Two-dimensional polyacrylamide gel analysis on nuclear proteins from human tumor cell lines

Two-dimensional polyacrylamide gel electrophoretic analysis was carried out on HeLa, KB, ALL and GW-39 cell "Chromatin Fraction II" proteins. Of the many proteins found, most were visualized in earlier studies on rodent tumors and normal tissues. Of these the greater density of protein CP and the presence of protein CG' differentiates tumors from the nontumor tissues. In samples of normal and mitogen stimulated human lymphocytes, protein CG' was absent and protein CP was present in small amounts. Two-dimensional patterns of 0.4N H2SO4 soluble nuclear proteins showed elevated amounts of proteins C16-18 in the GS-39 cell patterns. Proteins Hu1, G1, G2, G3 and G5 were detected only in human cell nuclei. The increased density of staining for protein CP and the presence of CG' are potential biological markers for neoplastic cells.     

Organization of F-actin filaments in human glioma cell lines cultured on extracellular matrix proteins

Extracellular matrix (ECM) constituents likely play an important role in cell proliferation and the invasion of malignant human gliomas. We examined the formation of stress fibers and the growth of the human glioblastoma cell lines A172 and T98G cultured on collagen types I, IV, and V laminin (LN), and fibronectin (FN). A172 cells cultured on LN and FN formed complete F-actin filaments after 24 h of culture and grew logarithmically after 48 h. In contrast, T98G cells on LN and FN reorganized only short F-actin filaments after 24 h of culture and grew rapidly after 72 h. However, on the collagen preparations, neither cell line formed definite stress fibers and both showed lower rates of cellular proliferation. Significantly positive correlation was observed between the relative intensity of F-actin filaments and the cell proliferation. The results indicate that the ability of ECM components to modulate the growth and differentiation of malignant glioma cells may be mediated, in part, by the assembly and disassembly of F-actin filaments.     

Comparative analysis of cellular and extracellular proteins secreted by two human tumor cell lines

Two human tumor cell lines, melanoma (TWI) and colon carcinoma (HT-29), replicate continuously in serum-free C-ITS medium [Chee's essential medium (CEM) supplemented with insulin (5 ng/ml), transferrin (5 micrograms/ml), and (5 ng/ml)]. The TWI cells assume normal morphology during growth, whereas the HT-29 cells become rounded and tend to aggregate in C-ITS medium. Under these conditions the two cell types synthesize a number of different cellular proteins, and TWI releases a number of proteins to the medium. These proteins include some factors which are needed for spreading and adhesion of the cells to the growth substrate as shown by the growth of HT-29 cells in serum-free C-ITS medium conditioned by TWI cells. HT-29 cells do not synthesize such factors or perhaps, do so in negligible amounts. The serum-free medium conditioned by the TWI cells may provide an approach for the cultivation of various human tumor cells in vitro.     

Extracellular proteins in breast tumor cytosol

Several proteins have been quantitated in cytosols prepared from benign and malignant tumors of the breast; orosomucoid, albumin, transferrin, complement 4, C-reactive protein alpha 2-macroglobulin and hemoglobin. With the exception of hemoglobin, concentrations of the proteins measured were significantly intercorrelated. Their relative abundance was close to that reported in blood, except for alpha 2-macroglobulin and hemoglobin which were present in lower amounts. From hemoglobin measurements it can be concluded that blood contamination contributed less than 10% to the cytosol proteins measured. When compared to albumin, none of the proteins investigated occurred in concentrations sufficient to indicate local synthesis of a magnitude that would significantly influence tumor environment. From the present data it can be concluded that extracellular proteins constitute about 50% of total cytosol protein. This indicates an exceptional capillary leakage in breast tumors possibly related to abnormal hormonal influences. There were, however, large individual variations and about 10% of the cytosols could be predicted to contain negligible amounts of cell-derived protein. There was a highly significant difference between benign and malignant tumors in their cytosol content of extracellular protein, benign tumors containing nearly twice as much albumin. It is suggested that measurement of an extracellular protein (albumin) should be included in tumor characterization to correct for cellularity and representativity of tumor samples used for steroid receptor determinations and for measurements of other parameters using "cytosol" protein to express specific activity.     

Extracellular matrix destruction by invasive tumor cells

Summary The invasion of normal tissues and penetration of basement membranes by malignant cells is likely to require the active participation of hydrolytic enzymes. The four major groups of connective tissue proteins, glycoproteins, proteoglycans, collagen and elastin, vary in their quantitative distributions between different tissues. With the exception of elastin, they also vary qualitatively within each class, so that there are no typical connective tissue barriers to tumor cell penetration. The matrix constituents are stabilized and organized by a variety of covalent and noncovalent interactions between the connective tissue proteins. These interactions play important roles in matrix integrity and may alter the susceptibilities of the constituents to degradative enzymes. It is likely that the complete degradation of the matrix will require the action of more than one enzyme because of differing susceptibilities to tissue proteinases. Primary and transplantable tumors produce well-characterized enzymes which may participate in invasion. These enzymes may also be involved in connective tissue turnover in other normal and pathological situations. The use of long-term tumor cell cultures has verified that tumor cells themselves are capable of producing these enzymes. However, there are many potential modulating influences opperative in vivo which are absent in culture so that details of actual mechanisms and control of digestion of complex substrates are not well understood. Recent work on the degradation by tumor cells of extracellular matrices previously produced by cultured cells is likely to shed more light on pathways of tissue destruction in vivo. Experiments with tumor cell variants of defined metastatic potentials will also be useful, but invasive and metastatic abilities are not necessarily correlated. It is unlikely that simple correlations can be drawn between the production of one particular degradative enzyme by all tumor cells and the complex biological mechanisms operative during tumor invasion.     

Development of alkylating agent-resistant human tumor cell lines

Summary Survival curves and dose escalation studies of four representative human tumor cell lines exposed to the various alkylating agents are presented. With HN2, at a level of one log of cell kill there was a fivefold range in drug concentration required to achieve this degree of cell kill among the cell lines, from 4.5 M for the SL6 lung adenocarcinoma to 22 M for the SW2 small-cell lung carcinoma. Four logs of SCC-25 squamous carcinoma cells were killed by 100 M CDDP; however, there was only about one log of SL6 cells killed by 500 M CDDP. To kill one log of G3361 melanoma cells required 380 M 4-HC and to kil one log of SCC-25 cells required 24 M, approximately a 16-fold difference. The curves for cell kill by L-PAM appeared to be biphasic, with a break at about 100 M. There was about a threefold range in drug concentration required to achieve one log of cell kill with L-PAM, from 60 M in the SCC-25 cell line to 18 M in the SW2 line. To kill one log of SCC-25 cells required 295 M BCNU and to kill one log of SW2 cell required 120 M, about a 2.5-fold difference. The range of maximally tolerated HN2 concentrations were from 1200M for the SL6 cell line, 48 times the initial concentration, to 300 M for the SCC-25 line, 16 times the initial concentration. The G3361 line tolerated the highest level of CDDP, 1900 M, 48 times the initial concentration. The SCC-25 line, on the other hand, tolerated only 600 M, 30 times the initial concentration. The SL6 cell line maximally tolerated 36 times the initial concentration of 4-HC (1450 M), whereas the SCC-25 cell line tolerated only 18 times the initial concentration (720 M). The G3361 melanoma tolerated 1555 M, 30 times the initial concentration of L-PAM, and the SCC-25 cell line tolerated 700 M, 14 times the initial concentration. The SL6 cell line tolerated the highest concentration of BCNU, 4200 M, 24 times the initial concentration. The SCC-25 cell line tolerated 1450 M, 8 times the initial concentration. In all cases, the SCC-25 cell line was least able to tolerate exposure to increasing concentrations of alkylating agents. The SL6 and G3361 cell lines showed the greatest tolerance for increasing concentrations of alkylating agents. With maximal selection pressure, in terms of intensity and duration, 5-to 15-fold resistance at best could be produced to these alkylating agents. This contrasts with other drugs, indicates that alkylating agents are more like X-rays, and has implications for high-dose clinical treatments. The importance of these findings to the clinical treatment of cancer is discussed.Abbreviations NH2 Nitrogen mustard (mustragen) - CDDP cis-diamminedichloroplatinum (II) (cisplatin) - BCNU N,N-bis(2-chloroethyl)-N-nitrosourea - L-PAM L-phenylalanine mustard (melphalan) - 4-HC 4-hydroperoxycyclophosphamide - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - PBS phosphate-buffered saline This work was supported by NCI grant #5PO1-CA38493 and a grant from the Mathers Foundation     

Nuclear matrix proteins associated with DNA in situ in hormone-dependent and hormone-independent human breast cancer cell lines

The nuclear matrix is a dynamic RNA-protein complex that organizes chromatin and regulates nuclear DNA metabolism. Nuclear matrix proteins informative in the diagnosis of cancer have been identified. Here, the nuclear matrix breast cancer proteins (NMBCs) cross-linked to nuclear DNA in situ with cisplatin in human breast cancer cell lines were analyzed by two-dimensional gel electrophoresis. We identified NMBCs that were differentially associated with nuclear DNA of hormone-dependent and -independent breast cancer cell lines. Three DNA cross-linked NMBCs were found to be exclusive to estrogen receptor-positive, hormone-dependent breast cancer cells, whereas two NMBCs were observed only in estrogen receptor-negative, hormone-independent breast cancer cells. Changes in these NMBCs were observed when hormone-dependent breast cancer cells became hormone independent. Furthermore, we show that the intermediate filament protein vimentin is associated with the nuclear DNA of MDA-MB-231 breast cancer cells, an estrogen receptor-negative, hormone-independent breast cancer cell line with high metastatic potential. These nuclear matrix DNA-binding proteins may play important roles in breast tumorigenesis.     

Extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases

Matrix metalloproteinases (MMPs) are endopeptidases that play pivotal roles in promoting tumor disease progression, including tumor angiogenesis. In many solid tumors, MMP expression could be attributed to tumor stromal cells and is partially regulated by tumor-stroma interactions via tumor cell-associated extracellular matrix metalloproteinase inducer (EMMPRIN). The role of EMMPRIN during tumor angiogenesis and growth was explored by modulating EMMPRIN expression and activity using recombinant DNA engineering and neutralizing antibodies. In human breast cancer cells, changes in EMMPRIN expression influenced vascular endothelial growth factor (VEGF) production at both RNA and protein levels. In coculture of tumor cells and fibroblasts mimicking tumor-stroma interactions, VEGF expression was induced in an EMMPRIN- and MMP-dependent fashion, and was further enhanced by overexpressing EMMPRIN. Conversely, VEGF expression was inhibited by suppressing EMMPRIN expression in tumor cells, by neutralizing EMMPRIN activity, or by inhibiting MMPs. In vivo, EMMPRIN overexpression stimulated tumor angiogenesis and growth; both were significantly inhibited by antisense suppression of EMMPRIN. Expression of both human and mouse VEGF and MMP, derived from tumor and host cells, respectively, was regulated by EMMPRIN. These results suggest a novel tumor angiogenesis mechanism in which tumor-associated EMMPRIN functionally mediates tumor-stroma interactions and directly contributes to tumor angiogenesis and growth by stimulating VEGF and MMP expression.     

Extracellular matrix modulates expression of growth factors and growth-factor receptors in liver-colonizing colon-cancer cell lines

Site-specific metastasis is determined by the extracellular matrix (ECM) of the colonized organ. We have shown that hepatocyte-derived ECM stimulated proliferation of colon-cancer cells via induction of autocrine growth factors and their receptors. The ECM component responsible was heparin proteoglycan. We therefore investigated the effect of exogenously added heparin on colon cell lines of varying liver-colonizing potential. The cells were grown on typical liver matrix components, such as fibronectin and collagens type I and IV. We assessed the effect of these matrix components on clonal growth, proliferation and expression of autocrine growth factors and their receptors. The clonal growth of the KM12 cells was not affected by heparin, while the other cell lines were inhibited by heparin. Cell proliferation in weakly metastatic KM12, but not in strongly metastatic KM12SM, was inhibited by heparin on plastic. Weakly metastatic LS174T, but not strongly metastatic LiM6, was inhibited by heparin on fibronectin. Expression of erb-B2 was also differently modulated by heparin in weakly metastatic vs. highly metastatic cells. In weakly metastatic cells, heparin reduced erb-B2 levels when cells were on plastic and fibronectin, while in strongly metastatic cells, erb-B2 was induced by heparin. In all 4 cell lines, mRNA for cripto was induced by heparin when the cells were grown on fibronectin. In KM12SM cells, amphiregulin was induced by heparin in cells on fibronectin and collagen IV. We show that soluble heparin, similar in its carbohydrate chemistry to liver heparin proteoglycan, regulates the growth of colon-cancer cells. This effect depends on other matrix components found in the liver and is mediated in part by EGF family members. Int. J. Cancer 77:295–301, 1998.© 1998 Wiley-Liss, Inc.     

Nuclear matrix protein composition of human lung carcinoma cell lines

The nuclear matrix is the RNA-protein skeleton within the nucleus that contributes to the structural and functional organization of DNA. Differences in the nuclear matrix protein composition between cancer and normal cells have been reported in various cell lines and tissues, suggesting altered gene expression. This study examined the nuclear matrix protein composition of various human lung cell lines. Using high resolution two-dimensional electrophoresis, at least ten common proteins, as well as specific differences, were identified in each category of lung cell lines. These protein differences may be responsible, at least in part, for the different phenotypes of human lung cancer.     

Anticancer drug activity in human bladder tumor cell lines

A panel of ten human bladder tumor cell lines were tested for drug sensitivity to ten standard or investigational anticancer drugs using a tumor colony assay. The activity of these anticancer agents in vitro was then compared with the clinical activity of these agents in bladder cancer. Drug activity was found in only five of the ten cell lines. In only 9 of 100 drug assays was the inhibition of colony growth lower than 30% of the controls. The activity of the more active anticancer drugs in bladder cancer (i.e., methotrexate and cisplatin) was not predicted using the tumor colony assay. Overall, the low level of activity of most anticancer drugs tested paralleled the clinical experience of drug resistance found in human bladder cancer.     

DNA mismatch binding in human lung tumor cell lines

Defects in mismatch repair (MMR) genes have been involved in several types of sporadic and hereditary cancers. In order to elucidate the role of MMR in human lung carcinogenesis we examined DNA mismatch binding in cell-free extracts of seven lung tumor cell lines and five corresponding lymphoblastoid cell lines from lung cancer patients. Using the technique of bandshift assay we have demonstrated that 2/7 of the tumor cell lines are aberrant in binding to specific DNA mismatches while all lymphoblastoid cell lines were proficient in binding to all tested mismatches. Both extracts were aberrant in binding to G/T mismatch whereas one of the cell lines showed deficiency in binding to the C:A mismatches as well. Immunoblotting analysis showed that all known DNA mismatch repair (MMR) proteins were present in these extracts. The cell line deficient in binding to both G:T and C:A mismatches showed microsatellite instability (MSI) in tumor DNA and higher resistance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This report indicates that DNA mismatch binding deficiencies may be implicated in at least a subgroup of human lung cancer.     

Loss of heterozygosity in cultured human tumor cell lines

One hundred and thirty-seven cultured human tumor cell lines derived from Caucasian patients were surveyed for ten of their polymorphic enzyme phenotypes. The gene frequencies in this cell line population were similar to those of normal Caucasian populations, although consistent differences in phenotype frequencies were detected at each of the loci. All 10 loci showed fewer heterozygous phenotypes and a correspondingly greater number of the common and rare homozygous phenotypes than occur in normal Caucasian populations. On the average, only 85% of the loci expressed in fully differentiated diploid cells were expressed in the neoplastic cell lines adapted to in vitro growth. There was no significant difference in the proportion of loci expressed in cells that had been passaged less than 10 times and in cells passaged more than 50 times. Consequently, it appears that there is a loss of expression in genetic marker loci from at least six different chromosomes. This loss occurs either in the in vivo tumor or in the very early stages of the cultivation of neoplastic cells derived from solid human tumors. Once the cells have become adapted to growth in vitro, the patterns of expression in their polymorphic loci remain stable for many passages.     

Distinctive banded marker chromosomes of human tumor cell lines

Nine human tumor cell lines (five breast carcinomas and four sarcomas) have been studied and each revealed groups of distinctive banded marker chromosomes which can serve to identify them and aid in monitoring cell line specificity. This was possible neither by conventional karyology in terms of numbers and morphology of chromosomes nor by glucose-6-phosphate-dehydrogenase mobility which was type B for all cultures. The significance of the clonal nature of the cell lines is discussed.     

神经元外单胺转运蛋白在人肿瘤细胞系的表达

我们先前发现的一个新的氯乙基亚硝脲的类似物２－氯乙基－３－肌氨酸酰胺－１－亚硝基脲, 是一种通过单胺递质的神经元外转运蛋白（ＥＭＴ）进入细胞内的选择性的细胞毒素。此药已进入１期临床试验。在本研究中,我们检测ＥＭＴ在２３个人类肿瘤细胞系里的表达水平,以证实ＥＭＴ的表达是否与ＳａｒＣＮＵ的细胞毒性有关。方法应用反转录多聚酶链反应（ＲＴ－ＰＣＲ）检测ＥＭＴ在肿瘤细胞系的表达。同时也用蛋白质印迹技术检测Ｄ     

Polymorphic enzyme analysis of cultured human tumor cell lines

Of the total of 137 cultured cell lines from a wide variety of human neoplasms now typed at a maximum of 16 of their polymorphic enzyme loci, the phenotype combinations for 72 are described in this paper. The phenotype frequency product of each line was less than 0.05, and only 148 of the 9,316 (1.6%) pairwise comparisons between lines had phenotypes that did not differ in at least one locus. Thus the probability of any two randomly selected lines having indistinguishable genetic signatures in this sample of 137 lines was 0.016. Fifty-two of the 137 lines (38%) had phenotype combinations distinguishable from each of the other 136 lines. The frequency products of the 85 lines that could not be distinguished from at least one other line were all sufficiently high to preclude a suspicion that contamination had occurred among them. No additional cell lines with phenotype combinations indistinguishable from the combination characteristic of HeLa cells were identified among these 72 cell lines.     

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.