Mutated and non-mutated TP53 as targets in the treatment of leukaemia

TP53 is mutated in 10-20% of cases of chronic lymphocytic leukaemia (CLL) and 3-8% of cases of acute myeloid leukaemia (AML). Recently, two classes of compounds that restore the function of p53 in tumours have been described. PRIMA-1 (p53-dependent reactivation and induction of massive apoptosis) restores the wild-type conformation of mutant TP53, whereas RITA (reactivation of p53 and induction of tumour cell apoptosis) increases intracellular levels of p53. We evaluated the effects of RITA alone and in combination with PRIMA-1 or conventional cytostatics on leukaemic cells isolated from AML and CLL patients. AML samples with -17, which are more resistant to daunorubicin and cytarabine compared with samples without -17, were effectively killed by PRIMA-1. RITA, which stabilizes the function of wild-type p53, induced apoptosis in AML cells. In contrast to that seen with PRIMA-1, AML patient samples without -17 were significantly more sensitive to RITA. Similarly, RITA exerted dose-dependent apoptosis and cytotoxicity in CLL cells, which was significantly more pronounced in samples without hemizygous TP53 deletion. Notably, a synergistic effect was observed in all CLL samples with RITA and fludarabine in combination. In both AML and CLL cells exposure to RITA resulted in induction of intracellular p53. We conclude that small molecules targeting p53 might be of clinical importance in the future for treating drug-resistant leukaemia.     

Synergistic antitumor effect of TRAIL and doxorubicin on colon cancer cell line SW480

AIM: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent.Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms.METHODS: SW480 cells were cultured in the regular condition and incubated with different levels of agents.Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT) assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detect apoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.RESULTS: (1) SW480 cells were not sensitive to TRAIL,with IC50>l mg·L^1 and dose-independent cytotoxicity. (2)SW480 cells were sensitive to doxorubicin at a certain degree,with dose-dependent cytotoxicity and IC50=65.25±3.48μmol·L^-1. (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC50 of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 μg·L^-1),combined with subtoxic doxorubicin (0.86 μmol·L^-1), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12±2.67 %, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82±1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (p>0.05).CONCLUSION: SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentra~on of doxorubidn to induce apoptosis effectively. The status of p53 protein is not involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubidn against colon cancers.     

Chronic doxorubicin cardiotoxicity is mediated by oxidative DNA damage-ATM-p53-apoptosis pathway and attenuated by pitavastatin through the inhibition of Rac1 activity

Doxorubicin is known to have cumulative dose-dependent cardiotoxicity, and a tumor suppressor protein p53 has been implicated in the pathogenesis of doxorubicin cardiotoxicity. However, how p53 is induced by doxorubicin and mediates the cardiotoxic effects of doxorubicin remains elusive. In cultured cardiac myocytes, doxorubicin induced oxidative stress, DNA damage, ATM activation, and p53 induction. A free radical scavenger NAC attenuated all of these events, whereas an ATM kinase inhibitor wortmannin attenuated doxorubicin-induced ATM activation and p53 induction but not oxidative stress. Doxorubicin treatment in vivo also induced oxidative stress, DNA damage, ATM activation, and p53 accumulation. These observations suggest that p53 induction by doxorubicin is mediated by oxidative DNA damage-ATM pathway. Doxorubicin-induced contractile dysfunction and myocyte apoptosis in vivo were attenuated in heterozygous p53 deficient mice and cardiac-restricted Bcl-2 transgenic mice, suggesting that myocyte apoptosis plays a central role downstream of p53 in doxorubicin cardiotoxicity. We also tested whether pitavastatin exerts protective effects on doxorubicin cardiotoxicity. Pitavastatin attenuated doxorubicin-induced oxidative stress, DNA damage, ATM activation, p53 accumulation, and apoptosis in vitro. Pitavastatin also attenuated myocyte apoptosis and contractile dysfunction in vivo. The beneficial effects of pitavastatin were reversed by intermediate products of the mevalonate pathway that are required for the activation of Rac1, and Rac1 inhibitor exhibited cardioprotective effects comparable to those of pitavastatin. These data collectively suggest that doxorubicin-induced cardiotoxicity is mediated by oxidative DNA damage-ATM-p53-apoptosis pathway, and is attenuated by pitavastatin through its antioxidant effect involving Rac1 inhibition.     

The human papilloma virus 16E6 gene sensitizes human mammary epithelial cells to apoptosis induced by DNA damage.

Programmed cell death (apoptosis) is a normal physiological process, which could in principle be manipulated to play an important role in cancer therapy. The key importance of p53 expression in the apoptotic response to DNA-damaging agents has been stressed because mutant or deleted p53 is so common in most kinds of cancer. An important strategy, therefore, is to find ways to induce apoptosis in the absence of wild-type p53. In this paper, we compare apoptosis in normal human mammary epithelial cells, in cells immortalized with human papilloma virus (HPV), and in mammary carcinoma cell lines expressing wild-type p53, mutant p53, or no p53 protein. Apoptosis was induced with mitomycin C (MMC), a DNA cross-linking and damaging agent, or with staurosporine (SSP), a protein kinase inhibitor. The normal and HPV-transfected cells responded more strongly to SSP than did the tumor cells. After exposure to MMC, cells expressing wild-type p53 underwent extensive apoptosis, whereas cells carrying mutated p53 responded weakly. Primary breast cancer cell lines null for p53 protein were resistant to MMC. In contrast, two HPV immortalized cell lines in which p53 protein was destroyed by E6-modulated ubiquitinylation were highly sensitive to apoptosis induced by MMC. Neither p53 mRNA nor protein was induced in the HPV immortalized cells after MMC treatment, although p53 protein was elevated by MMC in cells with wild-type p53. Importantly, MMC induced p21 mRNA but not p21 protein expression in the HPV immortalized cells. Thus, HPV 16E6 can sensitize mammary epithelial cells to MMC-induced apoptosis via a p53- and p21-independent pathway. We propose that the HPV 16E6 protein modulates ubiquitin-mediated degradation not only of p53 but also of p21 and perhaps other proteins involved in apoptosis.     

Synergistic antitumor effect of TRATL and doxorubicin on colon cancer cell line SW480

AIM: TRAIL (tumor necrosis factor-related apoptosisinducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent.Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms. METHODS: SW480 cells were cultured in the regular condition and incubated with different levels of agents.Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT)assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detectapoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.RESULTS: (1) SW480 cells were not sensitive to TRAIL,with IC50＞1 mg@L1 and dose-independent cytotoxicity. (2)SW480 cells were sensitive to doxorubicin at a certain degree,with dose-dependent cytotoxicity and IC50=65.25+3.48μmol@L-1. (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC50 of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 μg.L-1),combined with subtoxic doxorubicin (0.86 μmol@L1), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12+2.67%, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82±1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (P＞0.05). CONCLUSION: SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentration of doxorubicin to induce apoptosis effectively. The status of p53 protein i snot involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubicin against colon cancers.     

三氧化二砷对人体胃肠癌细胞凋亡及p53、survivin表达的影响

目的研究三氧化二砷（arsenic trioxide,As2O3）在人体内诱导胃肠癌细胞凋亡作用及对凋亡相关基因p53、survivin表达的影响,探讨其抗肿瘤作用及发生机制。方法采用原位末端转移酶标记技术（TUNEL）和免疫组化法分别检测As2O3用药前后胃肠癌细胞凋亡指数和凋亡相关基因蛋白P53、Survivin的表达变化情况。结果 As2O3用药后胃肠癌细胞凋亡指数为（17.04±3.67）%,与用药前（6.07±2.21）%相比差异有统计学意义（P〈0.01）;用药后胃肠癌细胞P53蛋白阳性表达率为（35.05±19.64）%,与用药前（34.80±16.48）%相比差异无统计学意义（P〉0.05）;用药后胃肠癌细胞Survivin蛋白阳性表达率为（15.59±3.94）%,与用药前（36.74±20.5）%相比明显下调,差异有统计学意义（P〈0.01）;As2O3作用后胃肠癌细胞Survivin蛋白表达下调时凋亡率升高,二者之间具有相关性（r=-0.47,P=0.04）。结论 As2O3在人体内可诱导胃肠癌细胞凋亡而发挥抗肿瘤作用,其机制可能与下调survivin基因的表达有关。     

Proteasome inhibitors induced caspase-dependent apoptosis and accumulation of p21WAF1/Cip1 in human immature leukemic cells

The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-L-valyl-L-alanyl-L-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.     

Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis

M1 myeloid leukemic cells overexpressing wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1β-converting enzyme (ICE)-like proteases, resulting in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these proteases and apoptosis were suppressed by the cytokine interleukin 6 or by a combination of the cytokine interferon γ and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were suppressed by some protease inhibitors. In a clone of M1 cells that did not express p53, vincristine or doxorubicin induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by interleukin 6. In another myeloid leukemia (7-M12) doxorubicin also induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by granulocyte–macrophage colony-stimulating factor. The results indicate that (i) overexpression of wild-type p53 by itself or treatment with cytotoxic compounds in wild-type p53-expressing or p53-nonexpressing myeloid leukemic cells is associated with activation of ICE-like proteases; (ii) cytokines exert apoptosis-suppressing functions upstream of protease activation; (iii) the cytotoxic compounds induce additional pathways in apoptosis; and (iv) cytokines can also suppress these other components of the apoptotic machinery.     

去C末端JAK3逆转录病毒载体对白血病细胞株NALM-6的影响

目的 :了解去C末端JAK3[JAK3(△C) ]基因对白血病细胞株NALM 6增殖和凋亡情况的影响 ,探索白血病基因治疗的新途径。方法 :用逆转录病毒作为载体 ,将JAK3(△C)基因导入NALM 6细胞 ,G4 18筛选阳性细胞株 ;用免疫沉淀 (IP)、SDS PAGE电泳、Westen blot检测JAK3(△C)对NALM 6细胞本身JAK3表达和磷酸化情况的影响 ;用MTT法检测NALM 6细胞和NALM 6 /JAK3(△C)细胞的增殖情况 ,了解JAK3(△C)对NALM 6细胞增殖的影响 ;用流式细胞术 (FCM )检测JAK3(△C)对NALM 6细胞凋亡的影响。结果 :JAK3(△C)能抑制NALM 6细胞本身JAK3的磷酸化活化 ;从生长曲线上可以看出 ,稳定表达JAK3(△C)的NALM 6 /JAK3(△C)细胞的增殖速率明显比NALM 6细胞阴性对照组低 ;从FCM图中可以看出NALM 6 /JAK3(△C)细胞出现了亚G1峰 (即凋亡峰 ) ,而NALM 6细胞未出现G1峰 ,同时NALM 6 /JAK3(△C)G0 /G1期细胞明显增多 ,而S、G2 /M期细胞相对减少 ,而根据其相应的软件计算出的这两组细胞的增殖指数和凋亡率也存在显著差异 (P<0 .0 5 ) ,说明导入了JAK3(△C)基因的NALM 6 /JAK3(△C)细胞凋亡增加、增殖能力明显降低。结论 :JAK3(△C)作为一种显性阴性分子 ,将为从细胞因子信号传导途径入手对白血病进行基因治疗提供一种新的途径     

Antiangiogenic activity of the MDM2 antagonist nutlin-3

Nutlin-3, a nongenotoxic activator of the p53 pathway, dose-dependently (range 0.1 to 10 micromol/L) inhibited the formation of capillaries in an in vivo matrigel assay, as well as the formation of capillary-like structures in an in vitro coculture system composed of endothelial cells surrounded by fibroblasts. In contrast to the chemotherapeutic agent doxorubicin, nutlin-3 showed no induction of apoptosis in vitro either in the cocultures or in isolated vascular endothelial cells, even when used at the highest concentration (10 micromol/L). However, treatment with pharmacological inhibitors of the nuclear factor kappaB and phosphatidylinositol 3-kinase/Akt pathways sensitized endothelial cells to nutlin-3-induced apoptosis. Although nutlin-3 and doxorubicin induced a comparable p53 accumulation in endothelial cells, nutlin-3 was significantly more efficient than doxorubicin in upregulating the p53 target genes CDKN1A/p21, MDM2, and GDF-15, as well as in inhibiting cell cycle progression. However, the predominant in vitro effect of nutlin-3 was its strong antimigratory activity observed at concentrations significantly lower (0.1 micromol/L) than those required to inhibit endothelial cell cycle progression. Taken together, our data suggest that the antiangiogenic activity of nutlin-3 observed in vivo was mainly attributable to inhibition of endothelial cell migration, to some extent attributable to cell cycle arrest, and to a lesser extent attributable to induction of apoptosis.     

Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L-induced apoptosis of human acute leukemia cells

In present studies, treatment with tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 microg/mL of Apo-2L (95.0% +/- 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53(wt) function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L-induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L-induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-x(L). Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L-induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L-induced apoptosis. (Blood. 2000;96:3900-3906)     

Lysosomal destabilization in p53-induced apoptosis

The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37 degrees C to 32 degrees C. We have now found that these cells showed an early lysosomal rupture after transfer to 32 degrees C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca(2+)-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders.     

CMA down-regulates p53 expression through degradation of HMGB1 protein to inhibit irradiation-triggered apoptosis in hepatocellular carcinoma

AIM To investigate the mechanism of chaperone-mediated autophagy(CMA)-induced resistance to irradiationtriggered apoptosis through regulation of the p53 protein in hepatocellular carcinoma(HCC). METHODS Firstly, we detected expression of lysosome-associated membrane protein 2a(Lamp-2a), which is the key protein of CMA, by western blot in Hep G2 and SMMC7721 cells after irradiation. We further used sh RNA Lamp-2a HCC cells to verify the radioresistance induced by CMA. Next, we detected the HMGB1 and p53 expression after irradiation by western blot, and we further used RNA interference and ethyl pyruvate(EP), as a HMGB1 inhibitor, to observe changes of p53 expression. Finally, an immunoprecipitation assay was conducted to explore the interaction between Lamp-2a and HMGB1, and the data were analyzed. RESULTS We found the expression of Lamp-2a was increased on irradiation while apoptosis decreased in Hep G2 and SMMC7721 cells. The apoptosis was increased markedly in the sh RNA Lamp-2a Hep G2 and SMMC7721 cells as detected by western blot and colony formation assay. Next, we found p53 expression was gradually reduced on irradiation but obviously increased in sh RNA Lamp-2a cells. Furthermore, p53 increased the cell apoptosis on irradiation in Hep3B(p53-/-) cells. Finally, p53 levels were regulated by HMGB1 as measured through RNA interference and the EP treatment. HMGB1 was able to combine with Lamp-2a as seen by immunoprecipitation assay and was degraded via the CMA pathway. The decreased HMGB1 inhibited p53 expression induced by irradiation and further reduced the apoptosis in HCC cells. CONCLUSION CMA pathway activation appears to down-regulate the susceptibility of HCC to irradiation by degrading HMGB1 with further impact on p53 expression. These findings have clinical relevance for radiotherapy of HCC.     

Contributions of p53 and PMA to g-irradiation induced apoptosis in Jurkat cells

Several mutations prevent the expression of p53 in the human lymphoblastoid T cell line Jurkat. Restoration of p53 in Jurkat cells had no effect on the cell growth but markedly increased the amount of apoptosis induced by g-irradiation. Inhibition of RNA synthesis using 5,6-dichlorobenimidizole riboside had little effect on apoptosis induced by irradiation in the presence of p53 and did not affect the p53-independent apoptotic pathway. Expression of p53 also had no effect on the expression levels of proteins such as Fas, GADD45, Bax, Bcl-2, Bcl-x_L or p53 induced proteins (PIGS) in resting cells or after irradiation. Activation of protein kinase C by phorbol 12-myristate 13-acetate produced an almost complete inhibition of p53-independent apoptosis following irradiation, whereas no significant effect was observed on the rate of p53-induced apoptosis. Although phorbol 12-myristate 13-acetate strongly induced p21 and stabilised p53 in the resting transfected Jurkat cells, neither apoptosis nor cell arrest was observed. In summary, this work shows that p53 enhances the radiosensitivity of Jurkat cells through an apoptotic process that is triggered by irradiation and is largely independent of RNA synthesis and protein kinase C activation. Apoptosis in p53- negative Jurkat cells is strongly inhibited by PMA indicating that the pathway triggered by p53 may be distinct from apoptotic pathways used in its absence.     

Rescue of mutants of the tumor suppressor p53 in cancer cells by a designed peptide

We designed a series of nine-residue peptides that bound to a defined site on the tumor suppressor p53 and stabilized it against denaturation. To test whether the peptides could act as chaperones and rescue the tumor-suppressing function of oncogenic mutants of p53 in living cells, we treated human tumor cells with the fluorescein-labeled peptide Fl-CDB3 (fluorescent derivative of CDB3). Before treatment, the mutant p53 in the cell was predominantly denatured. Fl-CDB3 was taken up into the cytoplasm and nucleus and induced a substantial up-regulation of wild-type p53 protein and representative mutants. The mutants, His-273 and His-175 p53, adopted the active conformation, with a dramatic decrease in the fraction of denatured protein. In all cases, there was p53-dependent induction of expression of the p53 target genes mdm2, gadd45, and p21, accompanied by p53-dependent partial restoration of apoptosis. Fl-CDB3 sensitized cancer cells that carried wild-type p53 to p53-dependent gamma-radiation-induced apoptosis. Although Fl-CDB3 did not elicit a full biological response, it did bind to and rescue p53 in cells and so can serve as a lead for the development of novel drugs for anticancer therapy.