Mass spectrometric high-throughput analysis of serum-free conditioned medium from cancer cell lines

Proteins secreted by cancer cells may be useful as tumor markers. We analyzed constituents produced by cells grown in serum-free conditioned medium to avoid confounding factors present in serum. Mass spectrometric techniques were used to obtain expression patterns of constituents between 2000 and 15 000 Da from as little as 1 μl of crude sample. This protocol can produce distinctive mass profiles from 16 cell lines within 1 h. Thus, differential display by mass spectrometry will expedite the discovery of peptides specifically secreted by particular cells. These data illustrate the advantage of this strategy over the conventional approach of electrophoretic separation of serum samples.     

Adaptation of cell lines to serum-free culture medium

The optimum conditions to allow proliferation of cells for the secretion of some growth factors and cytokines and the proliferation of cells in different media that do not contain proteins or serum from animals (serum-free media) were investigated. The culture of cell lines for the commercial production of products involves optimisation of cell proliferation and secretion in media from which the requisite proteins can be economically extracted. Some of these problems were addressed in this study. We used two different clones from a human myeloma cell line for adaptation to serum free medium in order to characterize long-term effects of the new medium. We gradually decreased serum content of medium and the results showed that cell mortality increased with serum reduction, antibody production lost by survived clones, and secretion of cytokines were always retained.     

Monoclonal antibodies reactive with components in serum-free conditioned medium from a human breast cancer cell line (MCF-7)

Approximately 10,000 primary hybridomas were generated after immunization with either serum-free conditioned medium (SFCM) or extracts from the human breast cancer cell line MCF-7. A total of 11 different monoclonal antibodies (MAbs; 8 generated against SFCM and 3 generated against cell extracts) were selected on the basis of high specificity in cell-binding ELISA for human breast cancer cell lines. The 8 different MAbs obtained by immunization with SFCM all reacted with secreted components in SFCM from MCF-7 cells and 4 of these MAbs reacted with glycolipids extracted from MCF-7 cells. 1 of these MAbs (S2) also recognized a secreted glycoprotein of approximately 77 kilodaltons (kDa). The remaining 4 MAbs did not show specificity solely for carbohydrate determinants. 1 of these MAbs (S7) recognized a secreted protein of approximately 41 kDa. The 3 MAbs raised against cell extracts from breast cancer cells reacted with cytoplasmic antigens in immunofluorescence but also reacted with a secreted component in SFCM from MCF-7 cells. Immunoblotting experiments with proteins from cell extracts and with proteins in SFCM showed that these antibodies all reacted with a protein of a molecular weight of approximately 40 kDa. Our results suggest that this component is cytokeratin 19 or proteolytically processed cytokeratin 19.     

Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium

We tested the ability of serum-free media to support the in vitro growth of human non-small cell lung carcinoma. A medium containing insulin, transferrin, sodium selenite, hydrocortisone, epidermal growth factor, and bovine serum albumin (1 mg/ml) with serum precoating of culture dishes (modified LA medium) supported three previously established cell lines of non-small cell lung cancer and prevented fibroblast proliferation in fresh tumor specimens but did not support long term tumor cell growth from fresh specimens. We added triiodothyronine, sodium pyruvate, and additional glutamine, insulin, and epidermal growth factor to modified LA medium, precoated with fibronectin and collagen instead of serum, and deleted bovine serum albumin, defining a new medium called ACL-3. ACL-3 medium alone supported the short term growth of 10 of 12 cell lines and the soft agarose cloning of 9 of 12 cell lines tested, and ACL-3 supplemented by an optimal concentration of bovine serum albumin (5 mg/ml) supported the long term growth of 10 of 12 cell lines tested. Moreover, we have grown tumor cells for more than 6 months from 11 of 33 (33%) consecutive fresh clinical specimens of human lung adenocarcinoma in ACL-3 with bovine serum albumin. ACL-3 medium provides a defined environment for the study of growth factor requirements of human non-small cell lung cancer and enhances our ability to grow human lung cancer, particularly adenocarcinoma, in vitro.     

人高、低转移肺大细胞癌细胞系的mRNA差异显示研究

目的 通过研究人高、低转移肺大细胞癌细胞系基冈差异表达谱，克隆与肿瘤转移相关的基因。方法 应用mRNA差异显示技术(DDRT-PCR)对高、低转移人肺大细胞癌细胞系的mRNA进行差异显示，切胶回收差异表达cDNA，克隆测序后与GenBank数据库进行Blast比对。结果 获得差异表达cDNA50条，其中已知功能基因18条，末知功能基因16条，DNA来源16条，EST4条。结论 人高、低转移肺大细胞癌细胞系的基因表达差异较大，转移的过程受基因的表达与调控的影响。应用DDRT-PCR技术有单找到调控肿瘤转移的基因，为肿瘤的诊断和治疗提供新的线索。     

A conditioned medium from a human liposarcoma-derived cell line induces p53-dependent apoptosis in several tumor cell lines

A novel cell line, named LSA, has been obtained, stabilized, and characterized from a human liposarcoma. These cells have morphological and biochemical features strongly resembling the adipocytes and were able to grow in the Ham's F12 medium, in presence or absence of FCS. A conditioned medium (LSA-CM) was obtained by growing the LSA cells in the F12 medium in the absence of FCS. LSA-CM had cytostatic and cytotoxic effects (apoptosis and necrosis) associated with down-regulation of c-myc and upregulation of p53 in several human cell lines (breast, lung, glioblastoma, etc. ). The MCF-7 and glioblastoma cells were killed by LSA-CM in 5-6 days, whereas the same cells were killed by LSA-CM co-incubated with low doses of cisplatin in 30 h. LSA-CM peri-tumoral injections for 15 days in Balb-c-fc3H mice affected by mammary tumors, resulted in the rapid disruption of tumors and absence of metastases. In contrast, in the untreated animals the tumor masses were 4 times larger than initial lesions, and numerous metastases were found in the lungs. The toxicity analysis of LSA-CM, performed on three different animal species, showed that LSA-CM is absolutely free of acute, subacute, and subchronic toxicity. The possible use of LSA-CM/cisplatin for cancer treatment is discussed.     

乳腺癌细胞条件培养基对成骨细胞增殖抑制作用的机制探讨

目的:探讨MDA-MB-231、MCF-7乳腺癌细胞条件培养基对hFOB1.19成骨细胞的增殖抑制作用与Notch信号通路的相关性。方法:MTT法检测MDA-MB-231、MCF-7乳腺癌细胞条件培养基对hFOB1.19细胞的增殖抑制效果;Western Blot与qRT-PCR分别检测hFOB1.19细胞的Notch1、Jagged1、Hes1蛋白与mRNA的表达。结果:MDA-MB-231、MCF-7条件培养基抑制hFOB1.19细胞增殖,DAPT（Notch阻断剂）可明显降低MDA-MB-231、MCF-7条件培养基对hFOB1.19细胞增殖的抑制作用。MDA-MB-231、MCF-7条件培养基上调hFOB1.19细胞Notch1、Jagged1、Hes1的mRNA和蛋白的表达,并随着条件培养基浓度的增加,以上指标的表达呈先上升而后下降的趋势。Notch阻断剂DAPT明显抑制两细胞条件培养基对hFOB1.19细胞的Notch1、Jagged1、Hes1 mRNA和蛋白表达的上调作用。结论:MDA-MB-231、MCF-7条件培养基对hFOB1.19细胞增殖抑制作用的机制与Notch信号通路相关。     

Autonomous proliferation of MeWo human melanoma cell lines in serum-free medium: secretion of growth-stimulating activities

The growth properties of a human melanoma cell line (MeWo) and of a variant (MeWo-LC1) endowed with higher metastatic potential in nude mice were compared using hormonally-defined serum-free media. The two cell lines failed to arrest in G0 following serum deprivation, and responded to INS and MSA but not to EGF, PDGF or FGF. Only MeWo-LC1 cells divided persistently in completely serum-free medium, and formed a high percentage of spheroids in agarose supplemented or not with serum or individual growth factors. The conditioned media of serum-free cultures of MeWo and MeWo-LC1 cells exhibited mitogenic activities. These were detected without prior concentration, fractionation or acid treatment. They stimulated DNA replication into sparse monolayers of autologous (MeWo, MeWo-LC1) or homologous (SK-MEL28 melanoma) cells and into NRK-49F normal rat fibroblasts, and acted in synergy with INS in a dose-dependent manner. Over a period of 5 days in culture, MeWo-LC1 cells produced bioactive material at a 2 to 3-fold higher rate than MeWo cells, in both the absence and presence of INS. MeWo-LC1-conditioned medium promoted or enhanced colony formation of MeWo and NRK-49F cells plated in serum-free (+/- INS) agarose. The two cell lines expressed the same amount of NGF and EGF receptors. On the basis of these results we suggest that: (i) MeWo and MeWo-LC1 melanoma cells have obviated allocrine stimulation of the division process characteristic of normal cells by responding to their own mitogens; (ii) some of these mitogens are akin to TGFs; (iii) the less efficient synthesis of autostimulatory factors by MeWo cells may account for their weaker proliferative capacity in vitro, and possibly in vivo.     

Continuous culture and soft agarose cloning of multiple human breast carcinoma cell lines in serum-free medium

We tested the ability of a serum-free medium containing insulin, transferrin, 17 beta-estradiol, dexamethasone, triiodothyronine, prostaglandin F2 alpha, and fibronectin (HBCA medium) to support the continuous growth and passage of five human breast carcinoma cell lines on a collagen matrix. Doubling times of the cell lines (20 to 44 hr) were similar in HBCA and serum-supplemented media. The gross morphology of the cell lines was not altered in the serum-free medium. Insulin, transferrin, and the collagen matrix were the most essential factors required for optimal growth of the cell lines. Estradiol appeared to stimulate the growth of cell lines, both with and without estrogen receptors. HBCA medium supplemented with low concentrations of bovine serum albumin, Fraction V (0.5%, v/v), supported the clonal growth of three cell lines in soft agarose with colony-forming efficiencies superior to that observed with standard serum-supplemented medium. Deleting estradiol from HBCA medium reduced the colony-forming efficiency of the three cell lines. HBCA medium may be useful in studying hormonal regulation and improving the in vitro growth of human breast cancer.     

Novel transmembrane GTPase of non-small cell lung cancer identified by mRNA differential display.

The technique of differential display was used previously to profile the gene expression patterns of non-small cell lung cancer, and several genes differentially expressed were thus identified. In this report, we demonstrate that a DNA fragment of 347-bp length, up-regulated in tumor tissues, showed 100% sequence similarity to human cDNA FLJ20693 for a 370-residue protein. The gene product of cDNA FLJ20693 was postulated to be a shorter isoform of transmembrane GTPase, termed TG370, based upon the results of searching for sequence homology. The nucleotide sequence alignment also indicated that the cDNA FLJ20693 and the cDNA for 741-residue human mitofusin 1 (TG741) possibly resulted from the event of alternative splicing from which a 127-bp region was retained in the latter. Analysis of the genome sequence confirmed the speculation that both cDNAs were mapped to the same chromosomal position composing of 18 exons, of which the 127-bp region of TG741 constituted exon 11. The alternative splicing in all lung cancer cell lines was also observed to occur nearly in all tissue specimens examined. The up-regulated expression of transmembrane GTPase was subsequently found in tumor tissues from at least five of seven non-small cell lung cancer patients. Also, a distinct PCR product was initially detected in cell line H520, and further sequence analysis identified the presence of the 86-bp region mapped to the genome sequence immediately followed by exon 10. To evaluate the retention of 86-bp region, it was found that, besides the predicted 486-bp product, an unexpected 332-bp product was concomitantly observed and identified as the result of exon 8 deletion. The expression and subcellular localization of the full-length TG741 and other shorter isoforms were detected by flow cytometry using three polyclonal antibodies. It was concluded that the full-length TG741 located at plasma membrane with its NH(2)-terminal domain exposed extracellularly and the shorter isoforms retained at cytosol. Finally, the up-regulation of transmembrane GTPase in tumor tissues was further illustrated using immunohistochemical staining.     

Mucin production by colon cancer cells cultured in serum-free medium.

Although many colon cancer cell lines are available for study, few of them exhibit differentiated properties. When cultured in medium containing fetal bovine serum, WiDr cells (WiDr-FBS) show an undifferentiated phenotype: growth as a multilayer of cells adherent to plastic and lack of polarization, brush border, and mucin vacuoles. In contrast, WiDr cells cultured in a chemically-defined serum-free medium containing insulin, transferrin and selenium (WiDr-ITS) grow as clusters of nonadherent cells with abundant desmosomes and tight junctions, microvilli and electron-lucid vacuoles. As WiDr-FBS cells, WiDr-ITS are not polarized. WiDr-ITS cells show a marked enhancement in mucin synthesis as demonstrated by: periodic acid-Schiff and Alcian blue stains, electron microscopy, immunohistochemistry using monoclonal antibodies (MAbs) reactive with mucin-associated epitopes, immune electron microscopy and immunochemical analysis using Western blots. In comparison with WiDr-FBS cells, WiDr-ITS cells showed strong expression of Tn, sialyl-Tn, blood group A and CEA. When mouse MAbs were used, higher levels of the MUCI gene product were detected in WiDr-ITS than in WiDr-FBS cells. The full spectrum of phenotypic changes was observed after I month of culture in ITS medium, and transfer of WiDr-ITS cells to FBS medium was accompanied by a partial phenotypic reversal, suggesting that these phenotypic changes result from an adaptative--rather than selective--process.     

Stimulation of low density lipoprotein receptor activity by conditioned medium from a human cancer cell line

Low density lipoprotein receptor activity in cultured rabbit aortic smooth muscle cells was enhanced by the addition of the conditioned medium derived from the serum-free culture of a human lung cancer cell line. By gel filtration, two peaks of low density lipoprotein receptor-enhancing fractions were obtained, and the molecular weights were 140,000 and greater than 850,000. These fractions also stimulated [3H]thymidine incorporation into DNA. Maximal stimulations of the low density lipoprotein receptor activity and [3H]thymidine incorporation were observed at 16 and 20 h after the addition of the conditioned medium, respectively. The apparent Km (about 10 micrograms/ml) was not changed by the addition of the conditioned medium. De novo cholesterol synthesis was also stimulated by the conditioned medium. It is speculated that certain factors released from the cancer cells affect cholesterol metabolism in normal cells.     

Metastasizing tumors from serum-supplemented and serum-free cell lines from a C57BL mouse lung tumor.

A tissue culture cell line was established from an alveologenic lung carcinoma from a C57BL/lcrf-at mouse. The cells can be maintained in a completely defined serum-free medium. Tumors derived from the tissue culture cells grown in serum-free or serum-supplemented medium give rise to lung metastases. The ultrastructure of the tissue culture cells in both media is similar to that of spontaneous or induced alveologenic mouse lung tumors.     

Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines.

We screened a panel of 103 human lung cancer cell lines for the presence of point mutations at codons 12, 13 or 61 of the human K-, H- and N-ras genes, using restriction fragment length polymorphisms (RFLP), created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. We found ras mutations in 22/61 (36%) non-small-cell lung cancer (NSCLC) cell lines, predominantly in K-ras codon 12. Identical mutations were present in uncultured tumor materials corresponding to 11 cell lines containing mutated ras genes. ras mutations were found not only in adenocarcinoma cell lines (9/32, 28%), but also in cell lines derived from other types of NSCLC (13/29, 45%). In contrast, none of 37 small-cell lung cancer (SCLC) cell lines and five extra-pulmonary small-cell cancer cell lines had ras mutations. ras mutations were not correlated with sex of the patients, tumor extent, prior therapy status or in vitro culture time. G to T or A to T transversions were the most common base substitutions, occurring in codons 12 and 61 respectively. We conclude that ras mutations play a role in the pathogenesis of a subset of NSCLC but are not involved in SCLC.     

肾癌细胞系SN12C 和786-O 无血清培养的干细胞球特性的比较*

目的:本研究通过比较SN12C 和786-O两株肾癌细胞系中肿瘤干细胞特征的差异性,初步探讨筛选肾癌干细胞更有效的方法。方法:以无血清培养法培养SN12C 和786-O细胞,比较其在不同时间形成肿瘤干细胞球的差异;应用流式细胞术分析SN12C 和786-O球体细胞中干细胞标志物的表达情况;利用裸鼠体内成瘤模型检测SN12C 和786-O球体细胞成瘤能力。结果:SN12C 较786-O更易形成肿瘤干细胞球且所需时间更短,即786-O在无血清培养的第7 天开始形成规则的球体,而SN12C 则在第5 天就已形成规则的球体。SN12C 和786-O球体细胞中干细胞指标表达量亦有显著性差异,在SN12C 球体细胞中CD133、CD44、Nanog和Oct3/ 4 比例明显高于786-O球体细胞,差异具有统计学意义（P < 0.05）。 裸鼠体内的成瘤能力实验发现SN12C 明显强于786-O球体细胞。结论:SN12C 肾癌细胞系通过无血清培养方法富集肿瘤干细胞球明显多于786-O,是获得肿瘤干细胞较好的研究对象。      

Metastasis from human breast cancer cell lines

Summary Immunodeficient animals, principally nude mice, when used in appropriately designed studies have been shown to be useful for the experimental analysis of human breast cancer metastasis. As with many other human tumors, the implantation of breast cancer cells into an anatomically appropriate tissue (the mammary fatpad) results in increased tumor take and incidence of metastasis for certain cell lines compared with subcutaneous injection. Testing a number of widely available human breast cancer cell lines identified the MDA-MB-435 cell line as the most metastatic, producing lung and lymph node metastases in a high proportion of nude and severe combined immunodeficient (SCID) mice after injection in the mammary fatpad. Mixing human breast cancer cells with normal fibroblasts or with Matrigel also increases the tumor incidence and growth rates in nude mice. Different routes of injection can be used to assess the ability of human breast cancer cells to form metastatic lesions in the lungs (i.v. injection), the liver (injection in the spleen), the brain (direct or intracarotid artery injection) and the bone marrow and bone (injection into the left ventricle of the heart). These different approaches demonstrate the potential of experimental studies of human breast cancer growth and metastasis using immunodeficient mice; this model is valuable for experiments that test the role of metastasis-associated genes and the efficacy of novel forms of therapy.     

Type-II insulin-like growth-factor receptor in conditioned medium from HT-29 human colon carcinoma cell line.

The HT-29 human colon cancer cell line has previously been shown to secrete high amounts of insulin-like growth factor II (IGF-II). The recent demonstration that soluble IGF-II/mannose 6-phosphate receptor was present in fetal serum prompted us to search for a release of type-II IGF receptor by these human colonic carcinoma cells. Serum-free conditioned medium from the HT-29 cell line was gel filtered on Sephadex G-200. There was significant binding of [125I]IGF-II to the void volume fractions in addition to binding to the 40-kDa IGF-binding protein (IGF-BP) fractions. Competitive binding studies using [125I]IGF-II and the void volume pool showed a pattern typical of the type-II receptor. It exhibited a high affinity for IGF-II (KD = 0.4 nM), but had a low affinity for IGF-I (KD = 6.8 nM), and no detectable affinity for insulin. Additional evidence was provided by affinity cross-linking of [125I]IGF-II to the same high-molecular-weight material which demonstrated a major specific band at 250 kDa after reduction of disulfide bonds. In contrast, the type-I IGF receptor was undetectable. The extracellular type-II IGF receptor was not a significant carrier for IGF-II since virtually all IGF-II secreted by HT-29 cells was associated with IGF-BP. The presence of a soluble IGF-II/mannose 6-phosphate receptor in the culture medium from colonic cancer cells suggests that it may play an important role in tumor pathogenesis.