D—苎烯对人胰腺瘤细胞法尼基蛋白转移酶,p21^ras结合的抑制作用 …

目的探讨ｄ－苎烯在化学诱发实验性肿瘤及人实体瘤的防治作用及其机制。方法 采用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ方法观察了ｄ－苎烯对胰腺癌细胞中与膜结合的ｐ２１＾ｒａｓ蛋白表达和法尼基蛋白转移酶活性的抑制作用。同时，采用免疫组化法观察了ｄ－苎烯对ｄ－苎烯对ｐ＾２１ｒａｓ在细胞膜上定位的影响。并以ｏｒｔｈｅｒｎ ｂｌｏｔｔｉｎｇ方法检测ｄ－苎烯对Ｈ－ｒａｓ癌基因在人胰腺癌细胞系中的表达。结果 ｄ－苎烯对人     

甲状腺癌组织中H-ras基因突变及蛋白表达的预后价值

对５４例甲状腺癌标本分别用ＰＣＲ－ＲＦＬＰ法检测Ｈ－ｒａｓ基因第１２位密码子的突变及免疫组化法检测ｐ２１ｒａｓ蛋白，并用时序检验分析ｒａｓ基因突变和ｐ２１ｒａｓ蛋白表达与甲状腺癌的关系。结果发现，甲状腺癌中１８例（３３．３％）有ｒａｓ基因突变和４９例（９０．７％）有ｐ２１ｒａｓ蛋白过度表达。临床分期晚、分化程度低的甲状腺癌突变率较高，具有Ｈ－ｒａｓ原癌基因突变和ｐ２１ｒａｓ蛋白阳性的病例有较高的复发率和死亡率。表明ｒａｓ基因突变及其蛋白过度表达在甲状腺癌的发生和发展过程中发挥作用，是预后不良的标志     

甲状腺组织中H—ras基因突变及蛋白表达的预后价值

对５４例甲状腺癌标本分别用ＰＣＲ－ＲＦＬＰ法检测Ｈ－ｒａｓ基因第１２位密码子的突变及免疫组化法检测ｐ２１ｒａｓ蛋白，并用时序检验分析ｒａｓ基因害变和ｐ２１ｒａｓ蛋白表达与甲状腺癌的关系，结果发现，甲状腺癌中１８例有ｒａｓ基因突变和４９例有ｐ２１蛋蛋白过度表达。     

睾丸癌组织C—myc蛋白及ras基因产物p21蛋白表达评价

本文探讨了人睾丸癌组织中Ｃ－ｍｙｃ蛋白及ｒａｓ基因产物Ｐ２１蛋白表达的临床意义。方法：应用Ｃ－ｍｙｃ和Ｐ２１单克隆抗体，通过免疫组织化学方法检测５例正常睾丸组织和２７例睾丸癌组织中ｒａｓ癌基因产物ｐ２１物Ｃ－ｍｙｃ蛋白的表达状况。结果：５例正常睾丸组织中未发现ｐ２１和Ｃ－ｍｙｃ蛋白阳性表达。阳性表达的ｐ２１和Ｃ－ｍｙｃ蛋白分别定位于肿瘤的细胞膜上和细胞核内。     

P糖蛋白在胰腺癌中的表达及其与p21^ras和p53蛋白表达的关系

为了解Ｐ糖蛋白在原发和复发胰腺癌组织中的表达及其临床意义，并分析其表达与Ｐ２１＾ｒａｓ和Ｐ５３蛋白表达的关系，采用免疫组化泽４８例原发胰腺导管癌和１５例复发胰腺癌的石蜡包埋标本进行Ｐｇｐ，ｐ２１＾ｒａｓ蛋白和ｐ５３蛋白单抗的免疫组化染色。     

睾丸癌组织中H－ras基因点突变及其蛋白表达的研究

目的研究睾丸癌组织中Ｈ－ｒａｓ基因点突变及其蛋白表达。方法应用聚合酶链反应结合寡核苷酸探针杂交技术及免疫组织化学方法检测睾九癌中Ｈ－ｒａｓ基因第１２位密码子点突变和蛋白产物ｐ２１表达水平。结果２７例睾丸癌中仅３例有这种点突变，免疫组织化学染色显示１２例标本ｐ２１蛋白表达阳性，且ｐ２１蛋白表达与睾丸癌的分化程度和临床分级有关。结论睾丸癌中Ｈ－ｒａｓ基因第１２位点突变较少见，ｐ２１超表达可能成为睾丸癌恶性程度判定的指标。     

2—乙酰氨基芴诱发大鼠肝癌变过程中rasp21,AFP的原位表达

目的 探讨ｒａｓｐ２１,ＡＦＰ在２－乙酰基芴诱发的大鼠肝癌变过程中的分布情况及关系。方法 ｒａｓｐ２１和ＡＦＰ采用免疫组化Ｓ－Ｐ法被检测及分析。结果 诱癌早期增生的肝细胞及变异肝细胞灶中即显示ｒａｓｐ２１和ＡＦＰ的过度表达,并随增生肝细胞结节的形成和演变二者过度表达细胞增多且相伴而存,由此提示,两者与大鼠肝癌发生有密切关系,ｒａｓｐ２１过度表达直接参与大鼠肝癌的启动和演进。     

乳腺肿瘤组织中Ha—ras基因蛋白表达与血清CA15—3水平的相关性及…

用免疫组化ＡＢＣ法和ＩＲＭＡ法分别检测了４２例乳腺癌和３０例良好性乳腺病患者组织中Ｈａ－ｒａｓ基因蛋白（ｒａｓｐ２１）的表达状况及血清ＣＡ１５－３，结果恶性病变组织中ｒａｓＰ２１呈过度表达，ｒａｓＰ２１的表达与细胞分化状态密切相关，不同淋巴吉转移状况的乳腺癌患者，其血清ＣＡ１５－３差异呈显著性，ＣＡ１５－３水平的变化与ｒａｓＰ２１表达状况显著相关，提示血清ＣＡ１５－３水平为监测乳腺癌病情变化的良性     

导入H—ras癌基因后肝癌细胞转移特性的变化

目的探讨Ｈ－ｒａｓ癌基因与肝癌细胞转移行为的关系。方法应用磷酸钙转染法将Ｈ－ｒａｓ癌基因导入人肝癌细胞株ＳＭＭＣ７７２１,通过进行粘附试验、运动试验、Ⅳ型胶原酶（ｃⅣａｓｅ）的分泌、表皮生长因子受体（ＥＧＦＲ）的表达等转移相关指标的测定,观察转染前后细胞转移特性的变化。结果转染Ｈ－ｒａｓ的ＳＭＭＣ７７２１细胞对胞外基质成分层粘连蛋白（ＬＮ）、纤维粘连蛋白（ＦＮ）的粘附能力提高,细胞的运动能力增强,ｃⅣａｓｅ的分泌明显增加,ＥＧＦＲ表达有中等程度的增加,且上述变化与Ｈ－ｒａｓ癌基因的蛋白产物ｐ２１的表达有明确的相关关系。结论Ｈ－ｒａｓ癌基因能在体外诱导人肝癌细胞的转移表型的产生,提高肝癌细胞的转移潜能。     

H—ras癌基因与肝癌浸润转移相关性的初步探讨

Ｈ－ｒａｓ癌基因与肝癌浸润转移相关性的初步探讨＊翁毅林芷英汪青作者单位：上海医科大学肝癌研究所（２０００３２）＊本研究系美国中华医学基金（ＣＭＢ）资助项目关键词肝肿瘤基因，ｒａｓ浸润肿瘤转移胶原蛋白免疫组织化学Ｃ－Ｈ－ｒａｓ癌基因及其蛋白产物ｐ２１广...     

EFFECTS OF LIMONENE,SALVIA MILTIORRHIZA AND TURMERIC DERIVATIVES ON H-RAS ONCOGENE EXPRESSION AND GAP JUNCTION INTERCELLULAR COMMUNICATION IN HUMAN SOLID TUMOR CELL LINES

Ｏｎｃｏｇｅｎｉｃｒａｓｐｒｏｔｅｉｎｓａｒｅｃａｕｓａｌｙｉｍｐｌｉｃａｔｅｄｃｅｒｔａｉｎｈｕｍａｎｍａｌｉｇｎａｎｃｉｅｓｗｉｔｈａｂｏｕｔ３０－４０％ｏｆｈｕｍａｎｌｕｎｇａｄｅｎｏｃａｒｃｉｎｏｍａｓ，５０％ｈｕｍａｎｃｏｌｏｎｃａｒｃｉｎ...     

Lovastatin, a cholesterol biosynthesis inhibitor, inhibits the growth of human H-ras oncogene transformed cells in nude mice

Post-translational modification of oncogenic p21ras proteins with farnesyl, a lipid intermediate in cholesterol biosynthesis, is required for p21ras membrane association and for the ability of p21ras to transform cultured cells. We have tested the ability of lovastatin, a specific inhibitor of cholesterol biosynthesis, to inhibit the growth of ras oncogene-transformed cells in vivo. Balb/c mouse 3T3 cells, transfected with H-ras oncogene from human EJ bladder carcinoma, were highly tumorigenic in nude mice. Immunoprecipitation studies with transformed EJ cells showed that lovastatin (1-100 microM) inhibited p21ras membrane association in a concentration-dependent manner and that a 10 microM concentration reduced the amount of p21ras bound to the membrane by 50%. Lovastatin also inhibited EJ cell growth in a concentration range that closely paralleled that required for inhibition of p21ras membrane association. Treatment of nude mice bearing subcutaneous (s.c.) EJ tumors with lovastatin (50 mg/kg) significantly inhibited the abilities of these tumors to grow as early as four days and, by day 12, the lovastatin treated group of animals had tumors with an average size that was 3-fold smaller than those in the saline treated group. Western blotting studies showed that lovastatin (50 mg/kg) was also able to inhibit p21ras membrane association in EJ tumors implanted s.c. in nude mice. These results demonstrate that lovastatin, an inhibitor of cholesterol biosynthesis, inhibited in vivo tumor growth of H-ras oncogene transformed cells. The results also suggest that inhibition of p21ras membrane association, an essential step in ras oncogene neoplastic transformation, is one mechanism by which lovastatin may express its antitumor activity.     

Inhibition of protein isoprenylation and p21ras membrane association by dehydroepiandrosterone in human colonic adenocarcinoma cells in vitro.

Treatment of mice and rats with the adrenal steroid, dehydroepiandrosterone (DHEA), protects against spontaneous and chemically induced tumors. The mechanism of the chemopreventive action of DHEA, however, remains uncertain. DHEA has been reported to inhibit cholesterol biosynthesis. Mevalonic acid constitutes the basic precursor not only for cholesterol but also for a variety of nonsterol isoprenoids involved in cell growth. Certain of these nonsterol isoprenoids are utilized for posttranslational modification of proteins including p21ras. We therefore investigated the effects of DHEA upon protein isoprenylation. Twenty-four-h exposure of HT-29 SF human colonic adenocarcinoma cells to 50 microM DHEA was associated with significant incorporation of products of [3H]mevalonate metabolism into several size classes of cellular proteins. The pattern of incorporation was similar to that obtained after treatment with 25 microM lovastatin, a specific 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor. Very little incorporation of label from [3H]mevalonate was observed in untreated cells. This suggests that [3H]mevalonate gains entrance to isoprenylation sites after treatment with DHEA or lovastatin because of depletion of endogenous mevalonate and subsequent inhibition of protein isoprenylation. Isoprenylation plays a critical role in promoting the association of p21ras with the cell membrane. Posttranslational processing and membrane association of p21ras were both found to be inhibited by DHEA. Thus, it is possible that the inhibition of isoprenylation of p21ras and other cellular proteins by DHEA may contribute to its anti-cancer effects.     

Inhibition of ras p21 membrane localization and modulation of protein kinase C isozyme expression during regression of chemical carcinogen–induced murine skin tumors by lovastatin

We investigated the ras p21 membrane localization and the expression and activation of protein kinase C (PKC) isozymes in activated ras oncogene–containing tumors and assessed whether these events were related to tumors growth. We used 7,12-dimethylbenz[a]anthracene–initiated and 12-O-tetradecanoylphorbol-13-acetate–promoted SENCAR mouse skin tumors, which were shown to contain Ha-ras oncogene activated by point mutation at codon 61, as an in vivo model for these studies. Compared with levels in epidermis, highly elevated levels of membrane-bound Ha-ras p21 were observed in growing tumors, which also showed strong expression and membrane translocation of PKC ζ and βII and weak expression of PCK α. However, when ras p21 membrane localization was blocked in vivo in growing tumors by lovastatin, opposite results were evident. Compared with saline-treated animals, in which tumor growth continued, lovastatin-treated animals had significantly inhibited tumor growth, which led to tumor regression with concomitant inhibition of Ha-ras p21 membrane localization. These regressing tumors from lovastatin-treated animals also showed a decrease in the expression and membrane translocation of PKC ζ and βII but increased expression of PKC α. Taken together, our results indicate that ras p21 membrane localization and the expression and activation of PKC ζ, βII, and α may be the critical events in the regulation of the growth of tumors that contain activated ras oncogenes. © 1995 Wiley-Liss Inc.     

Protection against malignant conversion in SENCAR mouse skin by all Trans retinoic acid: Inhibition of the ras p21-processing enzyme farnesyltransferase and Ha-ras p21 membrane localization

Many studies have shown that all trans retinoic acid (RA) exhibits significant protective effects against mouse skin tumor promotion and spontaneous as well as enhanced malignant conversion. In a recently completed study, we showed that under treatments in which papillomas on SENCAR mouse skin are induced at low and high probabilities to convert to malignant carcinomas, RA affords significant protection against both tumor promotion and subsequent malignant conversion. More than 95% of these mouse skin papillomas and carcinomas have been shown to contain point mutation at the 61 codon of Ha-ras oncogene. The ras oncogene encodes a p21 protein that, in its mutated form, transforms mammalian cells only when p21 is at the inner surface of the plasma membrane, by a series of enzymatic reactions in which the initial step is catalyzed by farnesyltransferase (FTase). In this study, we assessed whether the protective effect of RA against malignant conversion involves the inhibition of ras p21 processing in those tumors that contain the activated ras oncogene. The FTase activity and the levels of cytosolic and membrane-bound Ha-ras p21 were determined in all papillomas and carcinomas obtained from acetone- or RA-treated animals. No matter how the data were analyzed and what comparisons were considered, in all the protocols used, compared with controls, papillomas and carcinomas obtained from RA-treated groups showed significantly decreased (P < 0.01–0.001) FTase activity. Furthermore, the tissue samples from RA-treated groups in different protocols also showed significantly diminished membrane localization of Ha-ras p21, with a concomitant increase in cytosolic Ha-ras p21 levels. The analysis of these data also showed that in all the protocols used, the increased FTase activity and membrane localization of Ha-ras p21 were associated with the induction of papillomas and their subsequent malignant conversion to squamous cell carcinomas. Taken together, these results indicate a strong correlation between the inhibition of ras p21 farnesylation because of a decrease in FTase activity by RA and its protective effect against malignant conversion of papillomas to carcinomas. Based on the results of this study, it is tempting to suggest that clinical trials evaluating the preventive or therapeutic potential of retinoids may be directed more toward those clinical malignancies that are known to contain the activated ras oncogene. © 1996 Wiley-Liss, Inc.     

Modulation of the mevalonate pathway and cell growth by pravastatin and d-limonene in a human hepatoma cell line (Hep G2).

Modulation of cell growth by a combination of pravastatin [a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor] and d-limonene (an inhibitor of protein isoprenylation) was studied using Hep G2, a human hepatoma-derived cell line. Pravastatin, at 0.1 mM, produced 85% inhibition of cholesterol biosynthesis in Hep G2 cells. The combination of 0.1 mM pravastatin and 1.0 mM d-limonene had no further effect on the reduction seen with pravastatin alone. Addition of 0.1 mM pravastatin or 1.0 mM d-limonene did not significantly suppress DNA synthesis by the cells, whereas the combination suppressed it to 50% of the control level. Production of m-p21ras was markedly decreased to 35% of the control level by the combination of these two inhibitors. Both the reduction by pravastatin of farnesylpyrophosphate as substrate for protein:farnesyl transferase and inhibition of protein farnesylation by d-limonene seem to be responsible for the profound suppression of m-p21ras formation in the cells. However, dolichol synthesis was not suppressed by the combination of these inhibitors. In human fibroblasts, the combination suppressed m-p21ras production but not DNA synthesis. These findings suggest that the combination of pravastatin and d-limonene acts on cancer cell growth through inhibition of the post-translational processing of cellular proteins including p21ras, rather than through the suppression of cholesterol and dolichol biosynthesis. Thus, the combination of an HMG-CoA reductase inhibitor and an inhibitor of protein isoprenylation offers potential as a new approach for cancer therapy.     

Selective cytotoxicity of farnesylamine to pancreatic carcinoma cells and Ki-ras–transformed fibroblasts

Farnesyl protein transferase (FPTase) catalyses the post-translational modification of proteins by a farnesyl pyrophosphate. One of the substrates of this enzyme is p21^ras, the product of the ras oncogene. We examined whether farnesylamine, one of the FPTase inhibitors (FTI), is selectively cytotoxic in pancreatic carcinoma cells and Ki-ras–transformed fibroblasts. Furthermore, we investigated whether the cytotoxicity of farnesylamine is caused by the induction of apoptosis in these cells. Using the FPTase assay, we found that farnesylamine inhibited FPTase in vitro. Immunoprecipitation showed that farnesylamine inhibited farnesylation of p21^ras in vivo. In addition, 24 and 5 μM farnesylamine were required to achieve 50% cytotoxicity in pancreatic carcinoma cells containing activated Ki-ras and Ki-ras–transformed NIH/3T3 cells, respectively. The parental NIH/3T3 cells were resistant to the cytotoxic effect of farnesylamine at concentrations less than 100 μM. After incubation with farnesylamine, DNA fragmentation was observed in both pancreatic carcinoma cells and Ki-ras–transformed fibroblasts at cytotoxic doses of this compound but not in NIH/3T3 cells. These results indicate that the mechanism of cell death induced by farnesylamine is apoptosis, and this apoptosis occurred specifically in pancreatic carcinoma cells containing mutated Ki-ras and the Ki-ras–transformed cells. Because raf is downstream of ras (p21^ras) in the ras–raf–mitogen-activated protein kinase signaling pathway, we used c-raf-1–transformed fibroblasts, which proved to be resistant to apoptosis induced by farnesylamine. This supports the theory that inhibition of ras signaling may be related to the induction of apoptosis. These data further suggest that farnesylamine could be useful as a chemotherapeutic agent in cancers that very frequently contain a Ki-ras oncogene mutation, e.g., pancreatic cancer. Mol. Carcinog. 21:93–99, 1998. © 1998 Wiley-Liss, Inc.     

Immunohistochemical study of oncogene product ras p21, c-myc and growth factor EGF in breast carcinomas.

The expression of the oncogene products ras p21, c-myc and the growth factor EGF (epidermal growth factor) was studied immunohistochemically in the tissue of 119 benign and malignant human breasts. In most cases, histologically normal breast tissues and benign lesions were found to be negative or poorly-expressive for reactivity with each antibody. Similar findings were observed in carcinoma in situ. Invading breast carcinomas demonstrated a significantly higher percentage of stained cells than that observed in benign lesions or carcinoma in situ; forty-two of 66 invasive breast carcinomas (63.6%) were highly-expressive for ras p21, thirty-eight (57.6%) for c-myc and twenty (30.3%) for EGF, but overall correlations between each oncogene expression and the clinical stage, tumor size or degree of differentiation were not found. The overall 5-year survival rate was studied in 58 patients with Stage II and III in association with each oncogene or EGF expression. Their survival rate was significantly effected by the EGF expression (0.05 less than p less than 0.1) but not by ras p21 or c-myc expression. Analysis of 36 specimens available with ER (estrogen-receptor) level revealed a significant correlation between the ER status and c-myc or E2 (estradiol) and a significant inverse correlation between ER status and ras p21 or EGF expression (P less than 0.05). The expression of ras p21, EGF and c-myc was not associated with metastatic tumor progression.     

Dietary fish oil inhibits the expression of farnesyl protein transferase and colon tumor development in rodents

Although epidemiological and experimental studies indicate a strong relationship between different dietary fats and risk of colon cancer, the modulating effects of these nutritional factors at the molecular level are not fully elucidated. Activated ras genes have been implicated in the etiology of many human malignancies, including colon cancer. It is well established that the transforming ability of ras-p21 depends on its correct localization in plasma membrane. We have previously demonstrated that ingestion of a relatively higher amount of dietary fish oil leads to reduced plasma membrane levels of ras-p21 with concomitant increase in its cytoplasmic contents during the promotion and progression phases of chemically-induced colon tumorigenesis. In this follow-up experiment, we have found that intake of a high amount of corn oil, one of the most widely used fats in the American diet, enhances the expression of farnesyl protein transferase (FPTase). This enzyme catalyses farnesylation of ras precursors in a critical step during post-translational modification of ras oncoproteins, thereby enabling their anchorage to plasma membrane. In contrast, consumption of high amounts of fish oil, which is rich in omega-3 polyunsaturated fatty acids, reduces the levels of FPTase expression, thus inhibiting post-translational processing of ras precursors resulting in decreased ras function both in colonic mucosa as well as in colon tumors. These results correlate with increased incidence and multiplicity of grossly visibly colon tumors in carcinogen-treated animals fed a high corn oil diet versus decreased incidence and multiplicity of colon tumors in their counterparts fed the high fish oil diet. This dietary inhibition of FPTase may have a practical chemopreventive potential.      

Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumour cells

The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K-ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 +/- 0.11 microM and 2.68 +/- 0.20 microM, respectively, while the geranylgeranylation of p21 rhoA and p21rap1 was not affected. Manumycin dose-dependently inhibited (IC50 = 2.40 +/- 0.67 microM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21ras, as well as COLO320-DM cell growth (IC50 = 3.58 +/- 0.27 microM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 microM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1-25 microM for 24-72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity.