Differentiation and Growth Modulation of Myeloid Leukemia Cells by the Protein Kinase C Activating Agent Bryostatin-1

Bryostatin-1 (Bryo), a macrocyclic lactone of the sea water bryozoan Bugula neritina, is a potent activator of protein kinase C and was found to exhibit antineoplastic activity in several systems. We studied the effect of Bryo on differentiation and growth modulation of human myeloid leukemia cell lines and freshly explanted blood cells from patients with myeloid leukemia. Alterations at the molecular level and phenotypic changes triggered by Bryo were similar, but not identical, to those induced by phorbol esters. Bryo was able to inhibit cellular proliferation as evidenced by [³H]-thymidine uptake and induced morphological changes associated with monocytic differentiation. In studies using continuous cell lines, the glucocorticoid dexamethasone was unable to prevent the Bryo-induced growth inhibition or the induced phenotypic changes. However, in fresh myeloid Blood cells dexamethasone attenuated these Bryo-triggered effects. Our own data taken together with reports from the literature reviewed here suggest the following conclusions: (i) Bryo, while lacking tumor promoting activity, is able to induce differentiation in maturation arrested leukemia cells; (ii) it exhibits selective antiproliferative properties in normal or malignant hematopoietic cells and supports growth of multipotent stem cells. These features might qualify Bryostatin-1 as a potential candidate for promising research and possibly for future clinical applications.     

Lung Cancer Cells Often Express High Levels of Protein Kinase C Activity

We analyzed protein kinase C (PKC) activity in twenty-two tumor cell lines derived from lung, pancreas, stomach, tongue and vulva, and found that lung cancer cells often (9 out of 13) exhibit significantly higher PKC activity than other types of cancer cells. The PKC in these lung cancer cells was separated into one major and one minor peaks by a Mono Q column chromatography. The PKC in the major peak had an absolute requirement for Ca^2±, phosphatidylserine and 12-O-tetradeca-noylphorbol-13-acetate (TPA), as expected. However, the PKC in the minor peak did not require TPA for its activation. Hydroxyapatite column chromatography revealed that the PKC in the major peak is type III. These results indicate that in lung cancer cells type III PKC activity is often elevated compared to other types of cancer cells. The growth of many lung cancer cell lines was inhibited by TPA.     

Resveratrol Affects Protein Kinase C Activity and Promotes Apoptosis in Human Colon Carcinoma Cells

Background: Resveratrol has been reported to have potential chemopreventive and apoptosis-inducing properties in a variety of tumor cell lines. Objective: In this study, to investigate the effects of resveratrol on protein kinase C (PKC) activity and apoptosis in human colon carcinoma cells, we used HT-29 cells and examined the PKCα and ERK1/2 signaling pathways. Methods: To test the effects of resveratrol on the growth of HT- 29 cells, the cells were exposed to varying concentrations and assessed with the the MTT cell-viability assay.Fluorescence-activated cell sorter (FACS) analysis was applieded to determine the effects of resveratrol on cell apoptosis. Western blotting was performed to determine the protein levels of PKCα and ERK1/2. In inhibition experiments, HT-29 cells were treated with Gő6976 or PD98059 for 30 min, followed by exposure to 200 μM resveratrol for 72 h. Results: Resveratrol had a significant inhibitory effect on HT-29 cell growth. FACS revealed that resveratrol induced apoptosis. Western blotting showed that e phosphorylation of PKCα and ERK1/2 was significantly increased in response to resveratrol treatment. Pre-treatment with PKCα and ERK1/2 inhibitors (Gő6976 and PD98059) promoted apoptosis. Conclusion: Resveratrol has significant anti-proliferative effects on the colon cancer cell line HT-29. The PKC- ERK1/2 signaling pathway can partially mediate resveratrol-induced apoptosis of HT-29 cells.     

涎腺肿瘤中p53蛋白和血管内皮生长因子的表达及意义

 0引言涎腺肿瘤是口腔颌面部除牙源性肿瘤以外的另一类特征性肿瘤,是危害人类健康的一大类疾病,临床上尚缺少早期判断肿瘤性质和评估预后的指标。近期有关p53和VEGF与涎腺肿瘤关系的研究也有一些报道,但存在分歧。本研究通过免疫组织化学方法检测54例涎腺良恶性肿瘤标本p53蛋白、VEGF的表达,探讨二者与涎腺肿瘤生物学特性的关系及其表达的临床意义。     

Inhibition by Protein Kinase C Inhibitor of Expression of Leukocyte Function-associated Antigen-1 Molecules in Rat Hepatoma AH66F Cells

To examine the mechanism of inhibition by protein kinase C (PKC) inhibitors of the adhesion of highly malignant hepatoma AH66F cells to the mesentery-derived mesothelial cell (M-cell) layer through leukocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1, the effects of a PKC inhibitor, NA-382, on the expression of LFA-1 molecules in AH66F cells were examined and compared with those in thymocytes from normal rats. NA-382 inhibited the adhesion of AH66F cells to the M-cell layer and the expression of LFA-1 on the membrane of the hepatoma cells after treatment for more than 24 h. It was confirmed that AH66F cells express similar mRNAs for LFA-1 subunits to those of thymocytes, and their levels were also decreased after treatment with NA-382. On the other hand, the LFA-1-mediated adhesion and the expression of both protein and mRNA for LFA-1 subunits in thymocytes were not changed by the PKC inhibitor. These results suggest that the expression of LFA-1 molecules in AH66F cells may be regulated by PKC via quite different mechanisms from those in normal lymphocytes     

Mitogenic Properties of Insulin-Like Growth Factors I and II, Insulin-like Growth Factor Binding Protein-3 and Epidermal Growth Factor on Human Breast Stromal Cells in Primary Culture

Insulin-like growth factors I and II (IGF-I and IGF-II) are growth factors implicated in both normal mammary gland development and breast cancer. We have previously reported on the effects of components of the IGF system on breast epithelial cells. Since data suggests that stromal-epithelial interactions play a crucial role in breast cancer, we have now investigated the mitogenic properties of IGF-I, IGF-II, insulin-like growth factor binding protein-3 (IGFBP-3) and epidermal growth factor (EGF) on human breast stromal cells in primary culture. We show that, under serum-free conditions, stromal cells are stimulated to grow in response to IGF-I and IGF-II in a dose-dependent manner. IGF-I and EGF, a potent stimulator of human breast epithelial cell growth in primary culture and also associated with breast cancer, appear to stimulate stromal cell growth in a synergistic manner. IGFBP-3 does not inhibit the stimulation of growth by IGF-I, or IGF-I plus EGF. However, IGFBP-3 does inhibit the stimulation of growth by IGF-II. In contrast to our previous results with human breast epithelial cells, IGFBP-3 does not have an IGF-independent inhibitory effect on stromal cell growth. This study is the first to address the effects of IGF-I, IGF-II and IGFBP-3 alone and in combination with EGF on human breast stromal cell growth in primary culture. Characterizing the role of the IGF system in both normal breast epithelial cells and stromal cells will aid in our understanding of the mechanisms behind the role of the IGF system in breast cancer.     

Expression Behavior of 85-kDa Membrane Protein in Adriamycin-resistant Tumor Cells and the Inhibition of Human Tumor Growth in Athymic Mice by MRK-20 Monoclonal Antibody against the Protein

Treatment of tumors with monoclonal antibodies against tumor antigen is one of the selective modalities for cancer therapy. We examined the therapeutic effect of MRK-20 (IgG₁), a murine monoclonal antibody against resistance-associated 85-kDa membrane protein. The 85-kDa protein is expressed on the surface of multidrug-resistant cells induced by adriamycin. This protein is also expressed in some multiple-drug-resistant cells, including atypical multidrug-resistant cells. The protein, once lost during long-term culture without adriamycin, was rapidly induced by treatment with adriamycin but not with vinblastine or etoposide, suggesting a close relationship of the protein with adriamycin resistance but not with multidrug resistance. The antibody MRK-20 suppressed the growth of subcutaneously implanted tumors expressing the 85-kDa protein. Adriamycin-resistant human ovarian tumor 2780AD and innately resistant human erythroleukemia HEL cells in athymic mice were completely cured by treatment with MRK-20 antibody when the antibody was administered i.v. 2 days after s.c. tumor implantation. On the other hand, MRK-20 did not show any effect on the growth of the 85-kDa protein-negative A2780 human ovarian tumor. These results indicate that the effect of MRK-20 is highly specific to cells expressing 85-kDa protein.     

表皮生长因子与肿瘤坏死因子在胸腔积液中的表达

采用ＲＩＡ法对５８例良、恶性胸腔积液中ＥＧＦ与ＴＮＦ进行含量检测。结果显示２２例良性胸水中ＥＧＦ与ＴＮＦ含量分别为２．０３±０．３５μｇ／Ｌ，１．６３±０．５４μｇ／Ｌ。３６例恶性胸水中ＥＧＦ与ＴＮＦ含量分别为１．２５±０．３２μｇ／Ｌ，１．０２±０．２４μｇ／Ｌ。良性胸水组ＥＧＦ与ＴＮＦ浓度均明显高于恶性胸水组，两组间有显著性差异，（Ｐ＜０．０５）。结果表明ＥＧＦ与ＴＮＦ均参与了良、恶性胸腔积液的免疫病理生理过程。     

Protein Kinase C Inhibition by UCN-01 Induces Apoptosis in Human Glioma Cells in a Time-dependent Fashion

Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly. we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3–6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2–4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.     

脑肿瘤p53 蛋白和端粒酶活性表达及临床意义的研究

目的 :探讨p53基因蛋白产物和端粒酶活性在脑肿瘤中的表达情况及其相关性 ,和两者与脑肿瘤的病理类型、恶性程度之间的关系。方法 :采用免疫组化SABC法和改进的TRAP银染法对 4 8例脑肿瘤及 10例正常脑组织中p53蛋白和端粒酶活性进行检测。结果 :脑肿瘤中p53蛋白表达检测结果为70 8% (34/ 4 8) ,端粒酶活性检测结果为 77 1% (37/ 4 8) ,正常脑组织中两者均呈阴性表达。其中 2 9例脑肿瘤端粒酶活性与p53蛋白均有表达。结论 :p53和端粒酶参与脑肿瘤的发生发展 ,p53蛋白和端粒酶活性表达与脑肿瘤的病理类型、恶性程度密切相关 ,有可能成为恶性肿瘤的重要相关标志物 ,脑肿瘤中p53蛋白与端粒酶活性表达无相关性。     

Expression of Protein Kinase C Family in Human Hepatocellular Carcinoma

Protein kinase Cs (PKCs) play important roles in signal transduction, cell regulation, and tumor formation. In the present study, we analyzed the expression of PKCs in human hepatocellular carcinoma (HCC) tissues and explored their roles in the development of HCC. Real-time quantitative PCR and immunohistochemistry showed that PKCβ and PKCθ were down-regulated in HCC tissues. Reduced expression of PKCθ is well correlated with the grade of cancer cells (p = 0.009), and the down-regulated expression of PKCβII is associated with HBV infection (p = 0.035). Our findings suggest particular roles of the two PKC isoenzymes in the hepatocarcinogenesis of human HCC.     

Selenium Causes Growth Inhibition and Apoptosis in Human Brain Tumor Cell Lines

We examined the effect of the trace element selenium on human glioma cell lines: T98G, U373MG, and U87MG, in addition to dermal fibroblast cells. Cultures were incubated with sodium selenite, and the following parameters were studied: cell growth, mitochondrial function, and ultrastructure. Cell growth was assayed by counting the number of viable cells after treatment with selenium. Mitochondrial function was analyzed using the MTT (tetrazolium salt reduction) assay. Apoptosis was determined by evaluating nuclear chromatin condensation by electron microscopy.The results indicated that selenium had a significant inhibitory effect on the growth of the tumor cells but had little effect upon dermal fibroblasts which had been passaged numerous times. Selenium also induced mitochondrial damage as shown by MTT assay in two brain tumor cell lines and in minimally passaged fibroblasts, but it had little effect upon the high-passage fibroblasts. Ultrastructurally, mitochondria had electron-dense inclusions resulting from selenium treatment. High rates of apoptosis were induced by selenium in the tumor cell lines and in the minimally passaged fibroblasts, whereas the fibroblasts with a high number of passages had some resistance to selenium treatment. This study correlates the adverse effects of selenium on mitochondrial function, inhibition of cell growth, and apoptosis and shows that selenium similarly affects three different brain tumor cell lines and minimally passaged fibroblasts. Further, the results with fibroblasts show that some types of cells after repeated passages can develop resistance to selenium damage.     

蛋白激酶C与耐药性KB细胞的多药抗药性

目的 :研究蛋白激酶C(PKC)在肿瘤细胞多药抗药性的作用及机制。方法 :测定KB细胞及其耐药株的PKC活性及PKC调节剂对细胞药敏、药物代谢和耐药膜糖蛋白表达及其磷酸化的影响。结果 :耐药株胞质和胞膜的PKC活性分别是KB细胞的 6 6倍和 5 2倍 ,膜糖蛋白阳性率为 4 7 5 % ,明显高于KB细胞的 2 8% ;而且膜糖蛋白的磷酸化明显增强。耐药株对罗丹明 12 3的外排显著加快 ,蓄积明显减少。PKC激活剂佛波乙酯使耐药株的药物外排增加了 60 % ,蓄积减少了 70 % ;PKC抑制剂Stau rosprin使耐药株药物外排后潴留增加了 2 5倍 ,蓄积增加了 14倍 ,抗药性被逆转了 14倍 ,但未影响耐药膜糖蛋白的表达。结论 :PKC活性增高与耐药株抗药性密切相关 ;而且抗药性是通过膜糖蛋白磷酸化增强 ,增加药物外排 ,减少药物蓄积而实现的     

Synergistic Effect of Genistein and BCNU on Growth Inhibition and Cytotoxicity of Glioblastoma Cells

Objective: Recent experiments have shown that dietary soy isoflavones such as genistein can significantly suppress invasiveness and growth of a number of human malignancies. This study examined whether genistein, at a concentration typical of plasma levels following soy diet intake, in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine) exhibited an additive or synergistic inhibitory effect on the growth of glioma cells. Methods: The human glioblastoma multiforme (GBM) cell line U87 and the rodent C6 glioma were treated with genistein at 4M, combined with BCNU (0–50M). Monolayer cell growth and cytotoxicity, as measured by colonigenic survival in soft agarose, were then compared in control and drug-treated cultures. Presence of apoptosis, using the DNA ladder assay and laser scanning cytometry (LSC), was investigated in all cell lines at those concentrations where an enhancement of antiproliferative effect of BCNU in presence of genistein was observed. Results: A 32–41% increase in monolayer growth inhibition and a 28–42% increase in colony cytotoxicity in the U87 cell line were observed when genistein (4M) was added to BCNU in the 0–10M dose range. In the C6 cell line, a 30–36% increase in monolayer growth inhibition and a 39–54% increase in colony cytotoxicity were observed with the BCNU dose range of 0–50M. All experiments showed a significant increase in growth inhibition and a decrease in colonogenic survival (P<0.05). We were unable to detect apoptosis in any of the lines when genistein was combined with BCNU. Conclusion: These results indicate that genistein at typical adult dietary plasma levels can significantly enhance the antiproliferative and cytotoxic action of BCNU. The implication for treatment of GBM may be a reduction in the chemotherapeutic dose recommendations of these agents and subsequently a decrease in the risk of treatment sequelae for these patients.