Fas在6种泌尿生殖系统肿瘤细胞系中的表达

背景与目的：在肿瘤细胞中Fas蛋白的表达是研究治疗肿瘤及判断愈后的重要基础,本研究检测Fas在泌尿生殖系统恶性肿瘤细胞系中的表达情况。方法：采用流式细胞直接免疫荧光法、Western blot和Nortern杂交对6种泌尿生殖恶性肿瘤细胞系[膀胱癌（T24,EJ,BIU-87）、肾癌（GRC-1,RCC-949）、前列腺癌（PC-3M)和一种原代培养正常肾间质成纤维细胞Fas分子的表达进行了检测。结果：直接免疫荧光流式细胞术检测细胞表面Fas在6种泌尿生殖恶性肿瘤细胞系均有表达,阳性率波动于16.51%-49.13%,但与原代培养的正常肾间质成纤维细胞（阳性率77.98%)相比,其阳性细胞百分率较低,细胞表面特异性免疫荧光强度较弱;6种泌尿生殖肿瘤细胞系Fas蛋白的表达均可用Western blot法检测到一条分子量约48kDa的清晰条带,但表达强度不同,以BIU-87细胞最弱,而T24细胞最明显;只有在BIU-87细胞系中未能用Northern杂交检测到Fas mRNA的表达,其它细胞系均有不同程度的表达,以PC-3M、T24细胞系较强、GRC-1、RCC-949细胞系次之,EJ细胞仅有微弱表达,正常肾间质成纤维细胞RFb表达较强的Fas mRNA。结论：Fas广泛表达于泌尿生殖系恶性肿瘤培养细胞和正常细胞中,但在肿瘤中的表达情况明显低于正常细胞,这可能是泌尿生殖系统恶性肿瘤的产生和进展的重要原因之一。     

Alterations in glycoproteins and lipids in azaserine-induced acinar cell carcinoma of rat pancreas

Glycoproteins and lipids of rat pancreatic acinar cell carcinomas maintained in nude mice and in cell culture, were analyzed. The tumor contained significantly elevated levels of glycoproteins when compared with their normal counterparts. SDS-PAGE of tumor glycoproteins revealed that there were increased amounts of small molecular weight glycoproteins and the tumor also contained a 51,000 dalton glycoprotein which was not detected in the pancreas, liver or the sera of the control animals. The tumor in nude mice and cancer cells in culture had decreased lecithins and triglycerides, and increased amounts of free fatty acids, and both free and esterified cholesterols. The results indicate that altered glycoprotein and lipid compositions represent some of the characteristic features of the acinar cell carcinoma.     

Response of primary human lung carcinomas to autocrine growth factors produced by a lung carcinoma cell line

Medium conditioned for 48 to 72 h by A549-1 lung carcinoma cells was used to culture primary solid lung tumors on feeder layers of inactivated Swiss 3T3 cells. Of 22 cases placed into culture, primary cultures of carcinoma cells were obtained in 20. Subcultures were obtained in 18 cases, and cell lines were established in nine cases. The neoplastic origin of the cultured cells was demonstrated by several criteria: tumorigenicity in athymic mice; anchorage-independent growth; expression of altered lactate dehydrogenase isoenzyme profiles; and expression of the lung tumor marker pregnancy-specific glycoprotein 1. The epithelial nature of cultured carcinoma cells was demonstrated by expression of keratin. These characteristics were compared to normal epithelial cells established in culture from bronchial explants from the same donors as tumor tissue, or other donors. The growth-stimulating effect of conditioned medium toward primary or newly cultured tumor cells was quantitated by clonal assays in soft agar and in monolayer culture. Growth response in clonal assays of newly cultured carcinoma cells to the purified growth factors transforming growth factor alpha and insulin-like growth factor 1, two known components of medium conditioned by A549-1 cells, was also demonstrated.     

Differential tumorigenicity of two autologous human breast carcinoma cell lines, HMT-3909S1 and HMT-3909S8, established in serum-free medium

In a serum-free medium we have established two new human breast carcinoma cell lines from a single primary tumor. Cultures were maintained on chemically defined medium CDM3 or on minor modifications of this medium, Dulbecco's modified Eagle medium-Ham's F12 supplemented with epidermal growth factor, insulin, transferrin, estradiol, hydrocortisone, triiodothyronine, cyclic AMP, phosphoethanolamine, ethanolamine, fibronectin, fetuin, ascorbic acid, bovine serum albumin, and trace element salts including selenite (Petersen and van Deurs, Cancer Res., 47: 856-866, 1987). Primary cultures comprised both NADPH-neotetrazolium reductase-positive carcinoma cells and NADPH-neotetrazolium reductase-negative cells of stromal appearance, as well as normal epithelial cells (Petersen and van Deurs, Cancer Res., 46: 2013-2020, 1986). In subsequent passages the cells were monitored exclusively using the tumorigenicity assay on nude mice. Two cell lines, one nontumorigenic, HMT-3909S1, and one tumorigenic, HMT-3909S8, were selected from the primary cultures. Selection of S8 through subline S4 required transient supplementation of CDM3 with fetal calf serum. Permanent lines S1 and S8 were maintained on serum-free medium. Further characterization of the two cell lines in terms of normal breast gland differentiation (Petersen and van Deurs, Differentiation, 39: 197-215, 1988) was carried out using immunocytochemistry, immunochemistry, electron microscopy, and cytogenetics. S8 appeared to be identical with the NADPH-neotetrazolium reductase-positive carcinoma cells of the primary cultures, with a particular subpopulation of carcinoma cells in the tumor of origin, and with the tumorigenic cells of the nude mice. This subline was aneuploid, typically epithelial in morphology, and expressed keratins K8 and K18 and the glycoprotein MAM-6, typical of luminal epithelial cells in the normal breast gland. Subline S1 appeared more like the elongated cells in the primary cultures and like a second subpopulation of cells in the carcinoma of origin. However, S1 cells were in fact epithelial, since they expressed keratins. Also, S1 cells seemed to be a triploidation of a cell with close resemblance to S4, while only few cytogenetic differences were found between S4 and S8, suggesting an origin of S1 and S8 via S4 from a single hypothetical stem cell.     

Cytogenetic analysis of human renal carcinoma cell lines of common origin (NC 65).

Cytogenetic analyses were performed on 3 clonal cell lines derived from a human renal cell carcinoma and its lymph node metastasis, two long-term tissue culture cell lines (NC 65-Sp and NC 65-R) and a serially transplantable tumor line growing on nude mice and brought into culture at the fifth animal passage (NC 65-V). Karyotype were established using banding techniques. Most of the marker chromosomes could be identified and were derived by deletion, inversion, translocation, or isochromosome formation of Chromosomes 1, 3, 4, 5, 8, 9, and 17. These markers were different from HeLa markers. NC 65-Sp had a near diploid chromosome number, NC 65-R a hypotetraploid number, and NC 65-V had a bimodal chromosome number, and NC 65-V had a bimodal chromosome number. Three chromosome markers were shared by the three cell lines; NC 65-R and NC 65-V shared an additional set of four markers. Markers specific to each line were also observed; they demonstrated the independent derivation of the lines and eliminated laboratory cross-contamination. Common markers between the lines confirmed their common tumoral origin.     

Interleukin-6 functions as an autocrine growth factor in human bladder carcinoma cell lines in vitro

Interleukin (IL)-6 is reported to function as a growth factor for renal and prostatic carcinomas. We conducted the present study to define the role of IL-6 in the growth of normal and neoplastic urothelial cells. Human bladder carcinoma cell lines (253J, RT4 and T24) and primary cultured human urothelial cells derived from normal ureters were used. Recombinant human IL-6 stimulated the growth of bladder carcinoma cell lines far better than that of normal urothelial cells (p < 0.001). All carcinoma cell lines tested produced and released IL-6, whereas normal urothelial cells did so only at marginal levels. Furthermore, treatment with lipopolysaccharide derived from Escherichia coli, tumor necrosis factor-α or IL-1 increased IL-6 secretion by bladder carcinoma cell lines but not by normal urothelial cells. Growth of bladder carcinoma cells was significantly inhibited by anti-IL-6 neutralizing antibody or the anti-sense oligonucleotide for IL-6 cDNA. We conclude that IL-6 functions as an autocrine growth factor for bladder carcinoma cells but not for normal urothelial cells and that it may be a factor accounting for the marked enhancement of inflammation-associated bladder carcinogenesis and tumor growth. Int. J. Cancer 72:149–154, 1997. © 1997 Wiley-Liss Inc.     

Impaired expression of the cell cycle regulator BTG2 is common in clear cell renal cell carcinoma

The prognosis of patients with renal cell carcinoma (RCC) is poor. A full understanding of the molecular genetics and signaling pathways involved in renal cancer development and in the metastatic process is of central importance for developing innovative and novel treatment options. In this study, BD Atlas Human Cancer 1.2 cDNA microarrays were used to identify genes involved in renal tumorigenesis. By analyzing gene expression patterns of four clear cell RCC (cRCC) cell lines and normal renal tissue, 25 genes were found differentially expressed. To determine the relevance of these genes, RNA in situ hybridization was performed on a tissue microarray generated from 61 snap-frozen primary renal cell carcinomas and 12 normal renal cortex biopsies. B-cell translocation gene 2 (BTG2), a negative cell cycle regulator, which was expressed in normal renal tissue but down-regulated in cRCC cell lines and primary cRCCs, was selected for additional experiments. Quantitative BTG2 mRNA expression analysis in 42 primary cRCCs and 18 normal renal cortex biopsies revealed up to 44-fold reduced expression in the tumor tissues. Decrease of BTG2 expression was not associated with tumor stage, grade, and survival. Cell culture experiments demonstrated that BTG2 expression was weakly inducible by the phorbolester 12-O-tetradecanoylphorbol-13-acetate in one of four cRCC cell lines. In contrast, increasing cell density led to elevated BTG2 mRNA expression in three of four cRCC cell lines. In both experiments, BTG2 mRNA levels did not reach values observed in normal renal tissue. These data suggest that down-regulation of BTG2 is an important step in renal cancer development.     

Prolactin receptors in human breast cancer cells in long-term tissue culture.

Prolactin receptors have been identified for the first time in a number of human breast cancer cell lines and a normal human breast cell line maintained in long-term tissue culture. Optimal conditions for determining the binding of 125I-labeled human prolactin to these cells were established. Five different tumor cell lines have different content of prolactin receptors ranging from 2,300 to 26,000 sites/cell. All tumor cell lines contained more prolactin receptors than does one normal breast cell line (1700 sites/cell). The prolactin receptors in these human mammary tumor cells not only bind human prolactin but also recognize other lactogenic hormones such as human growth hormone, human placental lactogen, and sheep prolactin, but not animal growth hormone, which are not lactogenic. The affinity (Ka) of binding of human prolactin to these cells is 4 x 10(9) M-1 (Kd = 2.5 x 10(-10)M). The hormone specificity and affinity for hormone of these human mammary tumor cells are very similar to that found for the rabbit mammary gland. These human mammary tumor cell lines in long-term culture should prove very useful to study the biology of prolactin receptors in living human cells and the role of prolactin in the tumorigenesis of the human breast.     

Growth and metastasis of tumor cells isolated from a human renal cell carcinoma implanted into different organs of nude mice

The purpose of this study was to determine whether the methods for isolating tumor cells from a human renal cell carcinoma (HRCC) influence the biological behavior of the cancer cells. Renal cell carcinoma obtained from a surgical specimen was dissociated by enzymatic treatment and cells were plated into culture dishes or injected s.c. into the kidney of BALB/c nude mice. The resultant kidney tumor produced liver metastasis and ascites. All tumors growing in nude mice (s.c., kidney, liver, ascites) were also established in culture. The human origin of all five lines was ascertained by karyotypic and isoenzyme analyses. Cells from all lines were injected, s.c., i.p., i.v., intrasplenically, and beneath the renal capsule of nude mice. All the lines were tumorigenic after s.c. or renal subcapsule injection, although the rate of tumor growth varied among the five lines. The metastatic behavior of the HRCC cells was influenced by both the nature of the tumor cells and the route of injection into nude mice. In general, cells derived from the liver metastasis produced more metastases in nude mice than other lines. The lines established in culture from the primary HRCC and the ascites were poorly metastatic. Even with highly metastatic cells, i.v. injection did not yield significant metastasis, but the injection of cells into the renal subcapsule resulted in extensive metastasis to the lungs and in all peritoneal organs. These results indicate that nude mice can be used for the isolation of populations of HRCC cells with different growth and metastatic potential and that, of the organ sites tested, the renal subcapsule is the most advantageous site for implantation of HRCC cells.     

Detection of human lung epithelial cell growth factors produced by a lung carcinoma cell line: use in culture of primary solid lung tumors

Serum-free medium conditioned for 72 h by a human bronchioloalveolar carcinoma of lung, A549-1, stimulated the colony formation of normal human bronchial epithelial cells, newly cultured cells from human solid lung tumors, and established human lung tumor cell lines, including A549-1 cells themselves. This activity was concentration dependent and was stable to acid. Growth factors in A549-1 conditioned medium (CM) supported culture of solid lung tumors; primary cell cultures were obtained from nine of 10 solid lung tumors of non-small cell origin and from one small cell tumor using A549-1 CM. In addition, three cell lines have been established to date from these primary cultures. Gel filtration of concentrated A549-1 CM on Biogel P-10 separated the growth promoting activity into four regions of apparent Mr 70,000, 12,000, 8,000, and 6,000, and two broad regions of apparent Mr 3000-5000. All but the 12,000 Mr fraction contained activity which competed for specific binding of epidermal growth factor (EGF) to A431 cell membranes. CM was superior to both EGF and TGF alpha in stimulating growth of normal and neoplastic lung cells. EGF also was inhibitory to tumor cells while TGF alpha stimulated both normal and tumor cell growth. TGF beta was also found in CM but inhibited normal and neoplastic lung epithelial cell growth. Of other substances tested, ILGF-I stimulated colony formation. The results suggest that autocrine factors may be important in non-small cell lung tumor cell growth and that differences in response to EGF and TGF alpha may provide the basis for selective culturing of normal and neoplastic lung epithelial cells.     

人肾癌细胞原代培养方法比较及其生长特性观察

目的:分析比较三种方法分离人肾癌组织原代培养肾癌细胞及其传代能力,探讨建立理想的人肾癌细胞体外实验模型。方法:分别采用组织块贴壁法、机械分散法及差速贴壁分离法、混合酶消化法及差速贴壁分离法进行人肾癌细胞的体外培养,并从细胞形态学、表型、增殖生长曲线和细胞活力曲线等方面进行对比研究。结果:三种方法均可获得原代肾癌细胞,采用组织块贴壁法可获得较多数量的肾癌细胞,采用混合酶消化加分步贴壁后培养情况最好,数量较多,形态完整,第4代后肿瘤细胞状态稳定,增殖活跃,有明显的对数生长期,且与肾癌细胞系769-P细胞具有相似的表面抗原表达。结论:采用混合酶消化加分步贴壁法可获得较纯的肿瘤细胞,培养生长时间短,成功率高,适宜体外原代培养。     

Human renal carcinoma: characterization of five new cell lines

Five human renal carcinoma cell lines have been established in long-term tissue culture. Two of the cell lines, UM-RC-2 and UM-RC-3, produced clear cell tumors in athymic nude mice. The cell lines have been characterized by staining with oil red O, doubling time in vitro, and number of chromosomes. Although protein A assay reactivity of autologous combinations of patient's sera and tumor cells were seen with all five cell lines, similar binding was also found with autologous normal kidney cultures. However, the immune adherence assay demonstrated low titer autologous reactivity with two renal carcinoma cell lines but not with the corresponding normal kidney cultures. This strongly suggest host recognition of tumor-associated antigens. Characterization of cell surface antigens with murine monoclonal antibodies demonstrated shared reactivity between normal kidney tubular cells and renal carcinoma cells. Antibody A68.11 reacted strongly with all five cell lines. Antibody A80 bound to only UM-RC-3 and UM-RC-6.     

Membrane-associated antigens on tumor cells from transitional-cell carcinoma of the human urinary bladder. I. Immunological characterization by xenogeneic antisera

An antiserum was raised in rabbits by immunization with a human tumor cell line, T-24, derived from transitional cell carcinoma (TCC) of the urinary bladder. The specificity of the IgG fraction was assessed by antibody-dependent cellular cytotoxicity (ADCC), using purified blood lymphocytes from healthy human donors as effector cells and seven human cell lines as target cells (T-24 and two additional TCC-lines, two normal urothelial lines, one colon carcinoma and one malignant melanoma). The IgG induced strong lysis of all seven target cell types. However, lysis of the five urothelial lines was significantly stronger than that of the control tumors. Conversely, antisera to either of the control tumors induced a significantly stronger lysis of the homologous tumors than of the urothelial cells. All antisera contained antibodies to fetal bovine serum but removal of these by absorption did not change the specificity of the ADCC reactions. When the anti T-24 serum was exhaustively absorbed with glutaraldehyde-fixed spleen homogenate, the cytotoxicity to the control tumor targets was abolished. Although absorption reduced the antibody titer of the IgG preparation, ADCC to the TCC targets remained at a high level. There was no difference in the degree of lysis of the three TCC targets. Lysis of the two target lines from normal urothelium was slightly lower than that of the tumor cells. The results indicate that the anti-TCC serum contained antibodies to one or several antigens shared by five urothelial cell lines but not by the control tumors. Whether or not it also contained antibodies to TCC-associated antigens remains to be established. The molecular basis for these findings will be given in the accompanying paper.     

Expression of CD154 on renal cell carcinomas and effect on cell proliferation,motility and platelet-activating factor synthesis

CD40 activation by CD154 may trigger diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death, in normal and malignant cells. However, the pathophysiologic role of CD154 expressed by tumor cells remains unclear. We have investigated the expression of the CD40-CD154 system in 24 primary cultures derived from renal cell carcinomas, its correlation with tumor stage and its potential functional significance. We found coexpression of CD40 and CD154 in most of the renal carcinoma cell lines. CD154, but not CD40 expression, significantly correlated with tumor stage. Moreover, renal carcinoma cell lines also released the soluble form of CD154 into the supernatant. CD40 engagement by CD154 did not affect apoptosis or survival. On the contrary, CD154 stimulated cell proliferation, motility and production of PAF, a phospholipid mediator of inflammation with angiogenic properties. Furthermore, the renal carcinoma cell lines expressed PAF-R. Blockade of PAF-R by WEB-2170, a PAF-R antagonist, abolished the CD154-dependent motility, indicating a role for PAF synthesized after CD154 stimulation in renal carcinoma cell motility. In conclusion, this study identifies new functional properties for CD154, which are potentially relevant for the growth and dissemination of renal carcinoma cells.     

Tumor progression in four mammary epithelial cell lines derived from the same patient

Two primary and two metastatic cell lines with distinct phenotypes and genotypes have been established from a patient diagnosed as having infiltrating and intraductal mammary carcinoma (21T series). All four lines can be cultured in the same medium, DFCI-1, which also supports long-term growth of normal epithelial cells. Therefore, we have been able to compare normal and tumor cells at the cellular and molecular levels. The mammary origin of the 21T series was confirmed by using antibodies against the human milk fat globule antigen-2 epitope. The two primary tumor lines (21NT and 21PT) are both immortal and aneuploid, although only 21NT is tumorigenic in the nude mouse assay. The two populations derived from the metastatic pleural effusion (21MT-1 and 21MT-2) each exhibit distinct characteristics in morphology and growth factor requirements. The erbB2 gene is amplified and overexpressed in all of these cell lines compared to normal epithelial cell controls. These four tumor cell lines from a single patient represent a mammary tumor progression series that has been established in long-term cell culture.     

Heterogeneity of glycoprotein synthesis in human tumor cell lines

In order to screen human tumor cells for putative cell surface marker molecules, the glycoprotein composition of in vitro cultivated human tumor cell lines of different origin (12 carcinomas, one neuroblastoma, one melanoma and one sarcoma) was analyzed by metabolically labelling the cells with [³H]galactose, [³H]mannose and [³H]fucose and subsequently separating the labelled material by SDS-PAGE. The cell lines expressed their specific glycoprotein patterns. Strongly glycosylated proteins of apparent mol. wt 40–45 kD, 60–62 kD, 80–82 kD and 90–92 kD were shared by nearly all carcinoma cell lines studied. Apart from these glycoprotein clusters, a great diversity was observed between tumor cell lines derived from the same organ. Three bladder carcinoma cell lines had a 112–114 kD glycoprotein in common. Glycoprotein expression of these cell lines remained constant during 1 yr of in vitro culture. Hence, these glycoprotein patterns seem to be useful for monitoring the phenotypic stability of cell lines. A sarcoma cell line was deficient in incorporating fucose and showed strikingly different glycoprotein patterns compared to the other cell lines studied. The metabolic labelling procedure revealed a wide phenotypic heterogeneity of the human carcinoma cell lines concerning glycoprotein synthesis. This method contributes another parameter to map the major glycoprotein species of various types of carcinomas.     

Expression of thrombospondin-1 in human pancreatic adenocarcinomas: Role in matrix metalloproteinase-9 production

Human pancreatic adenocarcinoma, an aggressive malignant disease, shows a strong desmoplastic reaction characterized by a remarkable proliferation of interstitial connective tissues. Thrombospondin-1 (TSP-1), a 450 kDa platelet and matrix glycoprotein, has been implicated in tumor invasion, angiogenesis and metastasis. TSP-1 and MMP-9 expression in pancreatic adenocarcinoma and control pancreas tissues was measured by immunohistochemistry. TSP-1 expression in pancreatic carcinoma cell lines, fibroblasts, and endothelial cells was measured by a competitive TSP-1 enzyme linked immunosorbent assay (ELISA). The effect of TSP-1 on MMP-9 production in pancreatic carcinoma cell lines was measured by zymography and Western blot analysis. Eighty five per cent (23/27) of cases of pancreatic adenocarcinoma showed increased TSP-1 staining in the desmoplastic stroma adjacent to tumor cells. No specific positive staining for TSP-1 was observed in the normal pancreatic tissues and the inflammatory areas. TSP-1 localized in tumor stroma surrounding the tumor cells expressing MMP-9. Using TSP-1 competitive ELISA, the secretion of TSP-1 by different pancreatic cancer cell lines into culture medium varied from 11.45 plus minus 14.08 to 275.82 plus minus 45.56 ng/10 6 cells/24 hours. The amounts of TSP-1 detected in both culture media and cell extracts from fibroblasts or endothelial cells were at least 2-3 fold higher than those from pancreatic cancer cells. TSP-1 augmented the production of matrix metalloproteinase-9, a matrix degrading enzyme, in pancreatic cancer cells in vitro. Stromally-derived TSP-1 up-regulates the production of MMP-9 by pancreatic adenocarcinoma. These data are consistent with the conclusion that TSP-1-rich stroma is involved in regulating matrix remodeling in tumor invasion.     

Stimulation of thymidine uptake and cell proliferation in mouse embryo fibroblasts by conditioned medium from mammary cells in culture.

Undialyzed conditioned medium from several cell culture sources did not stimulate thymidine incorporation or cell overgrowth in quiescent, density-inhibited mouse embryo fibroblast cells. However, dialyzed conditioned medium (DCM) from clonal mouse mammary cell lines MCG-V14, MCG-T14, MCG-T10; HeLa cells; primary mouse adenocarcinoma cells; and BALB/c normal mouse mammary epithelial cells promoted growth in quiescent fibroblasts. The amount of growth-promoting activity produced per cell varied from 24% (HeLa) to 213% (MCG-V14) of the activity produced by primary tumor cells. The production of growth-promoting activity was not unique to tumor-derived cells or cells of high tumorigenicity. The amount of growth-promoting activity produced per cell in the active cultures was not correlated with any of the following: tumorigenicity, growth rat, cell density achieved at saturation, cell type, or species of cell origin. It is concluded that transformed and non-transformed cells of diverse origin, cell type, and tumorigenicity can produce growth factors in culture. The growth-promoting potential of the active media from primary tumor cultures accumulated with time of contact with cells and was too great to be accounted for entirely by the removal of low-molecular-weight inhibitors by dialysis. The results are consistent with the hypothesis that conditioned medium from the active cultures contained a dialyzable, growth-promoting activity. Different cell lines exhibited differential sensitivity to tumor cell DCM and fetal bovine serum. Furthermore, quiescent fibroblasts were stimulated by primary tumor cell DCM in the presence of saturating concentrations of fetal bovine serum. These observations support the notion that the active growth-promoting principle in primary tumor cell DCM may not be a serum factor(s).