硼替佐米对白血病细胞株HEL抑制作用的初步探讨

目的 探讨硼替佐米单独或联合化疗药物对急性白血病细胞株HEL生长的影响及其机制.方法 MTT法检测药物对HEL的生长抑制效应,流式细胞术检测细胞周期变化及凋亡,Western blotting检测凋亡及细胞周期相关蛋白表达.结果 硼替佐米对HEL细胞的生长抑制作用呈浓度依赖关系,其半数抑制浓度(IC50)为7.15 nmol/L;硼替佐米联合化疗药物明显增强抑制HEL细胞生长;流式细胞术检测可见时间依赖性的G2/M周期阻滞;Western blotting检测显示bcl-2表达下降,bax、p27表达增高,联合用药时具有相加效应,但不管是单独或联合用药,p53蛋白的表达均无明显改变.结论 硼替佐米可能是一种有效治疗急性白血病的药物,与柔红霉素联合应用有更强的抑制肿瘤细胞生长和诱导其凋亡的作用,其机制可能与bcl-2,bax及p27蛋白的调节有关,是非p53依赖性的.     

Bronchopulmonary dysplasia and oxidative stress: are we closer to an understanding of the pathogenesis of BPD?

OBJECTIVES: To determine if induction of heat shock protein 70 (HSP 70), a stress protein that plays a cytoprotective role and inhibits cell death in response to various stimuli, will protect thymocytes and T-cell clones from radiation-induced apoptosis, and to define the mechanism of such protection. DESIGN: Thymocytes from BALB/c mice or T-lymphocyte clones were incubated at 43 degrees C for 1 hour to induce HSP 70, then irradiated. Control cells were irradiated but not heated. Fragmentation of DNA was quantitated, and p53, bax, and bcl-2 expression was analyzed at various times by the Western blot method. RESULTS: Only heated cells expressed HSP 70. The induction of HSP 70 increased basal apoptosis but significantly decreased radiation-induced apoptosis. Furthermore, introduction of an HSP 70 antisense oligomer prior to heating reversed the protective effect of HSP 70. Induction of HSP 70 in T-cell clones with sodium arsenite had a similar protective effect against radiation-induced apoptosis. Irradiation induced p53 and markedly up-regulated bax. The expression of p53 peaked at 4 hours and preceded maximal bax induction. Induction of HSP 70 prior to irradiation suppressed p53 and significantly decreased bax levels. Levels of bcl-2 were unaffected. CONCLUSIONS: Our data show that HSP 70 induction protects thymocytes from radiation-induced apoptosis by down-regulating p53 and bax expression. The induction of HSP 70 may represent a novel mechanism by which the immunosuppressive effects and the associated infectious complications of radiation therapy can be minimized.     

Antitumor activity of bax and p53 naked gene transfer in lung cancer: in vitro and in vivo analysis

In vitro and in vivo data have demonstrated that virus-mediated p53 gene transfer can induce active cell death and lung tumor regression. In contrast, the therapeutic potential of bax, another apoptosis-inducing gene, has not been described. We compared p53 and bax cytotoxic effects by transient transfection of an average of 25 +/- 5% of the H-322 and H-358 bronchioloalveolar carcinoma cell lines in vitro. Under these conditions, bax expression killed 70 to 90% of the transfected cells whereas p53 killed only 40% of them. The killing activity of both genes involved apoptosis, as shown by TUNEL staining. Surprisingly, BrdU incorporation indicated that the cells that did resist Bax toxicity were blocked in the pre-S phase of the cell cycle, a result expected for p53 only. In vivo, repeated injections of naked DNA encoding Bax or p53 inhibited the growth of 4-mm preestablished H-322 tumors in nude mice. Growth retardation only, and not inhibition, was observed in H-358, a poorly transfectable and rapidly growing tumor. These results indicate that Bax and p53 share a similar, strong antitumor activity in vivo, even if the former is a more potent inducer of apoptosis in vitro.     

bcl-2 inhibits wild-type p53-triggered apoptosis but not G1 cell cycle arrest and transactivation of WAF1 and bax

We have earlier shown that wild-type (wt) p53 expressed from a temperature-sensitive construct (ts p53) triggers apoptosis in the v-myc retrovirus-induced, p53-negative T-cell lymphoma line J3D (Y. Wang et al., Cell Growth & Differ., 4: 467-473, 1993). We also found that constitutive bcl-2 expression inhibits wt p53-triggered apoptosis in these cells (Y. Wang et al., Oncogene, 8: 3427-3431, 1993). Here we demonstrate that more than 90% of the ts p53-transfected J3D cells were arrested in G1 at 18 h after induction of wt p53 expression by temperature shift to 32 degrees C. At this time, at least 80% of the cells remained viable. After 30 h at 32 degrees C, around 50% of the cells had died by apoptosis, while most of the remaining cells were still alive in G1, indicating that p53-induced apoptosis occurred following G1 arrest. The G1 cell cycle arrest at 18 h after temperature shift to 32 degrees C was reversible, as shown by the fact that the cells readily resumed exponential growth following temperature shift back to 37 degrees C, although viability dropped from around 80 to 65%. Expression of both WAF1 and bax mRNA was induced by wt p53 in both the ts p53 and ts p53/bcl-2 transfected cells. The kinetics of G1 cell cycle arrest at 32 degrees C was similar in both the ts p53 and the ts p53/bcl-2 double transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alphaVbeta3 during angiogenesis

Induction of p53 activity in cells undergoing DNA synthesis represents a molecular conflict that can lead to apoptosis. During angiogenesis, proliferative endothelial cells become apoptotic in response to antagonists of integrin alphavbeta3 and this leads to the regression of angiogenic blood vessels, thereby blocking the growth of various human tumors. Evidence is presented that administration of alphavbeta3 antagonists during angiogenesis in vivo selectively caused activation of endothelial cell p53 and increased expression of the p53-inducible cell cycle inhibitor p21WAF1/CIP1. In vitro studies revealed that the ligation state of human endothelial cell alphavbeta3 directly influenced p53 activity and the bax cell death pathway. Specifically, agonists of endothelial cell alphavbeta3, but not other integrins, suppressed p53 activity, blocked p21WAF1/CIP1 expression, and increased the bcl-2/bax ratio, thereby promoting cell survival. Thus, ligation of vascular cell integrin alphavbeta3 promotes a critical and specific adhesion-dependent cell survival signal during angiogenesis leading to inhibition of p53 activity, decreased expression of p21WAF1/CIP1, and suppression of the bax cell death pathway.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.     

Taxol-mediated augmentation of CD95 ligand-induced apoptosis of human malignant glioma cells: association with bcl-2 phosphorylation but neither activation of p53 nor G2/M cell cycle arrest

The anti-tumour alkaloid taxol shows strong cytotoxic and antiproliferative activity in two human malignant glioma cell lines, T98G and LN-229. CD95 (Fas/APO-1) ligand is a novel cytotoxic cytokine of the tumour necrosis factor (TNF) family that exerts prominent antiglioma activity. At clinically relevant taxol concentrations of 5-100 nM, taxol and CD95 ligand showed significant synergistic cytotoxicity and growth inhibition. High concentrations of taxol induced G/M cell cycle arrest in both cell lines. The synergy of taxol and CD95 ligand was independent of cell cycle effects of taxol as synergy was achieved at much lower taxol concentrations than G2/M arrest and as cell cycle effects of taxol were unaffected by co-exposure to CD95 ligand. Similarly, high concentrations of taxol were required to induce p53 activity in the p53 wild-type cell line LN-229. This effect was not modulated by CD95 ligand, suggesting that synergy is also independent of p53 activation. However, taxol induced a mobility shift of the bcl-2 protein on immunoblot analysis, indicative of bcl-2 phosphorylation. Bcl-2 phosphorylation on serine was confirmed by immunoprecipitation and phosphoserine immunoblot analysis. Considering (1) that phosphorylation of bcl-2 interferes with its heterodimerization with bax and (2) the inhibition of CD95-mediated apoptosis by bcl-2, we propose that taxol sensitizes malignant glioma cells to CD95 ligand by increasing the functional bax/bcl-2 rheostat in favour of bax and thus cell death.     

Resistance to apoptosis and elevated expression of Bcl-2 in clonally expanded CD4+CD28- T cells from rheumatoid arthritis patients

Patients with rheumatoid arthritis have a subset of CD4+ T lymphocytes that are characterized by a defect in CD28 expression. CD4+CD28- T cells frequently undergo clonal expansion in vivo. These clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4+CD28- T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4+CD28- T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28+ and CD28-CD4+ T cells could not be distinguished by the levels of bax or bcl-xL protein; however, CD4+CD28- T cells expressed higher amounts of bcl-2 protein than did CD4+CD28+ T cells. The increased bcl-2 expression in CD4+CD28- T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4+ T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4+ T cells. We propose that CD4+CD28- T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis.     

Activation of PKCalpha downstream from caspases during apoptosis induced by 7-hydroxystaurosporine or the topoisomerase inhibitors, camptothecin and etoposide, in human myeloid leukemia HL60 cells

We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKCalpha, betaI, betaII, delta, and zeta activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKCzeta and inhibited kinase activity of PKCbetaI, betaII, and delta. PKCalpha activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKCalpha activity during apoptosis in HL60 cells. However, CPT did not affect PKCbetaI, betaII and zeta, and activated PKCdelta. PKCalpha activation was not due to increased protein levels or proteolytic cleavage but was associated with PKCalpha autophosphorylation in vitro and increased phosphorylation in vivo. We also found that not only PKC delta but also PKC betaI was proteolytically activated in HL60 cells during apoptosis. The PKCalpha activation and hyperphosphorylation were abrogated by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKCdelta and betaI proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKCalpha and proteolytic activation for PKC delta and betaI.     

p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs

The p53 tumour suppressor protein plays a key role in the integration of stress signals. Multi-site phosphorylation of p53 may play an integral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-targeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence that the delta and epsilon isoforms of casein kinase 1 (CK1delta and CK1epsilon) show identical features to p53NK and can phosphorylate p53 both in vitro and in vivo. Recombinant, purified glutathione S-transferase (GST)-CK1delta and GST-CK1epsilon fusion proteins each phosphorylate p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK. Furthermore, p53NK (i) co-purifies with CK1delta/epsilon, (ii) shares identical kinetic properties to CK1delta/epsilon, and (iii) is inhibited by a CK1delta/epsilon-specific inhibitor (IC261). In addition, CK1delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1delta-specific monoclonal antibody. Treatment of murine SV3T3 cells with IC261 specifically blocked phosphorylation in vivo of the CK1delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1delta and/or CK1epsilon. Similarly, over-expression of a green fluorescent protein (GFP)-CK1delta fusion protein led to hyper-phosphorylation of p53 at its N-terminus. Treatment of MethAp53ts cells with the topoisomerase-directed drugs etoposide or camptothecin led to increases in both CK1delta-mRNA and -protein levels in a manner dependent on the integrity of p53. These data suggest that p53 is phosphorylated by CK1delta and CK1epsilon and additionally that there may be a regulatory feedback loop involving p53 and CK1delta.     

Chlorambucil in chronic lymphocytic leukemia: mechanism of action

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries but the clinical presentation and rate of disease progression are highly variable. When treatment is required the most commonly used therapy is the nitrogen mustard alkylating agent, chlorambucil (CLB), with or without prednisone. Although CLB has been used in the treatment of CLL for forty years the exact mechanism of action of this agent in CLL is still unclear. Studies in proliferating model tumor systems have demonstrated that CLB can bind to a variety of cellular structures such as membranes, RNA, proteins and DNA; however, DNA crosslinking appears to be most important for antitumor activity in these systems. In addition, a number of different mechanisms can contribute to CLB resistance in these tumor models including increased drug metabolism, DNA repair and CLB detoxification resulting from elevated levels of glutathione (GSH) and glutathione S-transferase (GST) activity. However, unlike tumor models in vitro, CLL cells are generally not proliferating and studies in CLL cells have raised questions about the hypothesis that DNA crosslinking is the major mechanism of antitumor action for CLB in this disease. CLB induces apoptosis in CLL cells and this appears to correlate with the clinical effects of this agent. Thus, alkylation of cellular targets other than DNA, which can also induce apoptosis, may contribute to the activity of CLB. Alterations in genes such as p53, mdm-2, bcl-2 and bax which control entry into apoptosis may cause drug resistance. Loss of wild-type p53 by mutation or deletion occurs in 10 to 15% of CLL patients and appears to correlate strongly with poor clinical response to CLB. The induction of apoptosis by CLB is paralleled by an increase in P53 and Mdm-2 but this increase in not observed in patients with p53 mutations indicating that with high drug concentrations CLB can produce cell death through P53 independent pathways. The level of Mdm-2 mRNA in the CLL cells is not a useful predictor of drug sensitivity. In addition, although Bax and Bcl-2 are important regulators of apoptosis and the levels of these proteins are elevated in CLL cells compared with normal B cells, the levels of Bax and Bcl-2, or the Bax:Bcl-2 ratio, are not important determinants of drug sensitivity in this leukemia. Finally, whereas CLB and nucleoside analogs may produce cell death in CLL by a P53 dependent pathway other agents, such as dexamethasone or vincristine, may act through P53-independent pathways.     

Social and reproductive conditions modulate urinary cortisol excretion in black tufted-ear marmosets (Callithrix kuhli)

The endogenous metabolite, 2-methoxyestradiol (2ME), is an inhibitor of tubulin polymerization and is therefore toxic to dividing fast-growing tumor cells. Transformed cells are not equally susceptible to the effects of 2ME. In this study the effects of 1-2 microM doses of 2ME on cell cycle progression, apoptosis induction and on p53 levels were evaluated using flow cytometry in cells with different p53 status. No effect of 2ME was seen in normal human skin fibroblast strain HSF43 with wild-type (wt) p53. However, in SV40 T antigen transformed HSF43 cells (line E8T4), 2ME caused a prominent G2/M arrest, with subsequent micronuclei formation followed by apoptosis. Increased p53 levels were present in the G2/M cells. Our results suggest that 2ME, being a microtubule poison, may release the bound p53 from T antigen, and that this p53 may enhance the apoptotic effects. Two lymphoblast cell lines derived from the same donor, TK6, expressing low levels of wt p53, and WTK1, expressing high levels of mutant p53, showed similar moderate responses to 2ME at 37 degrees C. The effects included enhanced apoptosis and a modest G2/M block. No increase in p53 levels was seen. However, at the permissive temperature of 30 degrees C marked increases in apoptosis and a prominent G2/M-phase block, similar to that seen in the E8T4 cells, were present in the WTK1 cells, indicating that the high levels of mutant p53 have now become functional, enhancing the apoptotic effects initiated by 2ME.     

Development of a small-scale bioreactor for drug metabolism studies maintaining hepatospecific functions

Regulation of apoptosis is an important component of multistage hepatocarcinogenesis. The objectives of the present study were to characterize apoptosis regulation in primary mouse hepatocytes and to determine whether nongenotoxic carcinogens alter apoptosis regulation. Bleomycin-induced apoptosis was accompanied by decreases in bcl-2 and bcl-xl and increases in p53, bak, and bax protein levels. Transforming growth factor (TGF)-beta-induced apoptosis was accompanied by decreased bcl-xL and increased bak. Bleomycin-induced apoptosis was partially dependent on p53, whereas TGF-beta-induced apoptosis was independent of p53. Phenobarbital inhibited both TGF-beta and bleomycin-induced apoptosis and the normal regulation of p53, bcl-2, and bax. Nafenopin inhibited apoptosis through a mechanism dependent on PPAR-alpha and inhibited the normal regulation of bcl-2 and bak. 2,3,7,8-Tetrachlorodibenzo-p-dioxin did not alter apoptosis or its regulation. Apoptosis was increased in hepatocytes from bcl-2-null mice, which indicated that the bcl-2 family contributes to hepatocyte apoptosis regulation. This study demonstrated that apoptosis regulation in mouse hepatocytes involves distinct pathways and that diverse nongenotoxic carcinogens differentially alter molecular pathways that represent targets for hepatocarcinogenesis.     

Activation of the K-ras oncogene in colorectal neoplasms is associated with decreased apoptosis

BACKGROUND: Recent in vitro data indicate that the oncogenic effects of activated ras genes may be mediated, at least in part, through inhibition of apoptotic cell death. To examine this proposition in vivo, the relationship between mutations of the K-ras gene and the frequency of apoptosis was studied in a series of 69 sporadic colorectal neoplasms (11 adenomas and 58 carcinomas). METHODS: Mutations in codon 12 of K-ras were determined by a single tube, enriched polymerase chain reaction. Apoptotic cells in tumor sections were identified by in situ end-labeling of fragmented DNA, whereas levels of bcl-2 and p53 proteins were determined by immunohistochemistry. RESULTS: Tumors with mutant K-ras had a significantly lower apoptotic index than those with the wild-type allele (P < 0.05). They were also more likely to exhibit positive bcl-2 staining (P < 0.05). Adenomas showed significantly greater bcl-2 positivity than carcinomas (89% and 51%, respectively; P < 0.05). The frequency of apoptosis in these tumors was not related to either bcl-2 positivity or p53 status. CONCLUSIONS: These findings suggest that activation of K-ras in colorectal carcinoma may inhibit apoptosis and thus favor tumor progression. Alternatively, this association may reflect an accumulation of K-ras mutations in cells in which normal apoptotic pathways have been impaired.     

p53 induces angiogenesis-restricted dormancy in a mouse fibrosarcoma

The p53 tumor-suppressor gene is inactivated in over 50% of all human cancers. In normal cells, p53 induces growth arrest and apoptosis in response to DNA damage. We show that p53 acts as potent tumor-suppressor gene independent of its well-documented effects on tumor-cell proliferation and apoptosis. p53 activates target genes in a murine fibrosarcoma cell-line but does not affect tumor cell-cycle progression or survival. Exogenous expression of wt-p53 does, however, block the angiogenic potential of the tumor cells resulting in formation of dormant tumors in vivo. These data provide evidence that: (1) p53 acts as a tumor suppressor gene independent of its anti-proliferative effects; (2) By inhibiting angiogenesis p53 can indirectly induce apoptosis in vivo but not in vitro; (3) p53-gene therapy which alters a tumors angiogenic potential, can revert tumors to a dormant phenotype.     

T-cell-rich large-B-cell lymphomas contain non-activated CD8+ cytolytic T cells, show increased tumor cell apoptosis, and have lower Bcl-2 expression than diffuse large-B-cell lymphomas

The factor(s) responsible for the reduced B cell number and increased T cell infiltrate in T-cell-rich large-B-cell lymphomas (TCRBCLs) have not been well characterized. We studied 18 TCRBCLs and 12 diffuse large-B-cell lymphomas (DLBCLs) to compare the 1) predominant T cell subpopulation(s), 2) expression of cytotoxic granule proteins (TIA-1 and granzyme B), 3) level of tumor cell apoptosis (Apoptag system, Oncor, Gaithersburg, MD), and 4) expression of Ki-67 (Mib-1) and apoptosis-related proteins (fas (CD95), bcl-2, and p53). T cells in TCRBCLs and DLBCLs were predominantly CD8+ T cells expressing alphabeta T-cell receptors and TIA-1 (16 of 18 TCRBCLs with >50% TIA-1+ small lymphocytes) but lacking granzyme B (16 of 18 TCRBCLs with <25% granzyme B+ small lymphocytes). Scattered apoptotic tumor cells (confirmed with CD20 co-labeling) were present in 15 of 18 TCRBCLs, with 14 of 15 cases having <10% apoptotic cells. No apoptotic cells were seen in 12 of 12 DLBCLs. In 16 of 16 immunoreactive TCRBCLs, <25% tumor cells were bcl-2+, whereas 6 of 12 DLBCLs had >50% bcl-2+ tumor cells. CD95 (fas) expression was also lower, with 3 of 18 (16.7%) TCRBCLs versus 4 of 12 (33%) DLBCLs having >25% CD95+ tumor cells. TCRBCLs and DLBCLs had similar levels of p53 and Ki-67 (Mib-1) expression. Thus, T cells in TCRBCLs are non-activated cytotoxic T lymphocytes (TIA-1+, granzyme B-). Tumor cell apoptosis (perhaps cytotoxic T cell mediated) may partly account for the decreased number of large (neoplastic) B cells in TCRBCLs, but other factors (ie, decreased bcl-2 expression) may also be needed.