5－氮－2＇－脱氧胞苷（5－aza－CdR）对HL－60细胞分化和DNA甲基化酶的影响

以５－氮－２＇－脱氧胞苷（５－ａｚａ－ＣｄＲ）为诱导物,在０．５μｍｏｌ／Ｌ的最佳浓度下,可诱导ＨＬ－６０细胞分化达１５％左右。同时,用［￣３Ｈ］－ｍｅｔｈｙｌ－ｓ－ａｄｅｎｏｓｙｌｍｅｔｈｉｏｎｉｎｅ（￣３Ｈ－ＳＡＭ）为底物,通过同位素参入法,测定了不同浓度诱导物对ＨＬ－６０细胞ＤＮＡ甲基化酶活力的影响,发现在最佳诱导物浓度下,可使ＨＬ－６０细胞ＤＮＡ甲基化酶活力明显下降,此外,也比较了不同分化水平的ＨＬ－６０细胞中具有不同甲基化水平的ＤＮＡ在体外接受甲基的能力,从而证明５－ａｚａ－ＣｄＲ诱导ＨＬ－６０细胞分化与其ＤＮＡ甲基化状态密切相关。     

TNF receptor－associated factor－2 binding site is involved in TNFR75－dependent enhancement of TNFR55－induced cell death

INTRODUCTIONTumor necrosis factor alpha (TNFa) is apleiotropic cytokine that is mainly produced by acti-vated macrophages and lymphocytes[1]. TNFa ini-tiates inflammatory, immune regulatory and patho-physiologic responses by binding to two distinct cellsurface receptors of TR55 (55 kDa) and TR75 (75kDa)[2]. Both receptors belong to TNF receptorsuperfamily because they share 28% homoIogy andcontain 4 conserved cysteine--rich subdomain in theirextracellular regions[3]. It is the uniqu…     

SH－SY5Y细胞胞内钙库特性研究

运用单细胞显微荧光测量技术测量了单个ＳＹ－ＳＹ５Ｙ细胞内游离钙离子浓度的变化。首次报道了ＳＨ－ＳＹ５Ｙ细胞内存在毒蕈碱敏感而非咖啡因敏感的钙库,并研究了它的动力学特征。     

大蒜油对人CFu－L与CFu－GM集落形成能力的影响

本研究用体外细胞培养技术观察了大蒜油对人白血病细胞集落（ＣＦｕ－Ｌ）及骨髓粒单细胞集落（ＣＦｕ－ＧＭ）形成能力的影响。结果证明大蒜油对白血病细胞集落生长有明显的抑制作用,而相同浓度的大蒜油对正常人骨髓（ＢＭ）粒单细胞集落的形成能力亦有一定程度的抑制作用,但抑制作用较弱。     

利用IL－3／GM－CSF融合蛋白（PIXY－321）扩增脐血造血细胞方法的初步建立

利用单克隆抗体免疫磁珠吸附方法分离脐血ＣＤ３４＋细胞,并观察了ＩＬ３／ＧＭＣＳＦ融合蛋白（ＰＩＸＹ３２１）对脐血ＣＤ３４＋细胞的刺激作用。ＰＩＸＹ３２１对脐血ＣＤ３４＋细胞扩增作用大于ＩＬ３和ＧＭＣＳＦ单独及联合应用。在液体培养条件下,每毫升２０ｎｇＰＩＸＹ３２１可有效地扩增脐血造血祖细胞,适宜扩增时间为５－８天,扩增后造血祖细胞的数量可达扩增前的８－１０倍,从而初步建立了一种简单可行的脐血造血细胞扩增方法。     

多胺生物合成的抑制对转化细胞c－Ha－ras癌基因表达的调控

抗多胺代谢剂──二氟甲基鸟氨酸（ＤＦＭＯ）作用于经含点突变的Ｈａ－ｒａｓ基因片段转染的转化细胞（ＨＲ－１细胞）引起细胞生长的抑制,其抑制率随ＤＦＭＯ浓度的增加而增大,此时细胞多停滞于Ｇ_１期;多胺合成的关键酶鸟氨酸脱羧酶（ＯＤＣ）活性显著下降;Ｈａ－ｒａｓ癌基因ｍＲＮＡ及ｒａｓＰ~（２１）蛋白的表达受到抑制;而外源性腐胺与ＤＦＭＯ的同时加入可防止上述一系列改变的发生,说明ＤＦＭＯ使ＨＲ－１细胞某些表型向亲本细胞逆转的作用是与细胞多胺生物合成的抑制直接相关。     

Par－4与阿尔茨海默病神经元凋亡

前列腺凋亡反应蛋白-4(prostate apoptosis response-4．Par-4)是一种小分子蛋白质．其生理功能尚不清楚,但能够促进多种细胞包括神经元的凋亡已得到证实。Par-4参与阿尔茨海默病(Alzheimer′s disease,AD)神经元凋亡过程复杂．且有望成为筛选治疗AD药物的作用靶点。     

β－连环素研究新进展

β-cat作为一种重要的细胞间黏附分子和信号转导分子。并通过与多种蛋白的结合参与细胞和组织的发育分化、损伤修复、肿瘤的发生发展及其它生物学过程。本文对近年来在β—cat的晶体结构特点、β-cat的降解与其丝／苏氨酸磷酸化的关系、及其在细胞内转运与APC基因的调控机制的联系、其出入细胞核及核内活化转录的分子机制以及β-cat研究的应用前景等方面的进展作一综述。     

Blockage of IGF－1R signaling sensitizes urinary bladder cancer cells to mitomycin－mediated cytotoxicity

INTRODUCTIONtransitional cell carcinoma (TCC) of the bladder represents the fifth most preValent malignancy inwestern population. A major problem in the management of TCC is the low sensitivity to chemotherapy and the high recu-rrence after transurethral resection, which occupies a large proportion (approximately 40%) among bladder cancer patients[1, 21. Sodrug resistance remains a major and difficult problem to resolye in TCC chemotherapy. This phenomenon has often been ascribed to so…     

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

INTRODUCTIONThaillll (TNF-related apoptosis inducing ligand)is a recently described member of the TNF family.Like other members of the TNF ligand family) availcould induce apoptosis of neoplastically transformedcells by priVating cell surface death receptors ThailRI and ThaiLR212].Trail has been demonstrated to play an important role in homeostasis of immune system includ.lug eradication of the old lymphocytel3], actiVationinduced T cell deathI41, regulation Of T cen eXPansion by…     

Protein Kinase C�� Modulates Ligand-induced Cell Surface Death Receptor Accumulation: A MECHANISTIC BASIS FOR ENZASTAURIN-DEATH LIGAND SYNERGY*

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.     

Phorbol 12-myristate 13-acetate inhibits death receptor-mediated apoptosis in Jurkat cells by disrupting recruitment of Fas-associated polypeptide with death domain.

Regulation of death receptor-mediated apoptosis is incompletely understood. Previous studies have demonstrated that phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, inhibits Fas (CD95)-mediated apoptosis in Jurkat (type II) cells but not SKW6.4 (type I) cells. In this study, we demonstrated that PMA also protects Jurkat cells from apoptosis induced by tumor necrosis factor-alpha and the tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). Interestingly, PMA failed to protect Jurkat cells from apoptosis induced by other agents, including etoposide, camptothecin, and gamma-irradiation. Analysis of the initial events induced by agonistic anti-Fas antibodies revealed that PMA inhibited Fas binding to Fas-associated polypeptide with death domain (FADD) in Jurkat cells but not in SKW6.4 cells. Although the protein kinase inhibitor bisindoylmaleimide VIII increased apoptosis induced by agonistic anti-Fas antibody, tumor necrosis factor-alpha, and TRAIL, these effects were not observed with the protein kinase C inhibitor H7 and were not associated with increased FADD recruitment to Fas. These results indicate that PMA inhibits death signaling induced by a number of discrete receptors and suggest that the effects are mediated at the level of receptor-mediated adaptor molecule recruitment.     

Tumoricidal activity of monocyte-derived dendritic cells: evidence for a caspase-8-dependent, Fas-associated death domain-independent mechanism

Monocyte-derived dendritic cells (DC) were found to be cytotoxic for several tumor cell lines including Jurkat cells, which were killed through a calcium-independent pathway. K562 cells were resistant, excluding a NK cell-like activity. DC-mediated apoptosis did not involve classical death receptors because it was not reversed by blocking TNF/TNFR, CD95/CD95 ligand, or TNF-related apoptosis-inducing ligand/TNF-related apoptosis-inducing ligand receptor interactions. Fas-associated death domain-deficient, but not caspase-8 deficient, Jurkat cells were killed by DC. Indeed, caspase-8 cleavage was demonstrated in Jurkat cells cocultured with DC, and the use of specific caspase inhibitors confirmed that apoptosis triggered by DC was caspase-8 dependent. Furthermore, the involvement of Bcl-2 family members in the control of DC-mediated apoptosis was demonstrated by Bid cleavage in Jurkat cells cocultured with DC and resistance of Jurkat cells overexpressing Bcl-2 to DC-mediated cytotoxicity. Overall, these data indicate that monocyte-derived DC exert a caspase-8-dependent, Fas associated death domain-independent tumoricidal activity, a finding that could be relevant to their therapeutic use in cancer.     

FADD deficiency sensitises Jurkat T cells to TNF-alpha-dependent necrosis during activation-induced cell death

Activation-induced cell death (AICD) in activated T lymphocytes is largely mediated by Fas/Fas ligand (FasL) interaction. The cytoplasmic adaptor molecule Fas-associated death domain protein (FADD) plays an essential role in the apoptotic signalling of the Fas death pathway. In the present study, we observed that FADD deficient (FADD(-/-)) Jurkat T cells undergo AICD to a similar extent as wild-type cells. AICD in wild-type Jurkat T cells is via apoptosis, whereas it is non-apoptotic in FADD(-/-) cells. The latter took up propidium iodide, exhibit a loss in mitochondrial membrane potential and have no detectable cleavage products of caspase-8 or -3 activation, suggesting that these cells die by necrosis. Wild-type Jurkat T cells undergo apoptosis when incubated with recombinant FasL and Trail but not with TNF-alpha. In contrast, FADD(-/-) Jurkat T cells are resistant to FasL and Trail but die of necrosis when incubated with TNF-alpha. We showed that neutralising anti-TNF-alpha blocked AICD as well as TNF-alpha-induced necrosis in FADD(-/-) Jurkat T cells. Furthermore, down regulating the receptor interacting protein, RIP, with geldanamycin treatment, which is essential for TNF-alpha signalling, markedly inhibited AICD in FADD(-/-) Jurkat T cells. In addition, caspase-8-deficient Jurkat T cells are resistant to Fas- and TNF-alpha-induced cell death. Taken together, our results suggest that a deficiency in FADD and not caspase-8 or the inhibition of the Fas signalling pathway sensitises Jurkat T cells to TNF-alpha-dependent necrosis during AICD.     

Molecular mechanism of the activation-induced cell death inhibition mediated by a p70 inhibitory killer cell Ig-like receptor in Jurkat T cells

In this study we investigated the molecular mechanism of the activation-induced cell death (AICD) inhibition mediated by a p70 inhibitory killer cell Ig-like receptor (KIR3DL1, also called NKB1) in Jurkat T cells. Using stable Jurkat transfectants that express KIR or CD8-KIR fusion proteins we have shown for the first time that KIR inhibits, in a ligation-independent manner, the AICD induced by PHA, PMA/ionomycin, or anti-CD3 Ab. The AICD inhibition mediated by KIR appears to result from the blockade of Fas ligand induction upon activation of the Jurkat transfectants. Moreover, the membrane-proximal 20 aa of the KIR cytoplasmic tail were determined to play a crucial role in this process. Since the membrane-proximal portion of the KIR cytoplasmic tail contains a putative protein kinase C (PKC) substrate site, we investigated the molecular interaction between KIR and PKC. Immunoprecipitation analysis demonstrated that KIR constitutively bound both to PKCalpha, a conventional Ca(2+)-dependent PKC, and to PKCtheta, a novel Ca(2+)-independent PKC. Furthermore, an in vitro kinase assay revealed that PKC activation was blocked after PHA stimulation in Jurkat transfectants expressing KIR. These observations were supported by the finding that a recombinant KIR cytoplasmic tail also appeared to inhibit PKCalpha activation in vitro. Taken together these data strongly suggest that KIR inhibits the AICD of T cells by blocking Fas ligand induction upon stimulation, in a process that seems to be accomplished by PKC recruitment to the membrane-proximal PKC binding site and subsequent inhibition of PKC activation against the activating stimuli.     

Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation. Role of Bcl-2 protein family.

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4 beta-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by gamma-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x(L) expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.     

Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases

The T cell receptor (TCR)-CD3 complex and the costimulatory molecule CD28 are critical for T cell function. Both receptors utilize protein tyrosine kinases (PTKs) for the phosphorylation of various signaling molecules, a process that is critical for the function of both receptors. The PTKs of the focal adhesion family, Pyk2 and Fak, have been implicated in the signaling of TCR and CD28. We show here evidence for the regulation of TCR- and CD28-induced tyrosine phosphorylation of the focal adhesion PTKs by protein kinase C (PKC). Thus, treating Jurkat T cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) rapidly and strongly reversed receptor-induced tyrosine phosphorylation of the focal adhesion PTKs. In contrast, PMA did not affect TCR-induced tyrosine phosphorylation of CD3zeta or the PTKs Fyn and Zap-70. However, PMA induced a strong and rapid dephosphorylation of the linker molecule for activation of T cells. PMA failed to induce the dephosphorylation of proteins in PKC-depleted cells or in cells pretreated with the PKC inhibitor Ro-31-8220, confirming the role of PKC in mediating the PMA effect on receptor-induced protein tyrosine phosphorylation. The involvement of protein tyrosine phosphatases (PTPases) in mediating the dephosphorylation of the focal adhesion PTKs was confirmed by the failure of PMA to dephosphorylate Pyk2 in cells pretreated with the PTPase inhibitor orthovanadate. These results implicate PKC in the regulation of receptor-induced tyrosine phosphorylation of the focal adhesion PTKs in T cells. The data also suggest a role for PTPases in the PKC action.     

Protein kinase C inhibits CD95 (Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells.

We have recently reported that activation of protein kinase C (PKC) plays a negative role in CD95-mediated apoptosis in human T cell lines. Here we present data indicating that although the PKC-induced mitogen-activated protein kinase pathway could be partially implicated in the abrogation of CD95-mediated apoptosis by phorbol esters in Jurkat T cells, the major inhibitory effect is exerted through a PKC-dependent, mitogen-activated protein kinase-independent signaling pathway. Furthermore, we demonstrate that activation of PKC diminishes CD95 receptor aggregation elicited by agonistic CD95 Abs. On the other hand, it has been reported that UV radiation-induced apoptosis is mediated at least in part by the induction of CD95 oligomerization at the cell surface. Here we show that activation of PKC also inhibits UVB light-induced CD95 aggregation and apoptosis in Jurkat T cells. These results reveal a novel mechanism by which T cells may restrain their sensitivity to CD95-induced cell death through PKC-mediated regulation of CD95 receptor oligomerization at the cell membrane.