The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.     

神经酰胺诱导药物敏感型和耐药型细胞凋亡机制的探讨

以药物敏感型细胞株Ｋ５６２／Ｓ和耐药型细胞株Ｋ５６２／Ａ０２为对象．观察原癌基因Ｂｃｌ－２的表达量在两种细胞中的差异,以及神经酰胺作为一个新的脂质第二信使诱导细胞凋亡的能力,并利用酪氨酸激酶抑制剂ｇｅｎｉｓｔｅｉｎ,酪氨酸磷酸酯酶抑制剂ｖａｎａｄａｔｅ,观察酪氨酸可逆磷酸化与细胞凋亡间的关系．结果显示：在Ｋ５６２／Ａ０２中Ｂｃｌ－２的表达量明显高于Ｋ５６２／Ｓ;外源性神经酰胺能成功地诱导Ｋ５６２／Ｓ,Ｋ５６２／Ａ０２细胞凋亡,凋亡细胞具有典型的形态学改变和ＤＮＡ“Ｌａｄｄｅｒ”形成,ＦＣＭ检测出现凋亡细胞峰,但在同样的诱导条件下,Ｋ５６２／Ｓ细胞凋亡明显高于Ｋ５６２／Ａ０２细胞．ＦＣＭ检测ｇｅｎｉｓｔｅｉｎ能显著改变这两种细胞生长周期,但细胞阻滞于Ｇ２／Ｍ期,便对神经酰胺诱导的细胞凋亡无明显作用,ｖａｎａｄａｔｅ单独对细胞地明显作用,但与神经酰胺共同作用能明显提高细胞凋亡率．以上结果表明在药物诱导的细胞调亡中Ｂｃｌ－２基因起重要作用,神经酰胺能诱导Ｋ５６２／Ｓ和Ｋ５６２／Ａ０２细胞调亡．     

Selective induction of apoptosis in Philadelphia chromosome-positive chronic myelogenous leukemia cells by an inhibitor of BCR - ABL tyrosine kinase, CGP 57148

The BCR - ABL tyrosine kinase has been implicated as the cause of Philadelphia chromosome (Ph1)-positive leukemias. We report herein that CGP 57148, a selective inhibitor of the ABL tyrosine kinase, caused apoptosis specifically in bcr - abl-positive chronic myelogenous leukemia (CML) cells, K562 and KYO-1. Upon treatment with CGP 57148, CRKL, a specific substrate for BCR - ABL that propagates signals via phosphatidylinositol-3' kinase (PI3K), was dephosphorylated, indicating inhibition of BCR - ABL tyrosine kinase at the cellular level. Likewise, MAPK/ERK, a downstream mediator of Ras, was also dephosphorylated. Caspase activation and cleavage of retinoblastoma protein (pRB) accompanied the development of CGP 57148-induced apoptosis. Inhibition of caspase suppressed apoptosis and the cleavage of pRB, and in turn arrested cells in the G1 phase. These results indicate that CGP 57148 shows apoptogenic and anti-proliferative effects on bcr - abl-positive cells by blocking BCR - ABL-initiated signaling pathways.     

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.     

Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance

The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.     

Purvalanol A enhances cell killing by inhibiting up-regulation of CDC2 kinase activity in tumor cells irradiated with high doses of X rays

To clarify the relationship between CDC2 kinase activity and radiation-induced apoptosis, we examined whether the cyclin-dependent kinase (CDK) inhibitor purvalanol A enhanced radiation-induced apoptosis in gastric tumor cells. MKN45 cells exposed to 20 Gy of X rays increased the CDC2 kinase activity and the expression of regulatory proteins (phospho-CDC2 and cyclin B1) of the G2/M phase, followed by activation of the G2/M checkpoint, whereas the treatment of X-irradiated MKN45 cells with 20 microM purvalanol A suppressed the increase in the CDC2 kinase activity and expression of the G2/M-phase regulatory proteins and reduced the fraction of the cells in the G2/M phase in the cell cycle. Furthermore, this treatment resulted in not only a significant increase in radiation-induced apoptosis but also the loss of clonogenicity in both MKN45 (p53-wild) and MKN28 (p53-mutated) cells. The expression of anti-apoptosis proteins, inhibitor of apoptosis protein (IAP) family members (survivin and XIAP) and BCL2 family members (Bcl-X(L) and Bcl-2), in purvalanol A-treated cells with and without X rays was significantly lower than for cells exposed to X rays alone. These results suggest that the inhibition of radiation-induced CDC2 kinase activity by purvalanol A induces apoptosis through the enhancement of active fragments of caspase 3.     

Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis.     

Influence of environmental pH on G2-phase arrest caused by ionizing radiation

We investigated the effects of an acidic environment on the G2/M-phase arrest, apoptosis, clonogenic death, and changes in cyclin B1-CDC2 kinase activity caused by a 4-Gy irradiation in RKO.C human colorectal cancer cells in vitro. The time to reach peak G2/M-phase arrest after irradiation was delayed in pH 6.6 medium compared to that in pH 7.5 medium. Furthermore, the radiation-induced G2/M-phase arrest decayed more slowly in pH 6.6 medium than in pH 7.5 medium. Finally, there was less radiation-induced apoptosis and clonogenic cell death in pH 6.6 medium than in pH 7.5 medium. It appeared that the prolongation of G2-phase arrest after irradiation in the acidic environment allowed for greater repair of radiation-induced DNA damage, thereby decreasing the radiation-induced cell death. The prolongation of G2-phase arrest after irradiation in the acidic pH environment appeared to be related at least in part to a prolongation of the phosphorylation of CDC2, which inhibited cyclin B1-CDC2 kinase activity.     

New alternative phosphorylation sites on the cyclin dependent kinase 1/cyclin a complex in p53-deficient human cells treated with etoposide: possible association with etoposide-induced apoptosis

Cell cycle arrest is a major cellular response to DNA damage preceding the decision to repair or die. Many malignant cells have non-functional p53 rendering them more “aggressive” in nature. Arrest in p53-negative cells occurs at the G2M cell cycle checkpoint. Failure of DNA damaged cells to arrest at G2 results in entry into mitosis and potential death through aberrant mitosis and/or apoptosis. The pivotal kinase regulating the G2M checkpoint is Cdk1/cyclin B whose activity is controlled by phosphorylation. The p53-negative myeloid leukemia cell lines K562 and HL-60 were used to determine Cdk1 phosphorylation status during etoposide treatment. Cdk1 tyrosine 15 phosphorylation was associated with G2M arrest, but not with cell death. Cdk1 tyrosine 15 phosphorylation also led to suppression of nuclear cyclin B-associated Cdk1 kinase activity. However cell death, associated with broader tyrosine phosphorylation of Cdk1 was not attributed to tyrosine 15 alone. This broader phosphoryl isoform of Cdk1 was associated with cyclin A and not cyclin B. Alternative phosphorylations sites were predicted as tyrosines 4, 99 and 237 by computer analysis. No similar pattern was found on Cdk2. These findings suggest novel Cdk1 phosphorylation sites, which appear to be associated with p53-independent cell death following etoposide treatment.     

The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior to the onset of apoptosis

In contrast to extracellular signals, the mechanisms utilized to transduce nuclear apoptotic signals are not well understood. Characterizing these mechanisms is important for predicting how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death domain-containing protein p84N5 can induce apoptosis that is inhibited by association with Rb. The pattern of caspase and NF-kappaB activation during p84N5-induced apoptosis is similar to p53-independent cellular responses to DNA damage. One hallmark of this response is the activation of a G(2)/M cell cycle checkpoint. In this report, we characterize the effects of p84N5 on the cell cycle. Expression of p84N5 induces changes in cell cycle distribution and kinetics that are consistent with the activation of a G(2)/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine blocks p84N5-induced G(2)/M arrest but not subsequent apoptotic cell death. The p84N5-induced checkpoint is functional in ataxia telangiectasia-mutated kinase-deficient cells. We conclude that p84N5 induces an ataxia telangiectasia-mutated kinase (ATM)-independent, caffeine-sensitive G(2)/M cell cycle arrest prior to the onset of apoptosis. This conclusion is consistent with the hypotheses that p84N5 functions in an Rb-regulated cellular response that is similar to that triggered by DNA damage.     

Fragments of ATM which have dominant-negative or complementing activity.

The ATM protein has been implicated in pathways controlling cell cycle checkpoints, radiosensitivity, genetic instability, and aging. Expression of ATM fragments containing a leucine zipper motif in a human tumor cell line abrogated the S-phase checkpoint after ionizing irradiation and enhanced radiosensitivity and chromosomal breakage. These fragments did not abrogate irradiation-induced G1 or G2 checkpoints, suggesting that cell cycle checkpoint defects alone cannot account for chromosomal instability in ataxia telangiectasia (AT) cells. Expression of the carboxy-terminal portion of ATM, which contains the PI-3 kinase domain, complemented radiosensitivity and the S-phase checkpoint and reduced chromosomal breakage after irradiation in AT cells. These observations suggest that ATM function is dependent on interactions with itself or other proteins through the leucine zipper region and that the PI-3 kinase domain contains much of the significant activity of ATM.     

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.     

Protein kinase Cdelta overexpression enhances radiation sensitivity via extracellular regulated protein kinase 1/2 activation, abolishing the radiation-induced G(2)-M arrest.

Protein kinase C (PKC) has been widely implicated in regulation ofcell growth/cell cycle progression and apoptosis. However,the role of PKCdelta in radiosensitivity and cell cycle regulation remains unclear. Overexpression of PKCdelta increased Ca2+-independent PKC activity without altering other PKC isoforms (PKCalpha, -beta1, -epsilon, and -zeta), and extracellular regulated protein kinase (ERK) 1/2 activity was also increased in PKCdelta-specific manner. A clonogenic survival assay showed that PKCdelta-overexpressed cells had more radiosensitivity and pronounced induction of apoptosis than control cells. Flow cytometric analysis revealed that PKCdelta made the cells escape from radiation-induced G(2)-M arrest. Moreover, p53 and p21(Waf) induction by radiation were higher in PKCdelta-overexpressed cells than control cells, and PKCdelta-mediated apoptosis was reduced, when radiation-induced ERK1/2 activity was inhibited by PD98059. Furthermore, PKCdelta antisense and rottlerin, PKC inhibitor-abrogated PKCdelta-mediated radiosensitivity and reduced ERK1/2 activity to the control vector level. These results demonstrated that PKCdelta overexpression enhanced radiation-induced apoptosis and radiosensitivity via ERK1/2 activation, thereby abolishing the radiation-induced G(2)-M arrest and finally apoptosis.     

PTPROt inactivates the oncogenic fusion protein BCR/ABL and suppresses transformation of K562 cells

Chronic myelogenous leukemia is typified by constitutive activation of the c-abl kinase as a result of its fusion to the breakpoint cluster region (BCR). Because the truncated isoform of protein-tyrosine phosphatase receptor-type O (PTPROt) is specifically expressed in hematopoietic cells, we tested the possibility that it could potentially dephosphorylate and inactivate the fusion protein bcr/abl. Ectopic expression of PTPROt in the chronic myelogenous leukemia cell line K562 indeed resulted in hypophosphorylation of bcr/abl and reduced phosphorylation of its downstream targets CrkL and Stat5, confirming that PTPROt could inactivate the function of bcr/abl. Furthermore, the expression of catalytically active PTPROt in K562 cells caused reduced proliferation, delayed transition from G0/G1 to S phase, loss of anchorage independent growth, inhibition of ex vivo tumor growth, and increased their susceptibility to apoptosis, affirming that this tyrosine phosphatase can revert the transformation potential of bcr/abl. Additionally, the catalytically inactive PTPROt acted as a trapping mutant that was also able to inhibit anchorage independence and facilitate apoptosis of K562 cells. The inhibitory action of PTPROt on bcr/abl was also confirmed in a murine myeloid cell line overexpressing bcr/abl. PTPROt expression was suppressed in K562 cells and was relieved upon treatment of the cells with 5-azacytidine, an inhibitor of DNA methyltransferase, with concomitant hypomethylation of the PTPRO CpG island. These data demonstrate that suppression of PTPROt by promoter methylation could contribute to the augmented phosphorylation and constitutive activity of its substrate bcr/abl and provide a potentially significant molecular therapeutic target for bcr/abl-positive leukemia.     

Inactivation of HDAC3 and STAT3 is Critically Involved in 1-Stearoyl-sn-Glycero-3-Phosphocholine-Induced Apoptosis in Chronic Myelogenous Leukemia K562 Cells

We here investigated the anticancer mechanism of 1-stearoyl-sn-glycero-3-phosphocholine (LPC), one of the lysophosphatidylcholines, in chronic myelogenous leukemia (CML) K562 cells. LPC significantly showed cytotoxicity at 80 μM and induced apoptosis by sub-G1 accumulation, increase in Annexin V positive and caspase activation. LPC enhanced histone H3 acetylation but decreased histone deacetylase (HDAC) activity and HDAC3 expression. LPC also inhibited phosphorylation of STAT3, its DNA binding activity and nuclear co-localization of HDAC3 and STAT3. In addition, LPC effectively attenuated the expression of survival genes such as Cyclin D1, Cyclin E, Bcl-x_L, Bcl-2 and survivin but did not affect COX-2 expression in K562 cells. Furthermore, LPC suppressed phosphorylation of Src and Janus activated kinase 2 while promoted the expression of tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1). Consistently, silencing SHP-1 and pervanadate, an inhibitor of protein tyrosine phosphatase, reversed inactivation of HDAC and STAT3, cleavages of caspase 3 and poly (ADP-ribose) polymerase in LPC-induced apoptosis. Of note, chromatin immunoprecipitation assay revealed that LPC suppressed the binding of HDAC3 and STAT3 to Bcl-x_L, Bcl-2 and survivin promoter. Overall, our findings indicate that inactivation of STAT3 and HDAC mediates LPC-induced apoptosis in CML K562 cells.     

Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase in Schwann cells.

In the nervous system, Src family tyrosine kinases are thought to be involved in cell growth, migration, differentiation, apoptosis, as well as in myelination and synaptic plasticity. Emerging evidence indicates that K+ channels are crucial targets of Src tyrosine kinases. However, most of the data accumulated so far refer to heterologous expression, and native K+-channel substrates of Src or Fyn in neurons and glia remain to be elucidated. The present study shows that a Src family tyrosine kinase constitutively activates delayed-rectifier K+ channels (IK) in mouse Schwann cells (SCs). IK currents are markedly downregulated upon exposure of cells to the tyrosine kinase inhibitors herbimycin A and genistein, while a potent upregulation of IK is observed when recombinant Fyn kinase is introduced through the patch pipette. The Kv1.5 and Kv2.1 K+-channel alpha subunits are constitutively tyrosine phosphorylated and physically associate with Fyn both in cultured SCs and in the sciatic nerve in vivo. Kv2.1- channel subunits are found to interact with the Fyn SH2 domain. Inhibition of Schwann cell proliferation by herbimycin A and by K+-channel blockers suggests that the functional linkage between Src tyrosine kinases and IK channels could be important for Schwann cell proliferation and the onset of myelination.     

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.     

Radiation could induce p53-independent and cell cycle--unrelated apoptosis in 5-fluorouracil radiosensitized head and neck carcinoma cells

The effect of chemoresistance induction in radiosensitivity and cellular behavior after irradiation remains misunderstood. This study was designed to understand the relationship between radiation-induced cell cycle arrest, apoptosis, and radiosensitivity in KB cell line and KB3 subline selected after 5-fluorouracil (5FU) exposure. Exposure of KB cells to 5FU led to an increase in radiosensitivity. G2/M cell cycle arrest was observed in the two cell lines after irradiation. The radioresistant KB cell line reached the maximum arrest two hours before KB3. The cellular exit from this arrest was found to be related to the wild type p53 protein expression induction. After irradiation, only KB3 cell line underwent apoptosis. This apoptosis induction seemed to be independent of G2/M arrest exit, which was carried out later. The difference in radiosensitivity between KB and KB3 subline may result therefore from both a difference in apoptosis induction and a difference in G2/M arrest maximum duration. Moreover, 5FU exposure has led to an increase in constitutive p53 protein expression, which may be associated with an increase in basal apoptosis cell fraction. Given the existing correlation between radiosensitivity and the percentage of basal apoptosis, the constitutive p53 protein expression may be related to intrinsic radiosensitivity in our cellular model.