Molecular addresses of tumors: selection by in vivo phage display

In vivo phage display has been used extensively to screen for novel targets of tumor therapy. Phage display peptide libraries can express random peptides or protein fragments and the aim of phage display is to identify peptide molecules that bind stably to a given target. Angiogenesis is essential to tumor development. Both blood and lymphatic vessels of tumors are different from those of normal tissues. Phage display has been used to analyze the structure and molecular diversity of tumor vasculature and to select tumor-specific antigens which have revealed stage- and type-specific markers of tumor blood vessels. Furthermore, peptides identified by in vivo phage display also work as vehicles to transport cargo therapeutic reagents to tumors. These peptides and their corresponding cellular proteins and ligands may provide molecular tools to selectively target the addresses of tumors and their pathological blood vessels and might increase the efficacy of therapy while decreasing side effects.     

Regulation of tumor growth as a 'total mass' in mice: Apoptosis as a major mechanism in altering growth rates of single and multiple coexisting tumor nodules

Earlier studies have suggested that a did tumor behaves, In Its general pattern of growth, like a normal Integrated organ. In this study, the growth patterns of spherically shaped tumor nodules are re-examlned using an accurate tumor volumemeasuring procedure, with the aim of lnveetlgattng the possible role of apoptosis in regulating tumor growth. Observations revealed at least three distinct phases at growth: rapid growth phase I, slower growth phase II and 'stationary' phase III. Transkion from one phase to the next was primarily due to an increase in the level of apoptosis and not to a decrease in the cell proliferation rate. The level of apoptosis, at a given phase, was similar in a single nodule and each of the multiple coexisting nodules of the same tumor line. However, temporal shifts in apoptosis levds caused early phase transition in coexisting nodules, such that their total volume was the same as that of a single nodule. it can be concluded that apoptasls appears to be a primary mechanism regulating tumor growth as a 'total mass', irrespective of whether the tumor exists in one or multiple nodules, if derived from the same tumor line.     

Modulation of the transcripts for tumor necrosis factor-α and its receptors in vivo

We investigated the effects of a single bacterial lipopolysaccharide (LPS) injection in vivo on the gene expression of tumor necrosis factor-α (TNF) and its receptors: TNF receptor type I (TNF-R 55 kDa or TNF-R1) and TNF receptor type II (TNF-R 75 kDa or TNF-R2) in various tissues and white blood cells. While TNF mRNA rapidly accumulated in most tissues, TNF-R1 and TNF-R2 mRNA levels were found to be differentially regulated in lung, spleen, lymph nodes and white blood cells. In most cases, TNF-R mRNA levels did not parallel TNF mRNA levels. These observations indicate that TNF-R of both types are capable of modulating the host response to LPS, not only by shedding of their extracellular domains, but also by strict regulation of their gene expression.     

The E1b oncogene of adenovirus confers cellular resistance to cytotoxicity of tumor necrosis factor and monoclonal anti-Fas antibody.

The cell lines KB8, 16, and 18 are KB cells which constitutively express adenovirus type 2 (Ad2) E1a, E1a plus E1b, and E1b genes, respectively. We show here that KB18 cells are completely resistant to cytolysis by tumor necrosis factor (TNF) or anti-Fas, although KB8 and KB or KB16 are highly and moderately sensitive, respectively. The levels of receptors for TNF and anti-Fas of KB18 were almost the same as compared with those of KB or other KB-cell lines. Expression of manganous superoxide dismutase (MnSOD) mRNA in KB18 was about 20-fold higher than that in KB or KB8 cells. KB, HT29, and A673 cells infected with dl337 (an Ad5 mutant defective in E1b function) are highly sensitive to TNF or anti-Fas, although wild-type Ad2-infected cells are resistant. Our results indicate that the E1b oncogene can confer cellular resistance to cytolysis by either TNF or anti-Fas in both KB cells and adenovirus-infected human cell lines through influencing intracellular events including regulation of MnSOD genes. Furthermore, we describe how anti-Fas mimics only the cytolytic activity of TNF, whereas TNF also has many other biological activities.     

Studies on the mechanism of rat natural cellular cytotoxicity and its inhibition by normal rat serum.

Non-immune rat spleen lymphoid cells exhibit the capacity to lyse normal dermal-derived fibroblast target cells in the presence of foetal bovine calf serum (FCS)-containing medium, using a 51Cr-release assay. The addition of as little as 1% normal rat serum (NRS) inhibits this reaction. The present study seeks to identify the conditions of factors that may induce or inhibit the expression of normal effector cell (NEC)-mediated lysis. Possible cytotoxicity promoting or inhibiting soluble products released from effector or target cells on their own or in combination could not be demonstrated. Also, the independence of NEC activity from stimulatory factors specifically present in FCS-supplemented medium was illustrated using a variety of heterologous serum supplements as well as purified bovine serum albumin (BSA). The effects of pre-incubating spleen cells in medium supplemented with FCS and NRS, and interchanging these sera in the assay system at various times, showed that the cytotoxic capacity of NECs develops from otherwise inactive cells, in a time-dependent way, immediately upon removal from the inhibitory influence of NRS. Identification of NRS inhibitory factor showed that it was a non-dialysable, heat-stable (at 56 degrees for 30 min) macromolecule of approximately 14,000 to 26,000 mol. wt.     

Expression and mechanism of action of the SARI tumor suppressor in prostate cancer

The objective of this study was to assess the expression of SARI (Suppressor of AP-1, Regulated by IFN) in prostate cancer (Pca) and explore the effects and possible mechanism of action of SARI in the occurrence and development of Pca. In the current study, the expression of SARI was detected using PCR in 40 patients with prostate cancer, 20 patients with prostatic hyperplasia, and prostate cancer cells (LNCaP. and DU145). In addition, the effects of the pro-inflammatory cytokine interferon (IFN)-β on the expression of SARI in DU145 prostate cancer cells and the possible potential signaling pathways activated by SARI were detected using RT-PCR. The expression of SARI protein was downregulated from 0.6957 ± 0.0104 to 0.1597 ± 0.0032 in prostate cancer cells compared with normal prostate tissues and cells. In addition, SARI gene expression increased from 0.0794 ± 0.0133 to 0.1232 ± 0.0162 significantly in a concentration- and time-dependent manner in DU145 cells treated with IFN-β (P<0.05). Finally, MTT assays demonstrated that DU145 cells growth slowed down, flow cytometry demonstrated that IFN-β induced apoptosis increased from 0.0343 ± 0.0039 to 0.0612 + 0.0025 in DU145 prostate cancer cells. In conclusion, the results of the current study suggest that SARI might play an important role in the occurrence and development of prostate cancer. In addition, IFN-β might inhibit the growth of prostate cancer and promote cellular apoptosis by inducing the expression of SARI.     

In vitro and in vivo induction of tumor necrosis factor alpha by Borrelia burgdorferi.

Tumor necrosis factor alpha (TNF-alpha) is an immunoregulatory cytokine with many biological activities including the mediation of inflammation. We examined sera and synovial fluids from patients seropositive for infection with Borrelia burgdorferi using a radioimmunoassay specific for TNF-alpha. Significant elevation of TNF-alpha was found in the sera and synovial fluids of patients examined, while controls showed no elevation. Sera of mice infected with B. burgdorferi contained elevated levels of TNF-alpha which varied during the course of a 24-day infection. To determine whether B. burgdorferi is capable of inducing TNF-alpha production, spirochetes were added to adherent human peripheral blood mononuclear cells or mouse peritoneal exudate cells and 24 h later supernatants were assayed. TNF-alpha induction occurred in a dose-dependent manner. The maximum stimulation occurred when a ratio of 1 to 10 spirochetes per mononuclear cell was used. At optimal concentrations, induction was not diminished by inactivation of spirochetes or pretreatment with polymyxin B. These results suggest that an increase in TNF-alpha production may occur as a result of infection with B. burgdorferi.     

Correlation between the number of viable tumor cells and immune cells in the tumor microenvironment in non-small cell lung cancer after induction therapy

This study aims to determine the correlation between the percent viable tumor cells (%VTC) and the tumor microenvironment in resected non-small cell lung cancer after induction therapy. We enrolled 72 patients with non-small cell lung cancer (NSCLC) who received chemoradiotherapy (CRT) or chemotherapy (CT) prior to surgery. The ratio of the area of viable tumor cells to the total tumor area was calculated to obtain the %VTC. We also examined the number of CD4 (+), CD8 (+), CD20 (+) and FOXP3 (+) tumor-infiltrating lymphocytes (TILs), podoplanin (PDPN) (+) cancer-associated fibroblasts (CAFs), and CD204 (+) tumor-associated macrophages (TAMs) by immunohistochemistry (IHC). In the CRT group (n = 37), the tumors had significantly lower %VTC than the CT group (n = 35) (P < 0.001). In both of the CT group and CRT group, the %VTC showed a significant positive correlation with the number of CD204 (+)-TAMs (P = 0.014 and 0.005, respectively). Only in the CRT group, a higher number of CD204 (+) TAMs was associated with a shorter overall survival (OS) (P = 0.007) and recurrence-free survival (RFS) (P = 0.015). In the CRT group, the number of CD204 (+) TAMs is associated with %VTC and prognosis, suggesting that these cells may have tumor-promoting effects on the residual lung cancer in specific microenvironments after CRT.     

Risk of tumor induction in vivo by residual cellular DNA: quantitative considerations

Certain genes, termed oncogenes, have the ability to transmit the transformed phenotype from tumor cells to hitherto normal cells in vitro and in vivo. This observation is the basis for the concern that medicinal products derived from tumor cell lines could contain possibly tumorigenic contaminating cellular DNA. A comparison between the number of oncogenes necessary for the induction of tumors in a suitable animal model and the quantity in 100 pg residual cellular DNA was carried out. It is shown that the latter contains less than a billionth of the number needed to induce tumors in half of the animals tested. Some factors implicit to this calculation such as the validity of the model system, the linearity of the dose response curve, the concept of multistep carcinogenesis, the amplification of oncogenes, and the transfection enhancing effect of other constituents of the drug are discussed. In summary, 100 pg residual cellular DNA per dose seems to be a safe and, therefore, an acceptable upper limit provided that some factors such as excessive amplification of oncogenes, co-purification of oncogenic DNA with the product or some transfection mediating activity can be excluded.     

逆转录病毒介导的人TNF在人肝癌细胞系（SMMC）中的表达

利用逆转录病毒载体ＬＸＳＮ构建了含人ＴＮＦａ基因的重组逆转录病毒载体Ｌ－ｔｎｆＳＮ。磷酸钙沉淀法将重组载体引入病毒包装细胞ＰＡ３１７，挑选Ｇ４１８抗性克隆，用ＮＩＨ３Ｔ３细胞测定病毒滴度，获得滴度为１×１０５ＣＦＵ／ｍｌ的细胞克隆。应用这一重组病毒感染人肝癌细胞ＳＭＭＣ７７２１，经Ｇ４１８筛选获得抗性克隆。ＰＣＲ可从转导的细胞ＤＮＡ中扩增出ＴＮＦａ基因片段，提示目的基因完整地整合在细胞基因组中。测定转导细胞培养上清中ＴＮＦａ活性，结果显示ＴＮＦａ有相对稳定的表达（１５０～３７０Ｕ／１０６ｃｅｌｌｓ／２４小时）。实验还表明转导细胞的体外生长能力无明显变化，但是其在裸鼠中的致瘤性却明显降低，提示逆转录病毒介导ＴＮＦａ基因对人原发性肝场可能有一定的治疗作用。     

肿瘤干细胞在肿瘤转移中作用与相关机制的研究进展

目前国内外对肿瘤转移的机制已进行了系统而深入的探索。随着肿瘤干细胞概念的提出和相关领域的深入研究,肿瘤干细胞作为肿瘤转移的最佳候选细胞的观点正逐渐被人们接受。目前和肿瘤转移过程相关的分子细胞机制包括:上皮间质转变(EMT)、肿瘤微环境及血管生成机制等。近年来许多研究成果显示上述机制在肿瘤干细胞转移过程中起到不可低估的作用。本文就肿瘤干细胞转移相关机制的进展做一简要综述。     

Spontaneous cytotoxicity of intestinal intraepithelial lymphocytes: clues to the mechanism.

Human intestinal intraepithelial lymphocytes (IEL) demonstrate target cell-restricted spontaneous cytotoxic (SC) activity that is due to CD2+CD3+CD8+CD16-CD56- effector cells; they kill epithelial cell (EC) tumours (such as DLD-1 colon cancer cells), but not natural killer (NK)-sensitive K-562 cells. The present study shows that the measured levels of SC activities by IEL correlated with those of autologous lamina propria lymphocytes (LPL), but not with those of peripheral blood lymphocytes (PBL). Also, the susceptibilities of DLD-1 cell clones to lysis by IEL and PBL effector cells did not correlate, suggesting different mechanisms of lysis. Antibody blocking experiments showed that the main surface molecules involved in lysis depended on the effector cell type: alpha E beta 7 (HML-1) on IEL and CD16 on PBL. No antibody-dependent cell-mediated cytotoxicity (ADCC) was demonstrated by IEL, even after stimulation with interferon-gamma (IFN-gamma). Few IEL expressed Fc receptors for IgG. This study describes further differences between the SC activities of IEL and PBL.     

Modulation of fibronectin production in normal human melanocytes and malignant melanoma cells by interferon-gamma and tumor necrosis factor-alpha.

Normal cultured human epidermal melanocytes and melanoma cells derived from three different malignant melanomas were examined for synthesis of extracellular matrix components before and after treatment for one day with interferon-gamma, tumor necrosis factor-alpha, or both. Treatment of the cells with either cytokine individually had minimal effects on fibronectin levels. Treatment of the cells with the two agents in combination greatly stimulated fibronectin production as indicated by biosynthetic labeling and immunoprecipitation and by enzyme-linked immunosorbent assay. Synthesis of laminin was decreased slightly by the same treatment whereas thrombospondin production was stimulated slightly. The same treatment reduced melanocyte viability slightly but significantly inhibited proliferation and altered the morphology of the melanocytes. The treated cells became flattened and polygonal in shape while the untreated cells exhibited a bipolar shape with one or more long dendritic processes. These morphologic changes were not seen in cultures treated with interferon-gamma or tumor necrosis factor-alpha individually. The effects of interferon-gamma and tumor necrosis factor-alpha on fibronectin production by epidermal melanocytes are in contrast to the effects of the same treatments on fibronectin production by epidermal keratinocytes where fibronectin production is decreased but are similar to the effects of transforming growth factor-beta on a number of other cell types in which increased synthesis of fibronectin occurs and is associated with decreased growth.     

Differential expression of tumor necrosis factor and interleukin-6 by peritoneal macrophages in vivo and in culture.

To investigate the differences in cytokine regulation in vitro as compared to in vivo, we examined the synthesis of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by peritoneal macrophages in response to lipopolysaccharide (LPS). Mice (CBA/J) were primed with an intraperitoneal injection of complete Freund's adjuvant and after 2 weeks, peritoneal cells were harvested for culture or mice were injected intraperitoneally with LPS for in vivo studies. In ascites fluid, TNF-alpha peaked 1 hour after LPS and returned to baseline levels by 4 hours. In contrast, TNF-alpha in the media reached maximum at 7 hours. Expression of TNF-alpha messenger (m)RNA in vivo was rapid but transient, as levels peaked at 15 minutes and returned to baseline 1 hour after LPS. In contrast, TNF-alpha mRNA in vitro became maximal at 1 hour, but remained elevated to 5 hours after LPS. In vivo, IL-6 in ascites fluid peaked at 2 hours, whereas in vitro, IL-6 continued increasing to 24 hours. In vivo, IL-6 mRNA reached maximum at 30 minutes, but fell below baseline by 1.5 hours after LPS. In contrast, IL-6 mRNA in vitro was sustained at maximal expression between 5 to 9 hours after LPS. These results demonstrate that both TNF-alpha and IL-6 synthesis is more rapid in vivo than in vitro. The rapid kinetics of cytokine expression in vivo must considered when designing strategies to inhibit cytokine action in vivo.     

Oxidative damage: The biochemical mechanism of cellular injury and necrosis in choline deficiency

Oxidative stress and damage are characterized by decreased tissue antioxidant levels, consumption of tissue α-tocopherol, and increased lipid peroxidation. These processes occur earlier than necrosis in the liver, heart, kidney, and brain of weanling rats fed a choline deficient (CD) diet. In tissues, water-soluble antioxidants were analyzed as total reactive antioxidant potential (TRAP), α-tocopherol content was estimated from homogenate chemiluminescence (homogenate-CL), and lipid peroxidation was evaluated by thiobarbituric acid reactive substances (TBARS). Histopathology showed hepatic steatosis at days 1-7, tubular and glomerular necrosis in kidney at days 6 and 7, and inflammation and necrosis in heart at days 6 and 7. TRAP levels decreased by 18%, 48%, 56%, and 66% at day 7, with t_1/2 (times for half maximal change) of 2.0, 1.8, 2.5, and 3.0 days in liver, kidney, heart, and brain, respectively. Homogenate-CL increased by 97%, 113%, 18%, and 297% at day 7, with t_1/2 of 2.5, 2.6, 2.8, and 3.2 days in the four organs, respectively. TBARS contents increased by 98%, 157%, 104%, and 347% at day 7, with t_1/2 of 2.6, 2.8, 3.0, and 5.0 days in the four organs, respectively. Plasma showed a 33% decrease in TRAP and a 5-fold increase in TBARS at day 5. Oxidative stress and damage are processes occurring earlier than necrosis in the kidney and heart. In case of steatosis prior to antioxidant consumption and increased lipid peroxidation, no necrosis is observed in the liver.     

Proliferative activity and tumor angiogenesis is closely correlated to stromal cellularity of fibroadenoma: Proposal fibroadenoma, cellular variant

Fibroadenoma (FA) is the most common benign tumor of the breast in adult women. Some FA have a highly cellular stroma, making it difficult to differentiate from phyllodes tumors (PT). Forty-three FA were grouped into: (i) 27 conventional type (FACT) median stromal cellularity (SC) of highest cellular area (HCA), < or = 125 cells/1 high-power field (HPF); and (ii) 16 cellular variant (FACV) median SC of HCA, > 125 cells/1 HPF. These were studied for the proliferative activity of their stromal cells. Expression of c-fos, p53, basic fibroblast growth factor (bFGF), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor (VEGF) in the stromal cells were examined in the FA and 12 PT to determine whether it is possible to separate FACV from FACT. The proliferative activity of stromal cells was evaluated by the labeling index (LI) of proliferating cell nuclear antigen (PCNA). Conventional type fibroadenoma stromal cells had the lowest frequency of c-fos, p53, bFGF, FGFR and VEGF protein expression; PT stromal cells had the highest frequency of expression; and FACV stromal cells had an intermediate frequency of expression. Multivariate analysis demonstrated that bFGF and FGFR expression are significantly correlated with SC of FA. Separation of FACV from FACT by SC seems appropriate in revealing the phenotypic and biological differences of FA. The SC of FA seems to be regulated by bFGF and FGFR expression.     

A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity

A simple way of measuring and evaluating lactate dehydrogenase release from lysed tumor cells is described. LDH activity was determined as NADH oxidation or INT reduction over a defined time interval, which was limited by stopping the enzymatic reaction with the inhibitor oxamate. Reaction products were then assayed using a microplate reader. The principle of measuring LDH activity of cellular culture supernatants as a measure of cytotoxicity was successfully applied to a number of murine and human effector-target cell combinations (macrophages, monocytes, NK cells and cytotoxic T cells with P815, A375, K562 and Yac-1 tumor cells) as well as to the determination of TNF activity on L929 cells. Comparison with 51Cr release assays suggests that LDH release assays are an appropriate and possibly preferable means of measuring cellular cytotoxic reactions. This LDH release assay combines the advantages of reliability and simple evaluation characteristic of radioisotope release assays with the convenience of speed and avoidance of radioactivity.     

死亡肿瘤细胞诱导分化的巨噬细胞体内抗瘤作用的研究

目的观察死亡肿瘤细胞诱导分化的巨噬细胞的体内抗瘤作用。方法采用死亡H22肿瘤细胞诱导单核-巨噬细胞的分化。取40只健康纯系的昆明小鼠，随机分为4组：①生理盐水接种组（A组）；②热灭活H22肿瘤细胞接种组（B组）；③液体石蜡诱生的巨噬细胞接种组（C组）；④死亡肿瘤细胞诱导分化的巨噬细胞接种组（D组）。A组、B组、C组和D组小鼠分别于左后肢皮下接种生理盐水、热灭活H22肿瘤细胞、液体石蜡诱生的巨噬细胞和死亡肿瘤细胞诱导分化的巨噬细胞，2周后各组小鼠分别于左前肢皮下接种生长良好的H22肿瘤细胞，6周后处死全部小鼠，并计算成瘤率、瘤重量与瘤体积；制备小鼠全脾细胞作为效应细胞，以H22肿瘤细胞为靶细胞，混合培养72h后MTT法检测肿瘤细胞杀伤率。结果D组的成瘤率、瘤重量与瘤体积分别为10％、0．12g和0．10cm^3，明显低于A组[80％、（2．41±0．72）g和（2．45±0．82）cm^3]、B组[40％、（0．51±0．36）g和（0．65±0．31）cm^3]和C组[60％、（1．87±0．67）g和（2．15±0．45）cm^3]，P〈0．05，差异有统计学意义；D组的肿瘤细胞杀伤率为（35．89±13．86）％，明显高于A组（12．10±2．74）％、B组（14．88±3．96）％和C组（24．54±4．18）％，P〈0．05，差异有统计学意义。结论死亡肿瘤细胞诱导分化的巨噬细胞具有明显的抗瘤作用。     

基因重组肿瘤坏死因子对LAK细胞体内外抗肿瘤作用的增强效果

TNF单独不能诱导LAK活性,也不能进一步提高最适剂量IL-2(1000U/ml)诱导的LAK活性,但能显著增强亚适剂量IL-2(10～100U/ml)诱导的LAK活性。抗TNF单抗和抗IL-2受体β链(p75)单抗显著抑制TNF/IL-2对LAK活性的协同诱导作用。实验证明,TNF能显著增强IL-2/LAK细胞的体内抗肿瘤效果,对于腹水型肝癌小鼠,以联合腹腔内注射TNF/IL-2/LAK细胞的体内抗肿瘤效果最佳。