Deregulated expression of<Emphasis Type="Italic"> KRAP</Emphasis>, a novel gene encoding actin-interacting protein,in human colon cancer cells

We have identified a novel gene, designated KRAP (Ki-ras-induced actin-interacting protein), encoding a protein of 1,259 amino acids with coiled-coil regions and transmembrane regions, from the cDNA library of human colon cancer HCT116 cells, as one of the genes upregulated by activated Ki-ras. While KRAP was rarely expressed in normal colon epithelium, deregulated constitutive KRAP expression was observed in some other colon cancer cells. In normal tissues, KRAP was strongly expressed in pancreas and testis. Anti-KRAP polyclonal antibodies detected endogenous KRAP as the molecular size of M_r 180,000, and immunofluorescence microscopy and cytochalasin E treatment revealed that KRAP was clearly associated with the actin filaments. Furthermore, KRAP was localized as a membrane-bound form with extracellular regions. These results together suggested KRAP might be involved in the regulation of filamentous actin and signals from the outside of the cells.     

Gastric adenoma — carcinoma sequence with special reference to p53 and Ki-ras gene alterations

With the aim of detecting the timing of p53 and Ki-ras gene alterations in the gastric adenoma-carcinoma sequence, 19 early gastric adenocarcinomas arising from adenomas were studied. Immunohistochemically, 5 adenocarcinomas were positive for p53; 3 focally and 2 diffusely. The p53 point mutations were detected in a focal area with p53 immunoreactivity in 2 of the 5 p53-positive adenocarcinomas. This indicated that p53 point mutations may play a less crucial part in malignant conversion of adenoma to adenocarcinoma in the stomach than in the colon. No Ki-ras gene mutations at codons 12 and 13 were detected in any lesion. These results suggest that the adenoma-carcinoma sequence in the stomach has a different mechanism from that in the colon.Supported in part by a Grant-in-Aid from the Ministry of Health and Welfare of Japan     

Augmentation of c-fos and c-jun expression in transgenic mice carrying the human T-cell leukemia virus type-Itax gene

To analyze the effect of human T-cell leukemia virus type I (HTLV-I) on cellular gene expression and its relation to tumorigenesis, two lines of transgenic mice carrying the long terminal repeat (LTR)-env-pX-LTR regions of the HTLV-I genome were produced. The transgene was expressed in many organs, including the brain, salivary gland, spleen, thymus, skin, muscle, and mammary gland. We found that the expression of the c-fos and c-jun genes, but not of thelyn and c-myc genes, was augmented 2- to 20-fold in histologically normal skin and muscle of these mice. The augmentation was tissue specific, suggesting the involvement of a cellular factor in the transgene action. In these mice, a three to seven times higher incidence of tumors was seen as compared with the control mice. These tumors included mesenchymal tumors, such as fibrosarcoma, neurofibroma, and lipoma, and adenocarcinomas of the mammary gland, salivary gland, and lung. The c-fos and c-jun genes were also activated in these tumors. The possible roles of elevated c-fos and c-jun gene expression in tumorigensis are discussed.The abbreviations used are ATL, adult T-cell leukemia; HTLV-I, human T-cell leukemia virus type I; IL-2, interleukin 2; IL-2R, interleukin 2 receptor; IL-6, interleukin 6; LTR, long terminal repeat.     

Expression of oncoproteins in primary human non-small cell lung cancer and incidence of metastases

In the current study the relationship between the incidence of metastatic spread and expression (at the protein level) of various proto-oncogenes was investigated in 217 human non-small cell lung carcinomas. Tumors with an overexpression of proteins encoded by the oncogenes c-jun and c-myc showed a significantly increased formation of metastases (c-jun: P = 0.008; c-myc: P = 0.018). No significant correlations were found between the expression of the c-fos, c-erbB1, c-neu and c-ras products and metastatic spread.     

Chromosomal location and structure of amplicons in two human cell lines with coamplification of c-myc and Ki-ras oncogenes

Gene amplification is a major mechanism through which oncogenes and genes responsible for drug resistance are overexpressed in neoplastic cells, and several models for structure of amplified units (amplicons) are postulated. In order to identify consistent changes associated with oncogene amplification, we analyzed chromosomal location and physical distance of amplicons of two independent human cell lines that have coamplified c-myc and Ki-ras oncogenes. In one cell line, KHC287, amplified c-myc genes were localized in two chromosomes and Ki-ras in three chromosomes. One marker chromosome was almost entirely encompassed by both amplified genes. In the other cell line, Lu-65, both of the amplified genes shared the same locus, on chromosome 12q+. The two genes, however, are more than 1500 kb apart in both cell lines. The above findings indicate that two different amplified genes became associated on one chromosome in two independent cell lines. This suggests that a common mechanism is associated with chromosomal rearrangements affecting different amplified genes.     

Studies of c-jun oncogene expression in human breast using a new monoclonal antibody,NCL-DK4

A new monoclonal antibody to human c-jun oncoprotein, designated NCL-DK4, has been produced. NCL-DK4 has been proved to be highly effective for use on formalin-fixed, paraffin-embedded tissues, enabling the study of c-jun expression at a cellular level in both normal and neoplastic human tissues. The expression of c-jun oncogene has been examined in normal, benign, and malignant breast tissues, and c-jun-specific immunoreactivity in carcinomas has been related to histological type, tumour grade, c-erbB-2, oestrogen receptor, progesterone receptor, and epidermal growth factor receptor expression. Normal and benign breast tissues showed c-jun-specific immunostaining, which was weaker and in fewer cells compared with the c-jun immunoreactivity observed in breast carcinomas. No relationship was found between the degree of immunostaining and the extent of proliferative changes in benign breast tissues. Ninety per cent of all breast carcinomas studied showed c-jun-specific nuclear staining. There were no statistically significant differences in the intensity of c-jun immunoreactivity among grade I, II, and III infiltrating ductal carcinomas. There was no significant relationship between c-jun oncoprotein expression and c-erbB-2, oestrogen, progesterone, and epidermal growth factor receptor immunoreactivity.     

A novel point mutation at codon 146 of the K-ras gene in a human colorectal cancer identified by the polymerase chain reaction

In this report, point mutations of the K-ras gene at codon 146 were analyzed in 25 cases of colon cancer, 4 cases of lung cancer, and 41 cases of lymphoid malignancy. A codon 146 mutation substituting threonine (ACA) for alanine (GCA) was detected in the tumor tissue of a patient with colon cancer and was not detected in the normal tissue of the same patient. Any additional mutations of theras gene family were not detected in this patient. These results suggest that the codon 146 mutation of the K-ras gene could be involved in the development of naturally occurring human malignancies.     

Undifferentiated carcinoma of the pancreas: analysis of intermediate filament profile and Ki-ras mutations provides evidence of a ductal origin

Undifferentiated carcinomas and osteoclast-like giant cell tumours of the pancreas commonly contain foci of neoplastic ductal glands. To test the hypothesis that undifferentiated carcinomas and osteoclast-like giant cell tumours have a ductal origin, the immunocytochemical cytokeratin pattern and the frequency and type of Ki-ras mutations at colon 12 were studied in a series of 17 undifferentiated carcinomas and two osteoclast-like giant cell tumours. The cytokeratin features of undifferentiated carcinomas and osteoclast-like giant cell tumours were compared with those found in 10 ductal adenocarcinomas, 20 acinar cell carcinomas, 25 neuroendocrine tumours, and 15 solid-pseudopapillary tumours. All undifferentiated carcinomas and osteoclast-like giant cell tumours stained with at least one cytokeratin antibody, and 13/19 of them with antibodies against cytokeratins 7, 8, 18, and 19. The latter cytokeratins were expressed in all ductal adenocarcinomas, but only in 15/20 acinar cell carcinomas, 2/25 neuroendocrine tumours, and 1/15 solid-pseudopapillary tumours. In addition to cytokeratin, 15/19 undifferentiated carcinomas/osteoclast-like giant cell tumours were positive for vimentin. Ki-ras mutations at codon 12 were found in 10 undifferentiated carcinomas and one osteoclast-like giant cell tumour from which DNA could be successfully amplified. The Ki-ras mutation patterns were analysed in six tumours and corresponded to those typical of ductal adenocarcinomas. In tumours with ductal and anaplastic components, both components revealed identical mutation patterns. From these findings, it is concluded that both undifferentiated carcinomas and osteoclast-like giant cell tumours belong to the pancreatic tumours that show a ductal phenotype. Since undifferentiated carcinomas and osteoclast-like giant cell tumours share the same cytokeratin and Ki-ras features, they are probably derived from the same cell lineage. © 1998 John Wiley & Sons, Ltd.     

Cytomorphological,cytogenetic, and molecular biological characterization of four new human renal carcinoma cell lines of the clear cell type

Four new permanent cell lines (RCC-A, -B,-C, and -D) derived from different human renal cell carcinomas of the clear cell type were established in tissue culture. The cell lines displayed characteristic differences in cell size and shape, which allowed individual identification by phase contrast microscopy. Ultrastructurally, the cell lines exhibited varying amounts of cytoplasmatic glycogen and lipid. Immunohistochemistry revealed co-expression of vimentin and cytokeratin in all cell lines. The mean population doubling time ranged from 27 h (RCC-A) to 104 h (RCC-D). RCC-B and -C cells produced slowly growing tumours after heterotransplantation into nude mice, whereas RCC-A and RCC-D cells were non-tumorigenic. The modal chromosome number was either near-diploid (RCC-A, -B, and -C) or near triploid (RCC-D). Clonal abnormalities affecting the short arm of chromosome 3 were seen in all cell lines. Northern blot analysis revealed no expression of the proto-on-cogenes c-fos, c-ros, and c-mos, whereas c-Ki-ras expression was observed in all cell lines. Expression of c-myc was observed in RCC-A, RCC-B, and RCC-D cells, whereas c-raf expression could be detected in RCC-B and RCC-D. Tumour suppressor gene p53 mRNA was observed in the cell line RCC-D.     

The silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway

Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66^Shc protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66^Shc in the progress of colon cancer still unknown. In this study, we found that p66^Shc highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO₂ cells. The silence of p66^Shc in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66^Shc siRNA. Furthermore, after HCT8 cells treated with p66^Shc siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66^Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell.     

转化生长因子β受体Ⅱ对微立星不稳定结肠癌细胞凋亡和迁移的影响

目的 研究转化生长因子β受体Ⅱ（TGFBRⅡ）表达对微卫星不稳定（MSI）结肠癌细胞凋亡和迁移的影响。方法 选择野生型PIK3CAHCT116细胞（HCT116wt）作为MSI结肠癌细胞模型。将TGFDRⅡ转染HCTll6wt细胞获得HCTll6wt—RⅡ细胞,并以HCT116wt空质粒细胞作为对照。通过生长因子缺失应激（GFDS）诱导细胞凋亡。采用Western印迹检测磷酸化Smad2（TGFD信号通路关键因子）、磷酸化AKT（P13K／AKT信号通路关键因子）、Bim（TGFB信号通路下游因子）和E-cadherin（诱导上皮向间质转化的标志物）表达。采用DNA碎片ELISA分析检测HCTll6wt—RⅡ细胞凋亡情况。通过Transwell实验检测HCT116wt—RⅡ细胞迁移活力。结果加入TGFB后,HCTll6wt—RlI细胞的TGFB信号通路得以启动,GFDS诱导的HCT116wt-RⅡ细胞凋亡受到显著抑制（DNA碎片值：0．69＋0．02比0．41±0．04,P〈0．01）;细胞迁移活力明显增强（2．10±0．15比4．03±0．48,P〈0．01）。P13K抑制剂（LY294002）可以逆转TGF[3对HCTll6wt—RⅡ细胞的凋亡抑制和迁移活力增强作用。TGFβ作用于HCT116wt．RⅡ细胞后,Bim和E—cadherin表达明显减少。结论TGFβRⅡ在MSI结肠癌细胞中的再表达可以增加MSI结肠癌细胞的存活能力和迁移活力,该作用依赖P13K／AKT途径,从而为MSI结肠癌患者的良好预后提供了一个分子水平的解释。     

激动型CD40单克隆抗体体外抑制结肠癌细胞增殖的实验研究

目的 探讨激动型CD40单克隆抗体在体外对结肠癌细胞增殖的抑制作用.方法 树突状细胞(DCs)经结肠癌冻融抗原致敏后予以不同条件激活,分为激动型CD40单克隆抗体组、阴性对照组及肿瘤坏死因子-α(TNF-α)阳性对照组,诱导培养至第7天,用流式细胞仪检测各组DCs表面分化相关抗原CD80、CD83、CD86和HLA-DR的表达,酶联免疫吸附测定法检测DCs培养液上清中白细胞介素-12(IL-12)的质量浓度,噻唑蓝比色法检测DCs体外刺激T淋巴细胞增殖的能力,进而检测DCs所诱导的肿瘤特异性细胞毒性T淋巴细胞(CTL)对人结肠癌细胞HCT116的杀伤作用.结果 与阴性对照组相比,激动型CD40单克隆抗体组活化的DCs表面抗原CD80、CD83、CD86和HLA-DR的表达率均显著升高(均P＜0.05),DCs上清中IL-12的质量浓度亦显著升高((716.80±53.43) pg/ml比(405.51±12.17) pg/ml,P＜0.05),活化的DCs具有更强的刺激T淋巴细胞增殖的能力(刺激指数2.006 2±0.438 3比1.365 0±0.209 8,P＜0.05),活化的DCs所诱导的CTL对HCT116细胞具有更强的杀伤作用(抑制率(66.08±0.41)％比(46.60±1.10)％,P＜0.05);而与TNF-α阳性对照组相比,其差异均无统计学意义(均P＞0.05).结论 激动型CD40单克隆抗体在体外可促进DCs的活化与成熟,进而诱导肿瘤特异性CTL的产生,从而抑制人结肠癌细胞HCT116的增殖.     

Curcumin induces permanent growth arrest of human colon cancer cells: link between senescence and autophagy

Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, is a potent anticancer agent, which restricts tumor cell growth both in vitro and in vivo. Thus far curcumin was shown to induce death of cancer cells. This study reports the induction of cellular senescence of human colon cancer cells HCT116 upon curcumin treatment. The SA-β-galactosidase activation was observed both in p53+/+ and p53−/− cells, however the latter ones were less sensitive to the prosenescent activity of curcumin. Upregulation of p53 and p21 proteins was observed in p53+/+ HCT116, while p53-independent induction of p21 was noticed in p53−/− HCT116. Moreover, the senescence of HCT116 cells was accompanied by autophagy, that was confirmed by electron microscopy observations of autophagosomes in the curcumin-treated cells as well as LC3-II expression, punctue staining of LC3 and increased content of acidic vacuoles. Inhibition of autophagy, due to the diminished expression of ATG5 by RNAi decreased the number of senescent cells induced by curcumin, but did not lead to increased cell death. Altogether, we demonstrated a new antitumor activity of curcumin leading to cancer cell senescence and revealed the presence of a functional link between senescence and autophagy in curcumin-treated cells.     

Degradation of endothelial cell matrix collagen is correlated with induction of stromelysin by an activated ras oncogene

A conditional expression system was established whereby the human K-ras, v-src, and v-mos genes were cloned into a conditional expression vector downstream of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Rat-1 fibroblasts were transfected with these constructs and selected in medium containing G418. Cloned transfectants were isolated and characterized for absolute dependence on dexamethasone for expression of oncogene products and anchorage-independent growth in soft agar. Expression of activated p21^K-ras(val12) enabled the fibroblasts to degrade extracellular matrix collagen secreted by murine microvessel endothelial cells. Concurrent with p21^K-ras(val12) induction a proteinase with the characteristic size and substrate specificity of transin, the murine homologue of the human matrix metalloproteinase stromelysin, was expressed and secreted. Induction of v-mos and v-src oncogenes resulted in little or no detectable transin expression respectively coinciding with a relative or absolute failure to increase degradation of extracellular matrix collagen. This study suggests that in this system the expression of the ras oncogene can contribute to the in vitro invasive behavior of tumor cells by upregulating the production of a metalloproteinase capable of degrading collagen synthesized by vascular endothelial cells.     

错配修复基因hMLH1在雌激素诱导结肠癌细胞凋亡中的作用

 摘 要 目的：探讨错配修复基因hMLH1在雌激素诱导结肠癌细胞凋亡中的作用。方法：将含野生型hMLH1-cDNA的质粒转入hMLH1缺陷的人结肠癌细胞株HCT116,在不同浓度的雌激素干预下,分别以流式细胞仪和活细胞计数试剂盒（cell counting kit-8）法检测转染后细胞的凋亡率和活性。结果：与空载组相比,转染hMLH1后雌激素能明显诱导HCT116细胞活性降低,且凋亡率增加。结论:hMLH1参与雌激素诱导的结肠癌细胞株HCT116凋亡。     

Oncogene-dependent expression of CD44 in Balb/c 3T3 derivatives: correlation with metastatic competence

Oncogene-dependent regulation and tumor relatedness of CD44 expression were investigated in Balb/c 3T3 cells and their derivatives transformed with different ras oncogenes (metastatic tumor model) or the human c-sis oncogene (non-metastatic model). Ras transformants using either the Harvey or Kirsten oncogenes expressed high levels of cell surface CD44 protein that bound fluoresceinated hyaluronan (HA). Much lower levels of CD44 were expressed in parental 3T3 cells, ras ⁻ revertants generated from Kirsten-transformed cells, or c-sis transformants, confirming the significance of the ras oncogene in this upregulation. To determine whether endogenous HA regulates these parameters, hyaluronidase treatment of ras transformants exposed more cell surface CD44 to anti-CD44 antibody and increased fluoresceinated HA binding; this did not occur with 3T3 or c-sis transformants. CD44 expression and its HA-binding function were conserved in a panel of in vivo primary and lung metastatic tumor cell lines derived from ras transformants. Ras transformants also retained the ability to downregulate CD44 protein levels in confluent cultures which occurred through a translational or post-translational mechanism (as CD44 mRNA levels were not reduced). These results taken together demonstrate that ras-dependent regulation of CD44 may correlate with tumor progression and metastasis in vivo, possibly (although not exclusively) supporting CD44's importance in metastatic progression.     

NIH3T3 transfectant containing human K-ras oncogene shows enhanced metastatic activity after in vivo tumor growth or co-culture with fibroblasts

A clone of NIH3T3 transformant (H-3), obtained by transfecting genomic DNA of a human colon carcinoma cell line, contains human K-ras oncogene and yields metastatic pulmonary nodules after intravenous injection of the cells into nude mice. This metastatic ability was enhanced remarkably after in vivo tumor growth (subcutaneous tumor formation in nude mice) accompanied by increased mRNA expression and gene amplification of the human-derived K-ras oncogene, while it declined gradually as the passage number increased in vitro, with corresponding decreases of gene amplification and mRNA expression. Six subclones were randomly selected from H-3 cells which had been subcultured to passage 22. All of the clones in culture showed almost the same low level of metastatic ability and exhibited little K-ras oncogene amplification with correspondingly low mRNA expression. However, after they formed tumors in nude mice, every clone acquired high metastatic ability and the gene amplification increased, with elevated mRNA expression. These experimental facts indicated that acquisition of metastatic ability coupled with the function of K-ras oncogene was conditional in nature, being strongly affected by in vivo tumor circumstances. The low metastatic and G-418-resistant H-3 cells were co-cultured with BALB/c3T3 fibroblasts for 2–4 weeks. After removal of fibroblasts by exposure to G-418, the tumor cells exhibited increased metastatic ability and human K-ras oncogene mRNA, suggesting an intimate interaction between H-3 cells and fibroblasts influencing the function of transfected human K-ras oncogene. Fibroblasts of the host animal may thus have an important role in generating enhanced metastatic activity of H-3 cells.