地塞米松诱导结肠癌细胞凋亡的实验研究

目的:研究糖皮质激素受体(GR)在结肠癌细胞株Lovo、HCT-116、HT-29和SW-480中的表达,观察地塞米松(Dex)在体外对结肠癌细胞株的增殖抑制和凋亡诱导作用。方法:采用免疫组化、RT-PCR检测糖皮质激素受体(GR)在结肠癌细胞株中的表达;MTT、DNALadder和流式细胞仪等方法进行增殖抑制和细胞凋亡检测。结果:在4种结肠癌细胞株中只有Lovo和HCT-116表达GR。地塞米松在体外作用后,对4种细胞的增殖均有抑制作用,其中对Lovo和HCT-116细胞作用最显著。在Lovo细胞可检测到特征性的凋亡细胞DNA梯带。在流式细胞仪检测显示经1×10-4mol/LDex诱导72h后,在Lovo和HCT-116细胞检测到细胞凋亡,分别为(34.8±1.9)%和(33.6±1.4)%,明显高于未加Dex对照组的Lovo(2.9±0.4)%和HCT-116(6.4±1.3)%,差异有统计学意义(t值分别为28.934和22.980,P值均为0.000)。结论:结肠癌细胞株Lovo和HCT-116表达GR,地塞米松体外抑制Lovo和HCT-116细胞增殖,并诱导其发生凋亡,可能与GR相关。     

Humanized anti-interleukin-6 receptor monoclonal antibody induced apoptosis of fresh and cloned human myeloma cells in vitro

We investigated the effect of anti-IL-6 receptor monoclonal antibody (hPM1) on the in vitro proliferation of cloned and freshly isolated myeloma cells from 20 patients with advanced stage multiple myeloma (MM). Humanized PM1 significantly inhibited the growth of a myeloma cell line in a dose-dependent manner and inhibited more than 30% of the proliferation of fresh myeloma cells in 10 of the 19 cases. Flow cytometric analysis using annexin V and 7AAD showed that hPM1 induced apoptosis of myeloma cells. These observations suggest the possibility of using hPM1 for treating some patients with MM whose growth depends on IL-6.     

The organization of intermediate filaments in normal human colonic epithelium and colonic carcinoma cells

The effect of EBV-conversion of two EBV-negative lymphoma lines (Ramos and BJAB) on agarose clonability and tumorigenicity in nude mice was explored. The cloning frequency was increased in all 9 sublines investigated, between 1.1 and 4.9 times compared to the original "parental" lines. Tumorigenicity was increased in one out of 2 BJAB-derived, EBV-positive lines and in 3 of 6 Ramos-derived lines, while it was decreased in 3 others. A strong positive correlation between the number of genomes/cell and the amount of EBNA/cell was detected, and in 7 of 9 converted lines the cloning frequency also correlated to the number of EBV genomes. On the other hand, no relation was established between the number of EBV-genomes/cell, the amount of EBNA/cell and tumorigenicity.     

地塞米松诱导结肠癌细胞凋亡的机制研究

目的：探讨地塞米松（Ｄｅｘ）体外抑制结肠癌细胞株的增殖并诱导其凋亡的作用机制。方法：采用ＭＴＴ及流式细胞仪检测细胞增殖抑制及凋亡,ＲＴ-ＰＣＲ检测ＧＲ、ＩκＢ及Ｂcl-２的表达改变,凝胶迁移或电泳迁移率实验（ＥＭＳＡ）检测Ｄｅｘ作用过程中的ＮＦ-κＢ活性改变。结果：Ｄｅｘ体外作用后,对４种结肠癌细胞株ＬｏＶｏ、ＨＣＴ-１１６、ＨＴ-２９及ＳＷ-４８０均有抑制作用,ＬｏＶｏ和ＨＣＴ-１１６细胞作用最显著。流式细胞仪检测显示,Ｄｅｘ（１×１０^-4mol/L）诱导７２ｈ后,在ＬｏＶｏ和ＨＣＴ-１１６细胞检测到细胞凋亡和坏死峰,明显高于未加Ｄｅｘ的对照组,具有显著性差异。ＲＴ ＰＣＲ检测显示Ｄｅｘ作用后ＧＲα、ＩκＢ表达升高,Ｂｃｌ-２无明显改变,ＥＭＳＡ结果也显示ＮＦ-κＢ活性明显降低。结论：地塞米松可能通过上调ＧＲα、ＩκＢ表达,抑制ＮＦ-κＢ活性来抑制ＬｏＶｏ细胞的增殖,并诱导其凋亡。     

VASP在结肠癌组织中的表达及其对预后的影响

目的 探讨血管扩张刺激磷蛋白(vasodilator stimulated phosphoprotein,VASP)在结肠癌中的表达及对预后的影响.方法 收集手术切除的60例结肠癌组织及对应癌旁组织,采用免疫组织化学法检测VASP蛋白的表达情况.随访3年,采用x2检验分析VASP蛋白与临床病理资料间的相关性,Kaplan-Meier生存曲线分析VASP蛋白表达对患者预后的影响.结果 VASP在结肠癌组织中表达水平高于对应癌旁组织(P＜0.05).结肠癌组织中VASP蛋白阳性表达与肿瘤直径增大(≥5 cm)、淋巴结转移及高TNM分期(Ⅲ～Ⅳ期)呈正相关(均P＜0.05).VASP蛋白阳性表达的患者3年总生存率及无瘤生存率均低于VASP蛋白表达阴性患者(均P＜0.05).Cox多因素回归分析证实,VASP阳性表达是结肠癌患者不良预后的独立危险因素之一(P ＜0.05).结论 VASP在结肠癌组织中表达上调,高水平VASP蛋白表达与肿瘤恶性临床病理特征及不良预后有关,VASP可能成为结肠癌患者诊断和预后评价的有效分子标志物.     

Expression of selenium-containing proteins in human colon carcinoma tissue

Selenium may be beneficial in reducing the risk of cancer incidence and mortality in many cancer types such as liver, prostate, colorectal and lung. However, despite the extensive recent research on selenium and selenium-containing proteins, there are still open questions concerning their expression in certain human cancer types, including colorectal carcinoma. Therefore, the expression level of the selenoproteins thioredoxin reductases 1 and 2 (TRXR-1 and TRXR-2) and glutathione peroxidases 1 and 4 (GPX1 and GPX4) in human colon carcinoma tissues was investigated. Up-regulation of TRXR-1 in the colon carcinoma specimens was found both in disease stage-dependent and independent analyses. No differences were found for TRXR-2 expression levels. GPX1 was up-regulated in carcinoma tissues at both the protein and mRNA levels. GPX4 was also up-regulated at the protein level, except for the samples derived from stage III patients. The expression of TRXR-1, GPX1 and GPX4, but not TRXR-2 is differently regulated in cancer as compared to healthy colonic tissue.     

Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro

Intratumoral injection of recombinant adenoviral type 5 (Ad5) vectors that carry prodrug-activating enzymes like DT-diaphorase (DTD) could be used to selectively target tumor cells for chemotherapy. To demonstrate the feasibility of this approach, Ad5 vectors were constructed, which express human DTD minigenes for both wild-type and mutant (C-to-T change in nucleotide 609 in DTD cDNA) DTD under the control of the cytomegalovirus (CMV) promoter. HT29 human colon carcinoma cells express wild-type DTD, whereas BE human colon carcinoma cells express mutant DTD, have low to undetectable DTD activity, and are 4- to 6-fold more resistant to mitomycin C (MMC) than HT29 cells. A test of the ability of Ad5 to infect these cells (using a beta-galactosidase CMV-driven minigene) indicated that 90-100% of BE cells were infected at a multiplicity of infection (MOI) of 100, whereas only 15-40% of HT29 cells were infected at this MOI. Infection of BE cells in vitro with recombinant Ad5 carrying a minigene for wild-type DTD at MOIs of 3-100 resulted in a progressive increase in DTD activity and a maximal 8-fold increase in sensitivity to MMC as measured by a colony-forming assay. HT29 cells were sensitized 2- to 3-fold following treatment with Ad5.DTD at an MOI of 100. These results indicate that adenovirus-mediated gene transfer and expression of wild-type DTD can sensitize resistant tumor cells to MMC and that this therapeutic strategy may exert a significant bystander effect.     

Amplification of myc-specific sequences in human colonic cancer

DNA samples obtained from tumors and adjacent mucosa of the large bowel of a patient with large bowel multiple neoplasia were examined after Southern. The procedure established amplification of v-myc oncogene-related DNA sequences in 1 out of 5 tumors tested. Restriction fragments of amplified myc-specific sequences and matching c-myc and N-myc loci of the human genome differed in size.     

Partial purification of human colonic carcinoma cells by sedimentation.

We have purified epithelial cells from human colonic tumours by velocity sedimentation in an isokinetic density gradient of Ficoll in tissue culture medium. In frozen sections of colonic carcinoma, histochemically demonstrable N-acetyl-beta-D-glucosaminidase (HDAG) was observed primarily in epithelial cells. We used this enzyme as a histochemical marker of epithelial cells. Initial suspensions of cells from colonic tumours suspended with 0-25% trypsin contained an average of 24% of the nucleated cells with HDAG. In the purest fraction obtained from gradient centrifugations, an average of 74% of the nucleated cells contained HDAG. After centrifugation, the quarter of the density gradient which contained the most rapidly sedimenting cells was purified 2-4-fold over that in the initial suspension. Cells in this zone of the gradient also gave rise to colonies in soft agar. Cells from initial suspension resulted in 15-25% as many colonies of 7 or more cells in cultures inoculated with the same number of nucleated cells. For the most part, cells obtained from the other zones of the gradient did not give rise to colonies in soft agar.     

Expression of gastric-associated antigens by human premalignant and malignant colonic epithelial cells

A rabbit antiserum prepared to 2nd-trimester fetal organ extracts and absorbed with adult tissue (anti-STFa) was used to detect antigens common to 2nd-trimester fetal large bowel, normal adult stomach, several colon carcinoma cells (in primary and established cultures) and epithelial cells cultured from benign colonic polyps. The frequency of anti-STFa-positive cells was highest in cultures derived from benign tumors known to be associated with a greater degree of premalignancy. Thus, the percentage of antigen-positive cells increased from 18 and 43% to 70% of cultures from tubular, villotubular and villous adenomas, respectively. Considerable heterogeneity in the distribution of antigen-containing cells was evident within any given area of a positive culture. Absorption experiments, using a spectrum of fetal and normal adult tissue extracts, indicated that the adenoma-carcinoma specificity resides in fetal, but not adult, large bowel and normal adult stomach.     

In vitro immune responses to PPD, extracts from Raji cells and nasopharyngeal carcinoma biopsies in NPC leucocytes

Peripheral leucocytes from nasopharyngeal carcinoma (NPC) patients and control subjects, which included healthy subjects and patients with other cancers, have been tested against PPD and a panel of extracts from Raji cells and pooled NPC biopsies, using the blast transformation and the macrophage migration inhibition assays. The results of both assays indicated that the in vitro cell-mediated immune (CMI) responses to the Raji-cell extracts and NPC-biopsy extracts were associated with NPC. However, the peripheral leucocytes from NPC patients and control subjects responded similarly to PPD. These results are in general accord with the skin tests reported by Levine et al. (1976) and Ho, Ng and Kwan (1977b). The antigenic specificity of the NPC-associated CMI responses remains, however, to be resolved, as the extracts used in these or in the in vivo CMI studies were heterogeneous mixtures.     

Expression of P-glycoprotein restricted to normal cells in neuroblastoma biopsies.

Immunohistological detection of P-glycoprotein (P-gp) with monoclonal antibody C219 was performed on serial sections of 37 neuroblastoma specimens representative of the different forms of the disease, from stage 1 ganglioneuroma to stage 4 neuroblastoma. Malignant cells, irrespective of their degree of maturation varying from neuroblasts to ganglion cells, were negative on all specimens. The expression of P-glycoprotein was detected in nine specimens, but it was restricted to normal cells within the tumour. In four specimens, C219 reacted with normal infiltrating cells in the stroma (i.e. monocytes, histiocytes or fibroblasts) representing 5 to 10% of the total population within the section; in three specimens, the residual adrenal gland was strongly positive, and in two ganglioneuromas, a weak reactivity of C219 was observed on a few satellite cells and schwann cells. Three of 15 biopsies obtained at diagnosis contained normal P-gp positive cells: two were classified as stage 1 ganglioneuromas; one was a typical stage 4 composite tumours with positive histiocytes and fibroblasts in the well-differentiated counterpart. Six of 22 biopsies obtained after patients had received our current protocol of chemotherapy contained normal P-gp positive cells: five were partially differentiated and necrotic under the effect of chemotherapy; only one positive specimen was classified as undifferentiated neuroblastoma. Among negative specimens from previously treated patients, one was obtained from a patient in relapse after high-dose chemotherapy and ABMT, two were obtained from patients who had not responded to induction therapy, and six from patients in partial remission after induction therapy. The clinical evolution was very similar in both groups of patients with P-gp negative or positive biopsies. These findings suggest that the quantitative assessment of MDR RNA by northern blotting on fresh homogenates is likely to overestimate its expression on neuroblastoma cells, and that the mechanism of chemoresistance in widespread neuroblastoma is less likely to be associated with P-gp expression.     

Biological behavior of cloned cells of human malignant fibrous histiocytoma in vivo and in vitro.

A human malignant fibrous histiocytoma cell line was established in vitro. Cells showed a wide variety of morphologies, although the karyotype study showed that the tumor was monoclonal in origin because of the presence of unique marker chromosomes in 100% of the cells examined (50 of 50). Cells were cloned according to their characteristic morphologies and biological behavior in culture. The cloned cells were sparse spindle, packed spindle, epithelioid, and lymphoid. In colonies, sparse spindle cells grew separately from each other without cell to cell contact but produced a cartwheel pattern at confluency. Packed spindle cells grew in a tightly packed fashion and produced a storiform pattern at confluency. Epithelioid cells were spindle shaped as individuals but became epithelioid when in contact with each other and produced many multinucleated giant cells at confluency. Lymphoid cells were spindle shaped as individuals but became spherical at confluency. When tumors were grown in nude mice after transplantation of these cloned cells, the histology was shown to be unrelated to morphology in culture and was epithelioid (histiocytic), as was the original tumor. These results show that (a) a single cell derived from malignant fibrous histiocytoma cells exhibits a wide range of phenotypical expression in vitro, (b) cells have their own morphological and biological characteristics in vitro, which (c) however, are easily influenced by environmental factors and (d) which are unstable and even interchangeable. These characteristics may contribute to the endless variety of cellular forms and growth patterns of malignant fibrous histiocytomas in humans.     

Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas

Protein kinase C (PK-C) seems to be involved in the regulation of growth and differentiation of normal epithelial cells. Colonic adenomas and carcinomas show increased proliferation and decreased differentiation. We investigated the activity and subcellular distribution of PK-C in biopsies of normal, neoplastic, and malignant colonic epithelium to evaluate alterations in enzyme activity. In the control group (n = 7), the activity of PK-C was highest in the distal ileum (597 pmol/min/mg protein) and declined to the lowest amounts in rectal mucosa (225 pmol/min/mg protein). In patients with colonic adenomas (n = 16), total PK-C activity was significantly reduced as compared to adjacent mucosa (146 versus 336 pmol/min/mg protein, P less than 0.05) and to values determined in the control group (372 pmol/min/mg protein, P less than 0.01). The reduction of total PK-C activity in the adenoma group was even more evident in intraindividual comparison to paired adjacent mucosa (41.8% of adjacent mucosa, P less than 0.001). Specific activity of membrane-associated PK-C was equally decreased in colonic adenomas (36.3 pmol/min/mg protein) when compared to adjacent mucosa (102 pmol/min/mg protein, P less than 0.05) or to the control group (107 pmol/min/mg protein). In patients with colonic carcinomas (n = 10), the amount of total PK-C activity was also decreased (198 pmol/min/mg protein) when compared to adjacent mucosa or to the control group (P less than 0.05). In addition, the amount of membrane-associated PK-C activity (89.1 pmol/min/mg protein) was significantly reduced in carcinoma when compared to adjacent mucosa (P less than 0.05). The ratio of membrane-associated/total PK-C was not altered in adenomas, while in patients bearing carcinomas the relative fraction of membrane-associated PK-C activity was increased in samples from carcinomas and equally from adjacent colonic mucosa (45.0 and 44.6 versus 28.9%, P less than 0.05) when compared to controls. These results indicate that alterations within the protein kinase C pathway occur as early events in the adenoma-carcinoma sequence of intestinal mucosa, suggesting an important role of PK-C in epithelial differentiation and growth.     

Effects of 5-fluorouracil on cytotoxicity and RNA metabolism in human colonic carcinoma cells

Summary The cytotoxicity of 5-fluorouracil (5-FU) is due in part to the incorporation of the base into RNA molecules. We assessed the cytotoxicity of 5-FU in human colonic carcinoma HT-29 cells and examined mRNA activity (measured by protein biosynthesis in vivo and in vitro) and the maturation of rRNA precursors as two possible modes of action of 5-FU. The rRNA processing pathways were studied using rDNA sequences as probes in blot hybridisation protocols and were specific for both the precursors and mature rRNA species of the maturation pathways. The conclusion from the studies was that although differences in mRNA activity were detected in vivo and in vitro, the significance of these changes are as yet unknown. In contrast, the effects on the pre-rRNA processing pathways proved to be highly significant cytotoxic consequences of 5-FU administration. We discuss the implications of this finding for an understanding of the mode of action of the drug and for the future monitoring of tumour sensitivity to 5-FU.     

Detection of human papillomavirus DNA in biopsies of human oral tissue

We have employed molecular probes produced from DNA fragments of human papillomavirus, cloned into prokaryotic vectors, to detect virus nucleic acid sequences in extracts of human oral tissues. The study was conducted with duplicate coded snap-frozen tissue biopsies from which frozen sections had been taken to accurately assess the pathology of each particular sample. The results show that a large proportion of the oral biopsies contained DNA which hybridized to the viral DNA probes, even under conditions of high stringency. The presence of virus did not correlate with neoplasia in the tissues examined, but HPV like sequences were found in a high proportion (80%) of biopsies taken from areas of keratosis and lichen planus and also in 41 to 46% of normal and tumour tissues.     

Selective nuclear protein phosphorylation/dephosphorylation in subpopulations of human colonic carcinoma cells

The nuclear protein and phosphoprotein profiles from 3 subpopulations of human colonic carcinoma cells which expressed different levels of neoplastic properties were characterized by two-dimensional electrophoresis. The silver stained nuclear protein profiles were found to be remarkably similar among the subpopulations. However, 2 types of nuclear proteins were found to be selectively modified by phosphorylation/dephosphorylation reactions. The dephosphorylation of type I and the phosphorylation of type II nuclear proteins were found to be associated with the HCT 116a subpopulation which expressed a high level of neoplastic properties. Conversely, the phosphorylation of type I and the dephosphorylation of type II nuclear proteins were found to be associated with the HCT 116b subpopulation, which expressed a low level of neoplastic properties. The HCT 116 subpopulation, which expressed an intermediate level of neoplastic properties, was found to possess an intermediate phosphoprotein profile relative to that of the other two subpopulations. Selective modification of cellular proteins by phosphorylation/dephosphorylation reactions may be involved in the generation of tumor cell diversity and heterogeneity.