to apoptosis and chromosomal damage induced by the drug, but did not affect clonogenic survival after bleomycin treatment. Disabling both p53 and MMR function led to abrogation of G1 arrest and to a moderate impairment of drug-induced apoptosis. Chromosomal damage was reduced in the MMR-deficient/p53 mutant clone with respect to the MMR-proficient/p53 wild-type one, when evaluated 48 h after bleomycin treatment, but was comparable in both clones 96 h after drug exposure. Clonogenic survival of the MMR-deficient/p53 mutant clone was similar to that of the MMR-deficient/p53 wild-type one. The effects of MMR-deficiency on cellular responses to bleomycin were confirmed using the MMR-proficient lymphoblastoid cell line TK6 and its MMR-deficient subline MT1, both expressing wild-type p53. In conclusion, our data show that loss of MMR and p53 function exerts opposite and independent effects on apoptosis and chromosomal damage induced by bleomycin. Moreover, inactivation of MMR confers resistance to the cytotoxic activity of the anticancer agent in cells expressing either wild-type or mutant p53.     

High level glucose increases mutagenesis in human lymphoblastoid cells

Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was also investigated by using human lymphoblastoid cell lines derived from same donor but differs in p53 statuses; TK6 has wild-type p53, NH32 has null p53, and WTK1 has mutant p53 (ile237). In addition, we studied the influence of antioxidant treatment on HG-induced mutagenesis. Mutation fractions at both loci increased significantly in all three lines at 21 and 28 days after HG treatments. At tk locus, the increase of a class of mutants with normal growth rate is mainly responsible for the overall increased mutant fraction. Compared to TK6 cells, both NH32 and WTK1 cells showed an early onset of mutagenesis. Treatment of cells with antioxidant N-acetyl-L-cysteine partially reduced HG induced mutagenesis. This study is the first to indicate that HG is able to induce gene mutation which may be one of the important mechanisms of diabetes-associated carcinogenesis.     

The effects of antioxidants on radiation-induced apoptosis pathways in TK6 cells

This study was designed to determine if radiation-mediated activation of the apoptotic pathways would be influenced by antioxidants and if a correlation would be found between radioprotection and changes in transduction pathways. Human lymphoblastoid TK6 cells, known to undergo apoptosis as a result of radiation, were irradiated (6 Gy) with and without antioxidants, and then whole-cell lysates were collected. Parallel studies were conducted to assess the survival (clonogenic assay) and apoptotic index. The impacts of two nitroxide antioxidants, tempol and CAT-1, differing in cell permeability, as well as the sulfhydryl antioxidant N-acetyl-L-cysteine (L-NAC), were estimated. Changes in apoptotic pathway proteins and p53 were assessed by Western blotting. Fraction of apoptotic cells was determined by flow cytometry. Tempol (10 mM), which readily enters cells, partially radioprotected TK6 cells against clonogenic killing, but had no effect on radiation-induced apoptotic parameters such as cleaved caspase 3 or cleaved PARP. Tempol alone did not induce cytotoxicity, yet did increase cleaved PARP levels. The radiation-induced increase in p53 protein was partly inhibited by tempol, but was unaffected by CAT-1 and L-NAC. Both CAT-1 (10 mM), which does not enter cells, and L-NAC (10 mM) had no radioprotective effect on cell survival. Although L-NAC did not protect against radiation-induced cytotoxicity, it completely inhibited radiation-induced increase in cleaved caspase 3 and cleaved PARP. Collectively, the results question the validity of using selected apoptosis pathway members as sole indicators of cytotoxicity.     

The histone deacetylase inhibitor sodium butyrate interacts synergistically with phorbol myristate acetate (PMA) to induce mitochondrial damage and apoptosis in human myeloid leukemia cells through a tumor necrosis factor-alpha-mediated process

Interactions between the histone deacetylase inhibitor sodium butyrate (SB) and phorbol 12-myristate 13-acetate (PMA) were examined in human myeloid leukemia cells (U937 and HL-60). Exposure of U937 cells to 1 mM SB and 1 nM PMA (24 h) markedly induced caspase activation and apoptosis, events accompanied by impaired differentiation induction (e.g., reduced plastic adherence and diminished expression of CD11b) as well as reduced clonogenic survival. The PKC inhibitor GF109203X blocked SB-/PMA-mediated apoptosis. Comparable results were obtained in HL-60 cells. Apoptosis was associated with early procaspase 8 activation and Bid cleavage, accompanied by pronounced mitochondrial damage (e.g., loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release). Neutralization of endogenous TNFalpha by a human soluble TNF receptor substantially blocked SB-/PMA-induced cytochrome c release and apoptosis. Consistent with this, ectopic expression of a mutant dominant-negative caspase 8 or CrmA resulted in a significant decrease in SB-/PMA-induced apoptosis, whereas Bcl-2 overexpression did not. SB/PMA treatment also triggered a decline in the S and G(2)M populations, and dephosphorylation of p34(cdc2). These results indicate that SB interacts with low concentrations of PMA to induce apoptosis in human leukemia cells and that this process proceeds through a PKC-/TNFalpha-dependent pathway in which procaspase 8 and Bid activation play key roles.     

Heterocyclic amine induced apoptotic response in the human lymphoblastoid cell line TK6 is linked to mismatch repair status.

The human lymphoblastoid cell, TK6, exhibited a dose-dependent cytotoxic and apoptotic response following treatment with the food borne heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Augmentation of the p53 protein and increases in p21-WAF1 levels were also observed. Comparison of the survival by clonogenic assays and the percentage of apoptotic cells (cells containing subG1 DNA or condensed nuclei) revealed that only 10-20% of the PhIP-induced cell death could be attributed to apoptosis that occurred in the first 24h after treatment. MT1, a derivative of TK6 that contains mutations in both alleles of its hMSH6 gene and is mismatch repair deficient, showed a decreased apoptotic response. A significant increase (P<0.05) in apoptosis was observed in TK6 and not in MT1 following treatment with 2.5microg/ml PhIP. A five- to six-fold increase and less than a two-fold increase in the fraction of apoptotic cells were observed in TK6 and MT1, respectively. Treatment with 5microg/ml PhIP resulted in significant increases in apoptosis (P<0.05) in TK6 and MT1. The percentages of apoptotic cells were, however, two- to three-fold higher in TK6 than in MT1. HCT116, a hMLH1 defective mismatch repair deficient colorectal carcinoma cell line, also exhibited lower PhIP-induced apoptosis than its mismatch repair proficient chromosome transfer cell line (HCT116+chr3) following PhIP treatment. These results show that PhIP-induced apoptosis is mediated through a mismatch repair dependent pathway. Accumulation of p53 in TK6 and MT1 were evident in samples taken 24h after PhIP treatment. Increases in p21-WAF1 were also observed in both cell lines confirming that the p53 was functional. The lower apoptotic response of MT1 but similar p53 accumulation in TK6 and MT1 suggest that the mismatch repair protein(s) are involved downstream of p53 or that PhIP-induced apoptosis is p53-independent.     

Caspase 2 is both required for p53-mediated apoptosis and downregulated by p53 in a p21-dependent manner

Upon treatment with some DNA damaging agents, human H1299 tumor-derived cells expressing inducible versions of wild-type or mutant p53 with inactive transactivation domain I (p53Q22/S23) undergo apoptosis. In cells expressing either version of p53, caspase 2 activation is required for release of cytochrome c and cell death. Furthermore, silencing of PIDD (a factor previously shown to be required for caspase 2 activation) by siRNA suppresses apoptosis by both wild-type p53 and p53Q22/S23. Despite the finding that caspase 2 is essential for DNA damage-facilitated, p53-mediated apoptosis, induction of wild-type p53 (with or without DNA damage) resulted in a reduction of caspase 2 mRNA and protein levels. In this study we sought to provide a mechanism for the negative regulation of caspase 2 by p53 as well as provide insight as to why p53 may repress a key mediator of p53-dependent apoptosis. Mechanistically, we show that DNA binding and/or transactivation domains of p53 are crucial for mediating transrepression. Further, expression of p21 (in p53-null cells inducibly expressing p21) is sufficient to mediate repression of caspase 2. Deletion of p21 or E2F-1 not only abrogated repression of caspase 2, but also stimulated the expression of caspase 2 above basal levels, implicating the requirement for an intact p21/Rb/E2F pathway in the down-regulation of caspase 2. As this p53/p21-dependent repression of caspase 2 can occur in the absence of DNA damage, caspase 2 repression does not simply seem to be a consequence of the apoptotic process. Down-regulation of caspase 2 levels by p53 may help to determine cell fate by preventing cell death when unnecessary.     

A radiation-induced acute apoptosis involving TP53 and BAX precedes the delayed apoptosis and neoplastic transformation of CGL1 human hybrid cells

Exposing CGL1 (HeLa x fibroblast) hybrid cells to 7 Gy of X rays results in the onset of a delayed apoptosis in the progeny of the cells 10 to 12 cell divisions postirradiation that correlates with the emergence of neoplastically transformed foci. The delayed apoptosis begins around day 8 postirradiation and lasts for 11 days. We now demonstrate that the delayed apoptosis is also characterized by the appearance of approximately 50-kb apoptotic DNA fragments and caspase 3 activation postirradiation. In addition, we confirm that stabilization of TP53 and transactivation of pro-apoptosis BAX also occurs during the delayed apoptosis and show that anti-apoptosis BCL-X(L) is down-regulated. To test whether the delayed apoptosis was due to a nonfunctional acute TP53 damage response in CGL1 cells, studies of acute apoptosis were completed. After irradiation, CGL1 cells underwent an acute wave of apoptosis that involves TP53 stabilization, transactivation of BAX gene expression, and a rapid caspase activation that ends by 96 h postirradiation. In addition, the acute onset of apoptosis correlates with transactivation of a standard wild-type TP53-responsive reporter (pG13-CAT) in CGL1 cells after radiation exposure. We propose that the onset of the delayed apoptosis is not the result of a nonfunctional acute TP53 damage response pathway but rather is a consequence of X-ray-induced genomic instability arising in the distant progeny of the irradiated cells.     

Conflicting effects of caffeine on apoptosis and clonogenic survival of human K1 thyroid carcinoma cell lines with different p53 status after exposure to cisplatin or UVc irradiation

Caffeine has been widely described as a chemo/radiosensitizing agent, presumably by inhibiting DNA repair, and affecting preferentially cells with an altered p53 status. We evaluated the effects of caffeine using isogenic and isophenotypic K1 cells derived from a papillary thyroid carcinoma and displaying either a wild type or a mutated p53 status. Apoptosis and clonogenic survival were examined after exposure of the cells to cisplatin or UVc irradiation. We find that at the most currently used concentration, 2mM, caffeine hinders cisplatin or UVc induced apoptosis in K1 cells. In addition, at this already barely achievable concentration in vivo, caffeine does not decrease their clonogenic survival. Hence in our cellular model, caffeine does not behave as a chemo- or a radiosensitizer. Although surprising, these results (1) are in agreement with the delayed G2/M block caused by caffeine that we previously observed in normal human fibroblasts and K1 cells and (2) allow us to elucidate some discrepancies concerning this molecule throughout the literature such as increase or decrease of apoptosis and clonogenic survival, activation or deactivation of molecules involved in DNA damage repair and proliferation inhibition but accelerated G2/M traverse.