The complexity of parathyroid hormone-related proteinsignalling

Our understanding of the mode of action of parathyroid hormone-related protein (PTHrP) has changed profoundly during the last decade. Most PTHrP activities are mediated by membrane receptors through autocrine/paracrine pathways. However, both endogenous and exogenous PTHrP also appear to have intracrine effects through translocation into the nucleus. The present review proposes unconventional PTHrP signalling, based on novel clues. First, PTHrP binding to its membrane receptor triggers internalization of the whole complex, mediated by beta-arrestin. There is growing evidence that the receptor and arrestin are the effectors of biological responses, rather than the ligand (or in addition to the ligand). Second, the existence of putative PTHrP targets within the cytoplasm is beginning to be supported. Recent findings of interactions between a COOH-terminus of PTHrP and beta-arrestin and between the PTHrP receptor and 14-3-3 proteins represent the starting point for identification of intracellular partners of both the hormone and its receptor.Received 19 June 2003; received after revision 10 July 2003; accepted 21 July 2003     

Differential effects of monastrol in two human cell lines

The kinesin-related protein HsEg5 plays essential roles in mitotic spindle dynamics. Although inhibition of HsEg5 has been suggested as an aid in cancer treatment, the effects of such inhibition on human cells have not been characterized. Here we studied the effects of monastrol, an allosteric HsEg5 inhibitor, on AGS and HT29 cell lines and compared them to those of taxol. While both cell lines were similarly sensitive to taxol, AGS cells were more sensitive to monastrol. The differences in sensitivity were determined by the degree of inhibitory effect on cell proliferation, reversibility of monastrol-induced G2/M arrest, intracellular phenotypes and induction of apoptosis. In both cell lines, monastrol-induced apoptosis was accompanied by mitochondrial membrane depolarization and poly-ADP-ribose polymerase 1 cleavage. In AGS, but not HT29 cells, monastrol-induced apoptosis involved a prominent cleavage of procaspases 8 and 3. While in AGS cells, monastrol induced the formation of symmetric microtubule asters only, in HT29 cells, asymmetric asters were also formed, which may be related to specific HsEg5 functions in HT29 cells.Received 18 February 2004; received after revision 30 May 2004; accepted 16 June 2004     

Administration of the antitumor drug mitoguazone protects normal thymocytes against spontaneous and etoposide-induced apoptosis

The suggestion has been made that polyamines may be involved in the control of cell death, since exceedingly high or low levels induce apoptosis in different cell systems. For a deeper insight into the relationship between apoptosis and polyamine metabolism, we investigated in vitro the effect on rat thymocytes of mitoguazone (MGBG, which inhibits S-adenosylmethionine decarboxylase, i.e. a key enzyme in the polyamine biosynthetic pathway). Thymocytes were selected as an especially suitable model system, since they undergo spontaneous apoptosis in vivo and can be easily induced to apoptose in vitro by etoposide, used here as an apoptogenic agent. MGBG protected thymocytes from both spontaneous and drug-induced apoptosis, and this protective effect was associated with a decrease in polyamine oxidase activity and total polyamine levels.Received 7 July 2004; received after revision 2 September 2004; accepted 9 September 2004     

Structure and function of bak gene and its apoptosis-inducing mechanism

The Bcl-2 family plays an important role in the regulation of apoptosis. Some members in the family prevent apoptosis while others promote it. The newly discovered bcl-2 homolog, bak, promotes the apoptosis induced by a variety of stimuli. It can interact with the anti-apoptotic members through its BH3 domain and antagonize their activity. Investigation results suggest that the expression of bak gene becomes altered in some diseases, such as cancer. The preferential stimulation of Bak-induced apoptosis, therefore, may be involved in the mechanisms of some an-titumor drugs.     

EGF-induced programmed cell death of human mammary carcinoma MDA-MB-468 cells is preceded by activation of AP-1

MDA-MB-468 is a human mammary adenocarcinoma cell line that overexpresses the epidermal growth factor (EGF) receptor and undergoes programmed cell death (apoptosis) in response to EGF treatment. Programmed cell death was shown to be greatly enhanced when cells were growth-arrested prior to EGF treatment. Apoptosis was characterized by an initial rounding up and detachment of the cells from their substrate starting about 12 h after EGF treatment, followed by chromatin condensation, nuclear fragmentation and oligonucleosomal fragmentation of the DNA at about 24 to 48 h. Cell death was dependent on de novo protein synthesis. We found a rapid induction of c-fos, c-jun and junB at the mRNA level after about 30 min of EGF treatment and a more delayed upregulation of fosB and fra-1. The junD gene was expressed in the absence of EGF, and it was moderately induced within 30 min of growth factor addition. The increase of the different fos and jun mRNAs were paralleled by an increase of activator protein-1 (AP-1) DNA binding activity. A characterization of the AP-1 complex revealed similar levels of several Fos and Jun proteins. Based on the kinetics of AP-1 accumulation and cell death, it seems likely that AP-1 contributes to the apoptotic cell death of EGF receptor-overexpressing MDA-MB-468 cells. Received 21 July 1997; received after revision 6 November 1997; accepted 6 November 1997     

敏感和抗性的白血病HL60细胞对staurosporine诱导凋亡的不同反应(英)

用对蛋白激酶具有强烈抑制、作用广泛的抑制剂staurosporine（Sta）,研究敏感和抗三尖杉酯碱的人白血病HL60细胞中凋亡和多药抗药性的关系．Sta均能诱导2种细胞发生典型的凋亡,但抗性细胞发生凋亡需更长的时间,凋亡的细胞数减少．Sta增加柔红霉素在抗性细胞内的积聚,说明其能逆转多药抗药性．在抗性细胞凋亡过程中,mdrl基因表达没有变化,c-myc基因表达稍有增加．结果显示：Sta能诱导敏感和抗三尖杉酯碱的HL60细胞发生凋亡,mdrl基因表达与凋亡过程无关．     

兔血清诱导Shope肿瘤细胞凋亡的实验研究

目的：前期实验结果已证明兔血清对Ｓｈｏｐｅ肿瘤细胞具有伤作用。本实验拟从细胞凋亡角度探讨兔血清杀伤该细胞的机理。方法：通过电子显微镜、激光共聚焦显微镜、琼脂糖凝胶电泳及流式细胞仪检测等方法,观察了兔血清诱导细胞凋亡的可能性。结果：经１０％兔血清处理３ｈ的中等分化程度的Ｓｈｏｐｅ肿瘤细胞（ＳＣＢ－６ａ）在电镜、激光共聚焦显微镜下均可见典型的细胞凋亡的形态学特征;１．５％琼脂糖凝胶电泳呈现明显的“阶梯状”图谱（ＤＮＡ ｌｅｄｄｅｒ）;ＰＩ染色后经流式细胞仪检测显示大量亚二倍体细胞（凋亡细胞）。结论：兔血清诱导Ｓｈｏｐｅ肿瘤细胞凋亡可能是其伤细胞的作用机理之一。     

反义基因逆转肿瘤细胞多药耐药性和诱导细胞凋亡的研究

探讨克服肿瘤细胞多药耐药（ＭＤＲ１）性的方法,提高化疗效果。本文采用多药耐药反义基因（ＭＤＲ１－ＲＳＰＳ－ＯＤＮ）逆转Ｋ５６２／ＡＤＭ肿瘤细胞的ＭＤＲ１,诱导肿瘤细胞凋亡,发现ＭＤＲ１－ＡＳＰＳ－ＯＤＮ诱导Ｋ５６２／ＡＤＭ细胞株细胞产生大量ＤＮＡ断片,ＦＡＣＳ检测发现几乎全部ＭＲＤ＋Ｋ５６２／ＡＤＭ细胞发生凋亡。其结果表明ＤＭＤＲ１－ＡＳＰＳ－ＯＤＮ能有效、特异地抑制ＭＤＲ１基因表达,逆转肿瘤细胞的ＭＤＲ１,促进阿霉素诱导ＭＤＲ＋１Ｋ５６２／ＡＤＭ细胞凋亡,为其临床应用提供理论依据     

Two tyrosines in CD3ε-ITAM are required to induce T lymphocyte apoptosis

CD3ε of T cell antigen receptor complex (TCR/CD3) plays an important role in the resembling of the complex and activation signaling through its conservative immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic tail. Previous study showed that a chimera molecule, consisting of the extracellular-transmembrane domain of human CD8α fused to the cytoplasmic domain of CD3ε, induced apoptosis of T lymphocytes, indicating that apoptotic signals were transduced through the CD3ε- ITAM. To elineate involvement of the two tyrosines in apoptotic signaling pathway, cDNAs with mutations at Y170F, Y181F and Y170F/Y181F in CD8-ε-ITAM were made by point mutation and PCR, and then cloned into pcDNA3 eukaryotic expression vectors. Stable expression cell lines were established after transfection of the expression vectors into CD8+- Jurkat T lymphocytes. Stimulation of these cell lines with anti-CD8 monoclonal antibody showed that only the cells with expression of wild type chimera CD8-ε died by apoptosis, but not those cells with expressions of mutated CD8-ε chimera, indicating that the two tyrosines in CD3ε-ITAM were required for the apoptotic signal transduction in T lymphocytes.