妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型,研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化.基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征——DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14、0.49和1.43,与妊娠早期相比,妊娠中、晚期胎盘基因组DNA断裂值有显著性增加.TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层.免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax∶Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势.实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax∶Bcl-2的比例相关.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型。研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化,基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征-DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14,0.49和1.43,与妊娠早期相比,妊娠中,晚期胎盘基因组DNA断裂值有显著性增加,TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层,免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax:Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势,实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax:Bcl-2的比例相关。     

Manganese-induced apoptosis in rat myocytes

Manganese can be toxic to the heart, causing dysfunction following long exposure. In our experiments, we examined the cytotoxicity of manganese in neonatal rat ventricular myocytes (NRVM) by MTT assays in vitro. Results showed that after incubation in the different concentrations of manganese for 24 h, apparent cytotoxicity was observed. At 500, 1000, and 1500 2 microM of manganese, the percentage of cell viability dropped to 82% +/- 6.13, 78% +/- 5.28, and 66% +/- 4.22, respectively. When cells were treated for 48 h, all concentrations tested exerted toxic effect; especially from 500 to 1500 microM the cell viability dropped from 67% +/- 4.84 to 37% +/- 3.25. Apoptosis in NRVM was then examined by flow cytometry. Results showed that the percentage of apoptotic cells treated with 500 microM of manganese for 24 h increased from 4% +/- 0.84 to 7% +/- 1.16. After 48 h of incubation, this percentage increased to 11% +/- 0.91. There was no significant difference between control groups (0 microM manganese) after 24 and 48 h incubation. The morphological changes of NRVM nuclei were visualized with the fluorescent DNA-binding dye Hoechst33342 after incubation in 500 microM of manganese for 48 h. Compared with normal nuclei, apoptotic nuclei showed the typical features of fragmentation and condensation. To investigate whether there are any apoptotic gene expression changes during apoptosis, we examined the expression level of Bcl-2, Bax, and P53 mRNAs after treatment with 500 microM of manganese for 48 h. The Bcl-2 mRNA expression decreased while the expression of Bax as well as P53 mRNAs increased. These results suggested that manganese cytotoxicity on NRVM could induce apoptosis in NRVM cells. The apoptosis process might involve, and be promoted by, the changes of the expression levels of P53, Bcl-2, and Bax proteins.     

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) mRNA expression and localization and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) in human placenta of the first, second, and third trimester

The cellular localization of protein tyrosine phosphatase 51 (PTPIP51) and its in vitro interacting partner protein tyrosine phosphatase 1B (PTP1B) was studied in human placentae of different gestational stages. The expression of PTPIP51 protein and mRNA was observed in the syncytiotrophoblast and cytotrophoblast layer of placentae from the first, second, and third trimesters. In contrast, PTP1B expression was restricted to the syncytiotrophoblast during all gestational stages. Cells of the cytotrophoblasts and parts of the syncytiotrophoblasts expressing high amounts of PTPIP51 were found to execute apoptosis as shown by TdT-mediated dUTP-biotin nick end labeling assay, cytokeratin 18f, and caspase 3 expression. PTPIP51 could also be traced in the endothelium and smooth muscle cells of placental arterial and venous vessels, identified by double immunostainings with antibodies directed against van Willebrand factor and alpha-smooth muscle actin. Some of these cells showing a high PTPIP51 reactivity were Ki67 positive, indicating proliferation. Additionally, a small population of placental CD14-positive macrophages and mesenchymal cells within the villous stroma were detected as PTPIP51 positive. Our data suggest that both proteins, PTPIP51 and PTP1B, play a role in differentiation and apoptosis of the cytotrophoblast and syncytiotrophoblast, respectively. Moreover, PTPIP51 may also serve as a cellular signaling partner in angiogenesis and vascular remodeling.     

Some oncogene and tumour suppressor gene protein products expression in B-cell chronic lymphocytic leukaemia

The expression of Bcl-2, P53 proteins and known markers of proliferation, namely proliferating cell nuclear antigen (PCNA) and Ki67, in 29 patients with B-cell chronic lymphocytic leukaemia (B-CLL) was investigated. All leukaemic patients were classified, and immunophenotyped by the two-colour immunofluorescence method with the use of fluorocytometry. B-CLL was heterogeneous in the range of biological parameters of tumour cells. B-CLL patients manifested 34% positive Ki67 and 61% PCNA expression, whereas Bcl-2 and P53 positivity was 81% and 42%, respectively. The level of intracellular expression of Bcl-2 and P53 proteins did not depend on the stage of disease estimated by routine methods. Ki67 and PCNA expression was significantly higher in B-CLL patients with more advanced stages of the disease. A statistically significant correlation was established between their mutual expression.     

The presence of 19-kDa Bcl-2 in dividing cells

The 26-kDa bcl-2 gene product inhibits apoptosis and cell proliferation. Cleavage of Bcl-2 into a 22-kDa fragment inactivates its anti-apoptotic activity and is a key event in apoptosis. Here, and in recent work, we describe massive 19-kDa Bcl-2 immunoreactivity in non-apoptotic cells, suggesting a link with viability rather than cell death. Loss of 19 kDa Bcl-2 in adriamycin-induced apoptotic cells underlines this. G2/M-phase accumulation of cells by nocodazole-treatment also results in loss of 19 kDa Bcl-2. Next to its well-documented cytoplasmic localization, a substantial pool of Bcl-2 resides in nuclei. Hampered nuclear localization of Bcl-2 leads to a loss of cell cycle repression. This has led us to point at a pivotal role for nuclear Bcl-2 in cellular proliferation. In this report, cellular fractionation of bcl-2 transfected cells in various phases of the cell cycle reveals a constitutive cytoplasmic pool of 19 kDa Bcl-2. Nuclear 19-kDa Bcl-2 immunoreactivity is far more pronounced in rapidly dividing nuclei compared with more quiescent nuclear fractions. This implicates that ongoing cell proliferation involves cleavage of nuclear Bcl-2 with a 19-kDa fragment.     

梓醇对氧糖剥夺诱导PC1 2 细胞凋亡的保护作用

目的：观察梓醇对氧糖剥夺（OGD）诱导PC12细胞凋亡的保护作用。方法：采用Hoechst 33258 DNA染色法,四甲基偶氮唑盐（MTT）检测细胞活性;化学比色法测定乳酸脱氢酶（LDH）的释放量,用流式细胞技术检测细胞凋亡比例以及P53和Bcl-2蛋白。结果：OGD可导致PC12细胞活力明显下降,LDH释放量增加、P53蛋白表达上升,Bcl-2蛋白表达下降。梓醇可明显改善细胞形态结构,显著降低LDH释放量、降低P53蛋白的表达,提高Bcl-2蛋白的表达,降低细胞凋亡率。结论：梓醇通过调节细胞凋亡相关基因的表达而抑制细胞凋亡。     

Impaired cytotrophoblast cell-cell fusion is associated with reduced Syncytin and increased apoptosis in patients with placental dysfunction

Preeclampsia (PE), Hemolysis Elevated Liver Enzymes and Low Platelets (HELLP)-syndrome, and intrauterine growth restriction (IUGR) are associated with abnormal placentation. In early pregnancy, placental cytotrophoblasts fuse and form multinuclear syncytiotrophoblasts. The envelope gene of the human endogenous retrovirus-W, Syncytin, is a key factor for mediating cell-cell fusion of cytotrophoblasts. This study investigated clinical parameters of PE and HELLP-associated IUGR and analyzed the cell-cell fusion index and beta-human chorionic gonadotropin (beta-hCG) secretion of cytotrophoblasts isolated and cultured from placentas of these patients. In addition, we performed absolute quantitation of Syncytin and determined the apoptosis rate in both cultured cytotrophoblasts and placental tissues. Cultured cytotrophoblasts from PE and HELLP-associated IUGR correlated with a pronounced lower cell-cell fusion index, 1.8- and 3.6-fold; less nuclei per syncytiotrophoblast, 1.4- and 2.0-fold; a significantly decreased beta-hCG secretion, 4.3- and 17.2-fold and a reduction of Syncytin gene expression, 8.1 (P = 0.019) and 222.7-fold (P = 0.011) compared with controls, respectively. In contrast, a significantly 2.3-fold higher apoptosis rate was observed in cultured PE/IUGR cytotrophoblasts (P = 0.043). Importantly, Syncytin gene expression in primary placental tissues of PE/IUGR was 5.4-fold lower (P = 0.047) and in HELLP/IUGR 10.6-fold lower (P = 0.019) along with a 1.8- and 1.9-fold significant increase in the apoptosis rate compared with controls, respectively. Low Syncytin expression in both cultured cytotrophoblasts and primary tissues from pathological placentas supports an intrinsic placenta-specific deregulation of cell-cell fusion in the formation of syncytiotrophoblasts leading to increased apoptosis. These processes could contribute to the development and severity of PE and HELLP-associated IUGR.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.     

Apoptotic signaling proteins: possible participation in the regulation of vasopressin and catecholamines biosynthesis in the hypothalamus

The role of apoptotic signaling proteins for long-lived neurons in the mature brain is poorly understood. Recently, we have shown that water deprivation leads to the activation of vasopressin (VP) secretion and expression of Bcl-2 and caspase-9 apototic proteins in the hypothalamus of the rat brain. In the present work, we continued to study a possible relationship between the functional activity of neurosecretory cells of the hypothalamus and apoptosis related proteins. We found that water deprivation leads to simultaneous activation of synthesis of VP and p53 and Bcl-2 apoptotic proteins in the mouse brain. To study a possible effect of apoptotic proteins on the functional state of hypothalamic neurons, the VP and tyrosine hydroxylase (TH) synthesis were analyzed in p53, p21^Waf1/Cip1 and Bcl-2 deficient mice. Loss of p53 and Bcl-2 significantly reduced VP synthesis in paraventricular and supraoptic nuclei and TH expression in arcuat, periventricular and zona incerta nuclei of the hypothalamus. Surprisingly, in contrast with the loss of p53, the inactivation of p21^Waf1/Cip1 up-regulates the expression of VP and TH. These data indicate that p53, p21^Waf1/Cip1 and Bcl-2 proteins, besides affecting cell cycle, tumor suppression and apoptosis, may act as modulators of neurosecretory activity of hypothalamic neurons; however, this problem remains to be determined more detailed.     

Epidermal growth factor-like growth factors prevent apoptosis of alcohol-exposed human placental cytotrophoblast cells

Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0-100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1-2 h of exposure to 50 mM alcohol. Exposure to 25-50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism.     

Differential Effects of Sodium Butyrate and Lithium Chloride on Rhesus Monkey Trophoblast Differentiation

Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O₂) and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of β-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of β-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/β-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNFα production. The results provide valuable tools to manipulate trophoblast differentiation in vitro and to better understand the differentiation pathways that occur during early gestation.     

Altered Bcl-2 and Bax expression is associated with cultured first trimester human cytotrophoblasts apoptosis induced by hypoxia

Preeclampsia is associated with placental hypoxia at early gestation. We therefore investigated the effect of hypoxia on the apoptosis of cultured first trimester human cytotrophoblasts and the expression of apoptosis relevant proteins, Bcl-2 and Bax. First trimester human cytotrophoblasts were isolated and cultured under either standard or hypoxic conditions. Cellular apoptosis was monitored by TUNEL and Annexin V binding, and the expression of Bcl-2 and Bax was determined by Western blot analysis. Apoptosis increased significantly in cytotrophoblasts cultured for 24 h under hypoxic conditions in contrast with those cultured under standard conditions, meanwhile expression of Bcl-2 reduced, and that of Bax increased. These changes suggested that hypoxia induced apoptosis in cultured first trimester cytotrophoblasts with altered Bcl-2 and Bax expression. Further study is needed to explore the role of cytotrophoblasts apoptosis in hypoxia-induced maternal and fetal diseases.     

Human chorionic gonadotropin contributes to maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system

The first known hormonal signal of the conceptus during implantation is human chorionic gonadotropin (hCG). Interestingly, increased apoptosis in human endometrium coincides with the implantation window. Factors from the fetal or placental origin as well as maternal hormonal factors are likely to have a potential role in the regulation of apoptotic signaling molecules. We hypothesized that hCG may be a placental link for the development of local maternal immunotolerance. Fas-Fas ligand (FasL) system is one of the apoptotic signaling pathways, shown to be important in the development of local immune tolerance during and after implantation. We report that hCG treatment decreases cell proliferation and increases apoptosis in endometrial cells. Moreover, hCG stimulates FasL mRNA and protein expression without affecting Fas mRNA in these cells. Interestingly, in coculture experiments, hCG-treated endometrial cells induce an increase in T cell apoptosis. Our in vivo results reveal that cells of early pregnancy decidua express strong FasL immunoreactivity, and decidual areas containing interstitial cytotrophoblasts have numerous TUNEL-positive cells. Compared with decidual areas devoid of interstitial cytotrophoblasts, we observed in decidual areas containing interstitial cytotrophoblasts clearly less amount of TUNEL-positive cells. These results suggest that hCG may be a link in the development of peritrophoblastic immune tolerance and may facilitate the trophoblast invasion by regulating proapoptotic molecules such as FasL in endometrial cells.     

Immunohistochemical expression of p53, p21/waf1, rb, p16, cyclin D1, p27, Ki67, cyclin A, cyclin B1, bcl2, bax and bak proteins and apoptotic index in normal thymus

The immunohistochemical expression of p53, p21, Rb, p16, cyclin D1, Ki67, cyclin A, cyclin B1, p27, bcl2, bax, and bak proteins and the apoptotic index (Al) were investigated in 20 normal thymuses (8 adults, 3 adolescents, 5 infants and 4 newborns). The expressions of Rb, Ki67, cyclin A and cyclin B1 were overlapping, being high in the cortex with a tendency for decreased expression toward the medulla. Apoptotic cells were mainly detected in the cortex and the corticomedullary junction, rarely being present in Hassall's corpuscles. The mean values of Ki67, cyclin A, and cyclin B1 expression in thymuses were 77.2%, 32.2% and 21.4% (newborns), 62.4%, 33.7% and 18.5% (infants), 56.9%, 23.4% and 18.9% (adolescents) and 38.7%, 21.7% and 14.6% (adults), respectively. The mean values of AI in thymuses from newborns, infants, adolescents and adults were 1.4%, 2.9%, 2.7% and 3.8%, respectively. This decrease in proliferation and increase in apoptosis may account for the process of thymic involution. P16 expression was widespread with most of Hassall's corpuscles being p16-positive. P16-positive cells and Hassall's corpuscles increased with the increase in age, in keeping with the suggested role of p16 in cellular senescence. P27 expression was undetectable in subcapsular thymocytes with a tendency for increased expression toward the medulla. The expressions of Ki67, cyclin A and cyclin B1 were inversly related with that of p27, consistent with previous evidence that p27 concentration is reduced when the cell-cycle progresses. P21 and much less frequently p53 proteins were mainly detected in a part of the subcapsular cortical epithelial cells. These findings suggest that a) in thymocytes, the apoptotic pathway is mostly p53-independent and the function of p21 as a negative regulator of the cell cycle must be redundant to other negative regulators, such as p16 and p27 which were abundantly detected in thymocytes and b) in some thymic epithelial cells, the p21 expression may be induced by p53, but in most of them seems to be p53-independent. Most of Hassall's corpuscles were p21-positive, consistent with previous evidence that these structures represent end stages of maturation of thymic medullary epithelium and that p21 protein is involved in the process of terminal differentiation. Cyclin D1 positivity was found in some macrophages. Bcl2 expression was mainly seen in medullary thymocytes, reflecting the surviving thymocytes in this region. The expressions of Bax and bak were more widespread in both the medulla and cortex, suggesting that these proteins play a broader role than bcl2 in the regulation of thymic apoptosis.     

Can transforming growth factor-beta1 and retinoids modify the activity of estradiol and antiestrogens in MCF-7 breast cancer cells?

Retinoic acid and transforming growth factor-beta (TGF-beta) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-beta1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-beta1 concentrations, a marked reduction in the stimulatory action of estradiol (10(-9) and 10(-8) M) was observed whereas in combination with tamoxifen (10(-7) and 10(-6) M) only 30 ng/ml TGF-beta1 caused a statistically significant reduction to approximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-beta1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 +/- 19% (control =100%) by 3 ng/ml TGF-beta1, and this dose was used throughout. It was found that addition of TGF-beta1 and isotretinoin to the culture did not decrease proliferation, while TGF-beta1 and tretinoin at low concentrations (3 x 10(-8) and 3 x 10(-7) M) reduced the percentage of proliferating cells by approximately 30% (67+/-8% and 67+/-5%, P<0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10(-9) M estradiol, attenuated by TGF-beta1. In addition, the retinoids in combination with TGF-beta1 and tamoxifen (10(-6) M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-beta1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying easier apoptotic induction. The majority of the examined compounds act via the p53 pathway although some bypass this important proapoptotic factor.     

Apoptotic index or a combination of Bax/Bcl-2 expression correlate with survival after resection of pancreatic adenocarcinoma

In the present study, the prognostic impact of factors involved in the apoptosis pathway were tested on 67 consecutive patients treated with surgical resection. Included in the study were all patients resected for pancreatic adenocarcinoma from 1988 to 2003. Expression analysis for p53, Bax, and Bcl-2 were performed by immunohistochemical staining. Apoptotic cells were identified by the TUNEL method. These data were correlated with survival. Sixty-seven tumor specimens were included in the study. A strong positive correlation was recorded between p53 overexpression and Bax expression levels (P < 0.001). By univariate analysis, overall survival seemed to be improved with Bcl-2 and Bax expression (respectively, P = 0.0379 and 0.0311). The median survival time in patients with low apoptotic index was better versus those with a high index (P = 0.0127). Lymph node involvement was the only clinico-pathologic parameter that significantly correlated with overall survival (P = 0.0202). By a multivariate Cox regression analysis, the only immunohistochemical parameter that influenced overall survival was the apoptotic index (P = 0.040). Tumor's overexpression of both Bax and Bcl-2 resulted the strongest independent prognostic factor (P = 0.013). This is the first study to report a statistically significant association of apoptosis to overall survival for pancreatic cancer patients treated with surgical resection. The contemporary overexpression of Bax and Bcl-2 represents the strongest prognostic factor.