Membranome – in primary tumors and immortalized in-vitro cultured tumor cells. 409 human samples, deriving from ten independent studies, were analyzed. These comprise normal tissues, primary tumors and tumor derived cell lines deriving from eight different tissues: brain, breast, colon, kidney, leukemia, lung, melanoma, and ovary. We demonstrated that the Membranome has greater power than the remainder of the transcriptome when used as input for the automatic classification of tumor samples. This feature is maintained in tumor derived cell lines. In most cases primary tumors show maximal similarity in Membranome expression with cell lines of same tissue origin. Differences in Membranome expression between tumors and cell lines were analyzed also at the pathway level and biological themes were identified that were differentially regulated in the two settings. Moreover, by including normal samples in the analysis, we quantified the degree to which cell lines retain the Membranome up- and down- regulations observed in primary tumors with respect to their normal counterparts. We showed that most of the Membranome up-regulations observed in primary tumors are lost in the in-vitro cultured cells. Conversely, the majority of Membranome genes down-regulated upon tumor transformation maintain lower expression levels also in the cell lines. This study points towards a central role of Membranome genes in the definition of the tumor phenotype. The comparative analysis of primary tumors and cell lines identifies the limits of cell lines as a model for the study of cancer-related processes mediated by the cell surface. Results presented allow for a more rational use of the cell lines as a model of cancer.Despite the wide use of cell lines in cancer research, the extent to which their surface properties correspond to those of primary tumors is poorly characterized. The present study addresses this problem from a transcriptional standpoint, analyzing the expression of membrane protein genes - the  They are composed of different cell types, interacting with each other, with the extracellular matrix (ECM) and the surrounding tissue through a complex network of signaling pathways, all mediated by cell surface proteins. In contrast, cell lines consist of homogeneous clonal populations generally lacking interactions with other cell types and instead interacting with an artificial support. Moreover, cell adaptation to in-vitro microenvironments involves recalibrations of many pathways involving the cell surface, for example by genetic and epigenetic alterations in-vitro cancer model. In fact, despite their wide use, the extent to which the surface properties of cell lines actually correspond to those of the corresponding tumor tissues of origin has been poorly characterized. We addressed this question from a transcriptional standpoint, by performing a meta-analysis of membrane protein gene-expression profiles from ten different studies Membranome the ensemble of all human genes coding for proteins integral to or covalently associated with the plasma membrane. First, we demonstrated that the Membranome expression data have greater power than the rest of the transcriptome when used as input for the automatic classification of tumor samples. This property suggests that most of the gene expression specificity of tumors of different origins resides into the genes codifying for cell surface proteins. This feature is maintained in tumor derived cell lines.Proteins associated with the cell plasma membranes mediate key processes such as molecular transport, cell adhesion, interaction with the extracellular matrix, signal transduction and cell-to-cell signaling. They have long been recognized to play a crucial role in the genesis and development of cancer, by mediating complex interactions between the tumor cells surface and the surrounding cellular environment Membranome expression in tumor and cell lines, using three different analytical approaches. The first one is based on the direct comparison of the Membranome expression values in primary tumors and cell lines, grouped by tissue of origin. The second focuses on pathways involving the Membranome and identifies those pathways differentially regulated in tumors and cognate cell lines. The third analysis quantifies the extent to which cell lines reproduce Membranome up- or down-regulation observed in primary tumors with respect to their normal tissue counterparts.Then we run a systematic comparison between the Gene expression data on tumor cell lines, primary tumors and normal tissues were integrated from ten independent studies, all based on the Affymetrix HG-U95Av2 array platform see  Table 1Table 1. Membranome the ensemble of all human genes coding for proteins integral or associated to the plasma membrane. All human genes reported in the NCBI Gene database were surveyed using a combined analysis of the available Gene Ontology annotations We defined as the The resulting human Membranome comprises 4,329 genes (about 17% of human genes) encoding for plasma membrane proteins, neglecting the additional complexity introduced by alternative splicing events or post-translational modifications. Of these genes, 1,701 are represented on the Affymetrix HG-U95A2 microarray platform, common to all data sets considered in the present study Membranome . The resulting groups of up- and down-regulated genes were analyzed separately by using a gene set enrichment approach , those Membranome genes up- or down-regulated in either primary tumor or cell line samples, as compared to normal samples with the same tissue origin. The analysis was restricted to those tissues for which cell lines, primary tumors and normal samples were available: brain, lung, colon, ovary and kidney. For each tissue, MTDG were identified in primary tumors and cell lines, using SAM (FDR<0.01) Membranome genes up-regulated in primary tumors therefore lose their de-regulation in cell lines, i.e. following immortalization and in the context of in-vitro growth conditions. Conversely, the majority of Membranome genes down-regulated upon tumor transformation maintain lower expression levels also in the cell lines. Noteworthy, tumors of different types always show the most significant overlap with the cell lines of same tissue origin . In particular, brain, leukemia, colon and ovary were the tumors with the most pronounced correspondence, suggesting their membrane composition being mostly preserved in the cognate cell lines. The lack of maximal correlation between breast and lung cell lines with their respective tumors can probably be ascribed to their heterogeneous gene expression patterns, already pointed out by previous clustering analysis, in this case performed at the whole-genome level In order to quantify the degree to which cell lines are representative of their tumor of origin, with respect to To understand which cell surface biological themes are differentially regulated between primary tumors and cell lines, a gene set enrichment analysis against a large sets of databases and cancer data extracted from the literature was performed.This type of analysis is significantly more interpretable than a standard gene-level approach as it allows for a global overview of the cell surface processes differentiating the two systems, potentially hidden from a gene-centric perspective.With gene set enrichment analysis lists of up- and down-regulated genes are translated into a more interpretable view of the biological pathways, which – as wholes - are differentially regulated in primary tumors and cell lines. Another important advantage lays in the fact that the perturbation of each pathway is quantified by an “aggregated” value, inferred from the statistical integration of dozens of genes taking part to the same pathway. This makes this analysis intrinsically more resistant to the presence of false positive/negative genes, which could potentially affect a “gene-centric” analysis, based on the evaluation of individual data points.Among the dominant themes up-regulated in primary tumors emerge those related to the immune response, pathways known to be up-regulated in all tumors, regardless of their tissue of origin in-vitro in defined cell-culture media Other themes generally up-regulated in primary tumors are those related to “cell adhesion”, “extracellular matrix”, “signal transduction” and “cell-cell communication” . The up-The “breast cancer mutated kinases” gene set – composed of kinases found to be genetically mutated in primary breast tumors Membranome tumor deregulated genes (MTDG) analysis discussed below, showing that a significant portion of the Membranome loses its up-regulated state passing from in-vivo to in-vitro conditions. Noticeably, the gene sets we identified as up-regulated in cell lines, have limited conservation across cell lines of different origin , “drug binding” (kidney) and “response to drugs” (ovary). The differential regulation of these pathways can possibly underpin the different anticancer drug sensitivities observed in-vitro and in-vivoOnly a limited number of pathways and gene sets were found to up-regulated in cell lines vs primary tumors. This is consistent with the results of the t origin . These iMembranome genes deregulated in primary tumor samples as compared to their normal tissue counterparts retain their altered state also in the cell lines. This information is of key importance when using the cell lines as an in-vitro model for surface cancer targets. Examples are the screening of anticancer therapeutics targeting cell surface receptors in-vitro cultured cells  can exist, their effects are expected to reciprocally compensate – and therefore to be strongly mitigated – in the context of a “global” scale analysis, one involving thousands of genes.As a further development of this study, the in-vitro tumor models  could add further interest to our observations.Additional comments need to be made regarding the samples we considered in the analysis. Our study has been constrained by the availability of transcriptional data sets publically available on a coherent microarray platform . As a result, we created a meta-dataset, all based on the Affymetrix HG-U95Av2 platform, which to our knowledge was the platform covering the broadest spectrum of tumor samples. It encompasses 10 independent studies, covering a total of 409 human samples deriving from 8 different tissues. Additional tumors  and Membranome genes in characterizing the tumor phenotype. The comparative analysis of primary tumors and corresponding cell lines reemphasizes the caution that should applied when using these model systems in the study of the cancer. The presented results contribute to a more informed use of cell lines and interpretation of results with regards to specific aspects of tumor biology involving the cell surface.Using transcriptional data from a large set of primary tumors, normal tissue and cell lines of different origin we have demonstrated a central role of Expression data for NCI60 cell lines were made publicly available through the Developmental Therapeutics Program of NCI/NIH. The NCI60 dataset includes data from 59 cell line. Cell culture growth conditions are described in “[…]RNA samples from two cell lines (MCF7 breast and K562 leukaemia) were collected on three different occasions (at different passage numbers), then labelled, hybridized and scanned independently. These replicates  clustered side by side, with approximately the same degree of similarity as shown by the MDA-MB435/MDA-N pair […]”No specific information is reported in the existing literature regarding the cell passage number at which cell lines were processed for microarray analysis. However, interesting information regarding this point can be found in the work of Ross and collaborators: These data, although limited to two cell lines only, point towards a relative transcriptional stability of these cell line samples across different passage numbers.The set of primary tumors included 21 classic glioblastoma and anaplastic oligodendroglioma http://dtp.nci.nih.gov/mtargets/download.html; http://www.broadinstitute.org/cgi-bin/cancer/datasets.cgi; http://www.mskcc.org/genomic/ccsmsp/; http://public.gnf.org/cancer/epican/.Normal tissue samples data were available for five different tissue types: brain The meta-dataset deriving from the integration of the individual data sets described above represent to our knowledge the largest study publicly available based on the Affymetrix HG-U95Av2 array. More detailed information on samples included in this study is provided in All datasets were processed using the MAS5 algorithm implemented in R Membranome, here defined as the ensemble of all human genes coding for proteins integral to or covalently associated with the plasma membrane,A semi-automated procedure was applied to identify the human All human genes reported in the NCBI CCDS database (NCBI Build 37.1) Membranome and Not-Membranome genes the PAM method  To compute the ‘discriminative power’ of Membranome and Not-Membranome genes, randomly chosen. For each list size  the analysis was run 1.000 times and the results of misclassification were averaged.PAM is based on nearest shrunken centroids classification and builds a classifier by identifying those genes that best characterize each group of samples. The size of the gene list used as the classifier, and the corresponding misclassification rate, depend on the shrinkage parameter Δ provided as input. PAM was run independently on primary tumors and cell lines using gene lists of decreasing sizes. Parallel analyses were performed using equally sized lists of Cell lines and primary tumors were grouped according to their origin: brain, leukemia, lung, melanoma, breast, colon, ovary and kidney. All possible pairs of tumor and cell line samples were compared using the Pearson's correlation coefficient as the metric of similarity. Pearson's correlation values were computed between all tumor samples and all cell lines of two given groups . The resulting distributions of correlation values were represented as a box plots in Membranome genes up- and down-regulated in the primary tumors as compared to the corresponding cell line, with the same tumor tissue origin (Membranome tumor deregulated genes) defined as Membranome genes up- or down-regulated in primary tumors or cell lines as compared to the normal samples of same tissue origin. Gene up- and down-regulations were in all cases assessed using the significance analysis of microarrays (SAM) For each of the eight tissues in analysis, we computed the list of e origin . For thep-value obtained.The significance of the overlap between pairs of the lists was computed by using the Fisher's exact test and are reported in p-values were computed according to the Fisher's exact test. Gene sets were obtained from publicly publically available sources Click here for additional data file.Table S2Detailed description of the 409 samples analyzed in this study.(0.10 MB XLS)Click here for additional data file.Table S3Membranome genes represented on the HG-U95Av2 array. For each probe the table shows (from left to right): Affymetrix ID; Entrez GeneID; whether the probe is differentially expressed  in tumor-to-cell line, tumor-to-normal and cell line-to-normal comparisons; whether the probe is consistently differentially expressed in tumor-to-normal and tumor-to cell line comparisons. CL: cell line, T: primary tumor, N: normal sample.Results of differential expression analysis . The file contains the complete list of 2,247 Affymetrix probes mapping to 1,701 (1.62 MB XLS)Click here for additional data file.Table S4Results of gene set enrichment analysis. The file includes the 639 gene sets significantly enriched (p<0.05) in at least one of the comparisons tumor-to-cell line, tumor-to-normal and cell line-to-normal. For each gene set the table shows (from left to right): gene set ID; source of the gene set; gene set name; p-value enrichment (Fisher's exact test) for the genes differentially expressed  in each comparison; the number of overlapping genes; the Entrez Gene IDs of the overlapping genes. CL: cell line, T: primary tumor, N: normal sample.(1.96 MB XLS)Click here for additional data file.


Three weeks after DEN treatment, Fas-mediated liver cell apoptosis induced by anti-Fas antibody resulted in a biphasic effect on induction of liver cell tumours, depending on dosage and time of antibody administration. Single or multiple treatment with high dose anti-Fas antibody , induced gross liver cell damage and decreased the incidence of liver cell tumours in DEN-treated mice. In contrast, five treatments with low dose anti-Fas antibody , induced dispersed localized liver cell damage and promoted the number of large-sized liver cell adenomas and hepatocellular carcinomas. These findings suggest that high dose anti-Fas antibody has a marked effect on the clearance of DEN-initiated liver cells, whereas repeated administration of low dose anti-Fas antibody promotes hepatocarcinogenesis. It is concluded that Fas-mediated liver cell apoptosis has a biphasic effect on hepatocarcinogenesis. © 2000 Cancer Research CampaignThe present study was designed to investigate the effect of Fas-mediated liver cell apoptosis, induced by a hamster monoclonal antibody against mouse Fas antigen, on diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. DEN (10 μg g


We have shown that CAL 29 cells were tumorigenic in mice and expressed characteristic features of both cell scattering and transition from epithelial to mesenchymal phenotype (EMT) after triggering by the EGF receptor ligands, TGFα and EGF. At the opposite, the CAL 185 cells were not tumorigenic in mice and neither scattered nor expressed vimentin intermediary filaments in the presence of growth factors. We further demonstrated that CAL 29 cell scattering was reversible after growth factor removal and that both scattering and EMT were markedly impaired after treatment with MEK, Src and PI3-kinase inhibitors suggesting that these kinases might be important components of the cellular responses to EGF and TGF-α leading to scattering and EMT. These agents could help to understand the intracellular pathways involved in invasiveness and to find new targets for limiting metastasis. In conclusion, these two new cell lines could be good models to dissect the molecular mechanisms involved in invasion and metastasis development in human bladder cancer. © 2001 Cancer Research Campaign


A cell line, TCCSUP, derived from an undifferentiated, Grade IV transitional cell carcinoma is described. The karyotype showed an abnormal distribution of chromosomes, with no obvious modal number. Distinct marker chromosomes were observed in both early and late in vitro passages. These cells have been subcultured over 50 times during a 20-month period. TCCSUP differs in certain morphological and immunological features from other cell lines from transitional cell carcinomas.


Drug resistance is generally considered to be a major impediment to successful cancer chemotherapy, yet it is generally not possible to predict the degree or timing of the emergence of tumour resistance in most chemotherapy protocols. Recent developments with the single-cell gel electrophoresis or 'comet' assay for DNA damage at the single-cell level suggest that this technique might provide a method for identifying and potentially monitoring tumour cell responsiveness to many anti-cancer agents in situ. In principle, this assay could be applied to any accessible tumour being treated with chemotherapeutic agents that cause overt DNA damage. We have investigated that supposition using several rodent and human tumour cell lines exhibiting a spectrum of resistance to the DNA strand-breaking drug, etoposide. By assessing cells grown as monolayers, spheroids and xenografted tumours in immunodeficient mice, we found that the comet assay can provide not only an index of sensitivity to etoposide, but, additionally, can demonstrate the efficacy (or lack thereof) of multidrug resistance (MDR) reversing agents for cells in vitro, and tumours in vivo.


Well-characterized cell lines established from primitive neuroectodermal tumours (PNETs) were examined by proton nuclear magnetic resonance (1H-NMR) spectroscopy and chromatographic analysis of perchloric acid extracts, following amplification in cell culture. A characteristic 1H-NMR spectroscopic metabolite pattern was found for medulloblastoma cell lines, which clearly discriminates these cells from PNETs of other locations in the central nervous system (CNS), on the basis of their N-acetyl aspartate (NAA) and aspartate expression. Medulloblastoma cell lines were heterogeneous in respect of their metabolite expression, possibly owing to the heterogeneity in their differentiation along lineages of the CNS. All PNET spectra displayed similar features, including decreased NAA and creatine peaks and increased signals from choline compounds (Cho) compared with normal cerebellum. The expression of NAA by the medulloblastoma lines was in the opposite order to the extent of neuronal differentiation, which may indicate their origin from a progenitor cell with the phenotype of an oligodendrocyte-type-2 astrocyte cell.


Lines of fibroblasts have been established from normal individuals and from patients diagnosed as suffering from one of the mucopolysaccharidoses or mucopolysaccharide-storage diseases. Transformation of these lines with SV40 virus has been found to reduce their capacity to secrete sulphated mucopolysaccharides into the growth medium. No differences were detected between the individual cell types in their secretory capacity, either before or after viral transformation. A direct relationship was found to exist between the rate of acid mucopolysaccharide production and cell-doubling time. The level of sulphated mucopolysaccharide detected within the cell was also reduced for all cell types after transformation by SV40. Transformed fibroblasts from mucopolysaccharidosis patients, however, showed a relatively greater reduction in storage capacity than those derived from normal individuals.


Sir,Cancer Lett245(1–2): 350–352) and KB cells are not oral carcinoma but were cross-contaminated with HeLa many years ago (European Collection of Cell Cultures (2002). HeLa contamination – an old problem that has not gone away! ECACC Newsletter, March 2002; Stacey GN, Masters JRM, Hay RJ, Drexler HG, MacLeod RAF, Freshney RI (2000). Cell contamination leads to inaccurate data: we must take action now. Nature403: 456). Clearly, these authors (like many others!) are unaware of this.Although the role of this compound in multidrug resistance may well be important and the observations valid, the two cell lines that they have used are not as described in their article. MCF-7-ADR is not a drug-resistant variant of the MCF-7 breast carcinoma cell line but is, in fact OVCAR-8 an ovarian carcinoma cell line . www.ecacc.org.uk) or ATCC (www.atcc.org) where information can be obtained as to the authenticity of cell lines in common use. Authors should also be asked to provide hard evidence of the authenticity of the cell lines that they use as a condition for accepting a manuscript for review.I suggest that future instructions to reviewers contain advice on the scrutiny of cell lines used in such publications to ensure that similar errors do not occur in the future. It is a simple matter for a reviewer to access the website of a recognised cell bank, such as ECACC (


The ease of access allows direct monitoring of tumour growth by biomicroscopy and the possibility to screen many samples in an inexpensive way. The CAM model provides a powerful tool to study effects of molecules, which interfere with physiological angiogenesis, or experimental tumours derived from cancer cell lines. We therefore screened eight osteosarcoma cell lines for their ability to form vascularized tumours on the CAM.The chick chorio-allantoic membrane (CAM) assay is a commonly used method for studying angiogenic or anti-angiogenic activities in vivo biomicroscopy at different time points and tumours were fixed in paraformaldehyde seven days after cell grafting. The tissue was observed, photographed and selected cases were further analyzed using standard histology.We implanted 3-5 million cells of human osteosarcoma lines  on the CAM at day 10 of embryonic development. Tumour growth was monitored by From the eight cell lines the MNNG-HOS, U2OS and SAOS were able to form solid tumours when grafted on the CAM. The MNNG-HOS tumours showed the most reliable and consistent growth and were able to penetrate the chorionic epithelium, grow in the CAM stroma and induce a strong angiogenic response.Our results show that the CAM assay is a useful tool for studying osteosarcoma growth. The model provides an excellent alternative to current rodent models and could serve as a preclinical screening assay for anticancer molecules. It might increase the speed and efficacy of the development of new drugs for the treatment of osteosarcoma. It is expected that anti-angiogenic agents will have clinical benefits for patients with osteosarcoma, probably by reducing metastasis[Osteosarcoma is the most common primary malignant bone tumour that usually affects children and adolescents. After the introduction of cytotoxic polychemotherapy to the treatment of osteosarcoma tremendous advances were achieved, increasing the survival rate from under 20% to over 60%. But oveetastasis. Thus esetastasis.in vivo assay to serve these purposes is the chick chorio-allantoic membrane (CAM) assay. The CAM is formed on embryonic day 4 - 5 by fusion of the chorion with the allantoic vesicle[An c vesicle. The resc vesicle. On the c vesicle. The CAMc vesicle.Since its use to demonstrate normal embryonic blood vessel development more than 100 years ago, the CAM served as a host for transplantations of embryonic tissue as well as for bacteria and viruses. During the last 20 years the CAM is extensively used in angiogenesis research but a reliable bone tumour model has not yet been reported.Here we present the results of the screening of the human osteosarcoma cell lines HOS, MG63, MNNG-HOS, OST, SAOS, SJSA1, U2OS and ZK58 for their use in the CAM assay and provide evidence that the MNNG-HOS cell line reproducibly simulates key features of human osteosarcoma growth.2 incubator. Prior implantation, cell suspensions were prepared by detaching cells with trypsin/EDTA . Cells were centrifuged at 1200 rpm for 5 min, washed twice in culture medium and resuspended in medium at a final concentration of 3 to 5 million cells per 20 μl[Eight osteosarcoma cell lines  were used in this study. All cell lines were cultured in RPMI 1640  supplemented with 10% Fetal Bovine Serum  and 1% penicilline/streptomycine  at 37°C in a humidified 5% COin ovo with a digital camera (Olympus E330) attached to a stereomicroscope. CAMs were checked for tumour growth. Positive tumour development ("+" tumours) was reported when tumour angiogenesis was visible and when tumours were bigger than 2 mm. All other tumours were classified as ("-"). The estimated tumour volume was calculated . Frequently several independent tumour nodules developed. However for quantification only the biggest nodule was taken. Only CAMs still alive at day 17 were included in this analysis.Fertilized white leghorn chicken eggs  were incubated at a humidity of 70% and 37°C. On day 3 of incubation, a round window was cut into the shell after removal of 2 - 3 ml of albumen allowing detachment of the embryo from the eggshell. Normal development was verified and embryos with malformations or dead embryos were excluded. The window was sealed with tape and the eggs were returned to the incubator. On day 10 of development, small plastic rings made out of Thermanox™ discs were placed on the CAM and 25 μl of medium containing different cell lines were deposited into the rings after gentle laceration of the CAM surface. The number of CAMs implanted for each cell line is indicated in Table To determine if osteosarcoma growth on the CAM was associated with increased embryonic death, a 2 × 2 contingency table test (Fisher's exact test) was used for each cell line compared to control CAMs.On embryonic day 17, a few ml of 4% paraformaldehyde were put onto the CAM after photo documentation. After 20 min the window was carefully enlarged using scissors without destroying the CAM. Areas containing the tumour were cut out and transferred into culture discs. All tumours were evaluated and relevant samples were embedded in paraffin and processed for sectioning. Embedded tumours were cut into 10 μm sections, stained with hematoxylin-eosin and further analysed by standard light microscopy (Leica DM2500 with Leica EC3 camera).From the eight cell lines tested only the MNNG-HOS, U2OS and SAOS line consistently  formed vascularized tumours of more than 2 mm in size  its rarin vivo. The expin vivo,17 potenAnother group inoculated single cell suspensions of LNCaP, PC-3 and Tsu-Pr1 human prostatic cancer cell lines on the chorio-allantoic membrane. They demonstrated a reliable tumour growth allowing the evaluation of proliferation and apoptosis induction after intravascular or topic application of anticancer drugs.There are very few publications about the use of the CAM assay in human bone or soft tissue sarcoma,20. The in vivo[The MNNG-HOS cells might be most suitable for further inhibition studies in our model, since this line reliably simulated key features of human osteosarcoma growth such as angiogenesis, necrosis and haemorrhage. Osteoid formation however, did not occur in this model. This might be due to the lack of mature bone in the developing chick embryo or due to the short time span of only 7 days of tumour growth. The experimental osteosarcoma described here are hybrid tumours: they develop out of human cells and secondarily get invaded by newly formed chicken capillaries and stromal cells, therefore allowing further studies aiming at the molecular characterization of osteosarcoma angiogenesis. It might for example be possible to separately study gene expression in the newly formed vessels using Affymetrix chicken GeneChips, in parallel with human GeneChips, which will measure gene expression in the tumour cells. This could lead to the development of new antitumour drugs. The CAM model also is an attractive tool to follow the fate and visualize microscopically the behaviour of the grafted cells. In vivoin vivo. This miin vivo such as in vivo.A limit of this model is the short time span of only 7 days for tumour growth which might not have been long enough for the other cell lines to grow to bigger tumours. However, especially this time frame might be of importance for the development of metastases or tumour recurrence. These first steps are heavily dependent on invasion and angiogenesis, which is a promising target for new drugs against solid tumours, including osteosarcoma,33.Our model could serve as a preclinical screening assay for anticancer molecules against osteosarcoma and therefore increase the speed and efficacy of the development of new drugs. More, the model is an excellent alternative to rodent models and fits perfectly into current recommendations of an ethically appreciable use of live animals in cancer research.Last, the experimental tumour model on the CAM might be adapted for other malignant bone and soft-tissue tumours such as chondrosarcoma, Ewing's-sarcoma, malignant fibrous histiocytosis, and benign but locally aggressive lesions such as giant cell tumour of bone.The authors declare that they have no competing interests.MB performed all CAM experiments and drafted the manuscript. AN did all culturing of the cells and helped in drafting of the manuscript. CK and HB did the histology and analysis of the tumours. KA helped with analysis and interpretation of data. MH critically revised the manuscript and together with MB is responsible for the study design. CG and GG participated in the study design and coordination and helped in interpretation of the data. All authors read and approved the final manuscript.


However, the mode of action is uncertain. Our previous studies showed no direct effect of GnRHa on endometrial cancer cell growth in vitro. We have now examined the effect of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on endometrial cancer cell growth. The aim was to determine whether suppression of pituitary LH and FSH by GnRHa could explain the tumour regression seen in up to 44% of patients treated with this drug. We show that recombinant human LH and FSH (rhLH and rhFSH) produce a concentration dependent stimulation of the endometrial cancer cell line HEC-1A, in serum-free medium (maximum increase of 62 and 50% respectively relative to untreated controls). This increase is equivalent to that obtained by addition of 10% newborn calf serum. Growth of the Ishikawa cell line in culture increases in the presence of rhLH (maximum increase of 67%) but not with rhFSH. Using RT-PCR, we show that the Ishikawa cell line intermittently expresses receptor mRNA of LH but not of FSH; there is no expression of either mRNA by HEC-1A. Classically, both LH and FSH act via cAMP linked membrane receptors. However, neither rhLH nor rhFSH elicit cAMP production in either of our endometrial cancer cell lines. Thus, although a growth response to LH and FSH can be shown, and some cells express the LH receptor, stimulation appears to be via a pathway separate from that of the classical gonadotrophin receptor. © 2000 Cancer Research Campaign


The histological structure of 58 primary tumours derived from spontaneously transformed tissue culture cell lines from embryonic (14-16 days), young (3-20 days) and old (28-34 months) C3H and C57 mice is described. Although the cell lines were derived from a number of different organs the tumours were similar in morphology. The pattern was mixed, with “fibrosarcomatous”, “myxoid”, “epithelioid” and giant cell areas. The tumours resemble some types of haemangiopericytoma.


Three cell lines derived from adult rat liver have been used to study changes in levels of gamma-glutamyl transferase (GGT), a possible marker for premalignant transformation in liver in vivo. None of the cell lines was able to metabolize aflatoxin B1 (AFB1) and treatment with AFB1 alone did not influence GGT activity. However, treatment with microsomally activated AFB1 increased the level of activity in a cell line (BL8L) derived from normal liver with very low levels of GGT, by as much as 10-fold, and 5-fold in a cell line (ARL) also isolated from normal rat liver, but which had subsequently undergone spontaneous transformation. Microsomes from rats pretreated with phenobarbitone were compared with those from 3-methylcholanthrene-treated animals. AFB1 activated by the former produced larger increases in GGT activity, but in no case did the enzyme levels approach that in a cell line (JBI) derived from a hepatoma in the liver of an AFB1-fed rat. Treatment of JBI cells with microsomally activated AFB1 produced no further increase in activity. Histochemical staining indicated an uneven distribution of enzyme in all cell populations, both before and after treatment. This cell-culture system is useful for further studies on the role of GGT in carcinogenesis.


The SCC-25 cell line is a well-established line derived from a human squamous carcinoma of the head and neck. The capacity of this cell line for recovery from potentially lethal damage following X-ray treatment has been documented. The survival curve of stationary phase SCC-25 cells exposed to various concentrations of bleomycin is biphasic with an initial sensitive phase and a less sensitive second phase as is common for many cell lines. Stationary phase SCC-25 cells were exposed to 100 mU ml-1 of bleomycin for 1 h. The drug was removed and the cells were allowed various periods to recover from potentially lethal damage. After 24 h, the SCC-25 cells showed a recovery ratio (R/R0) of 7.0 which corresponded to an immediate survival at a drug level of 27 mU ml-1, a dose 3.7-fold less than the exposure concentration of 100 mU ml-1. Over the course of the first 4 h following bleomycin exposure, 0.5 microM CDDP was a very effective inhibitor of potentially lethal damage repair, giving a R/R0 of 1.1 or nearly complete inhibition of recovery. Between 2 and 4 h the R/R0 was 1.6-1.8 with CDDP and 4.1-5.3 without CDDP indicating appreciable inhibition of recovery. Plant (10 microM) and Plato (10 microM) produced potentially lethal damage recovery inhibition patterns very similar to that of CDDP. After 1 h the recovery ratios in the presence of Plant and Plato were 1.1-1.3. Between 2 and 4 h, Plato and Plant gave recovery ratios of 1.8-2.3 and 1.6-1.9, respectively. NIPt and Pt(terpy) were examined at both 10 microM and 25 microM for their ability to inhibit potentially lethal damage recovery after bleomycin treatment. After 1 h, NIPt gave a recovery ratio of 1.3-1.4, and after 2-4 h the recovery ratio was 1.7-2.6. Pt(terpy) gave recovery ratios of 1.3-1.6 after 1 h and 1.5-1.8 after 24 h.


Early clinical results are encouraging, but the variable response may partly depend on local pharmacokinetics. This study evaluated the in vitro cytotoxic effects of 131I-Lipiodol on human hepatocellular carcinoma (Hep-G2), human colorectal metastatic cancer (SW620), human colorectal hepatic cancer (LoVo) and human umbilical vein endothelial cells (HUVEC) cell lines. The cell cultures were exposed to 131I-Lipiodol for 48 h, following which cell counts and viability were assessed by haemocytometer, S-Rhodamine uptake and radioactivity assay. The effect of exposure to control Lipiodol, 131I-Lipiodol and 131I alone was evaluated. 131I-Lipiodol was cytotoxic against all the cancer cell lines but not against the non-malignant (HUVEC) cell line. The cytotoxicity effects were very similar in all the cancer cell lines. There were no cytotoxic effects following exposure to plain 131I in any of the cell lines . A similar trend was seen with radioactivity counts using a gamma counter. The cytotoxic effect of 131I-Lipiodol had a graded effect with an increase in cytotoxicity following the increase in the radioactive dose. This study showed that there was a marked cytotoxic effect by 131I-Lipiodol on all the cancer cell lines. There was no difference between the controls and the 131Iodine. This suggests that effective 131I-Lipiodol targeted therapy is dependent on the uptake and retention of Lipiodol by malignant cells. © 1999 Cancer Research CampaignIntra-arterial Lipiodol has been used to deliver targeted therapies to primary, and some metastatic, liver cancers. Targeted radiotherapy has been used by substituting the iodine in Lipiodol with


The effect of taxol (TX) and cisplatin (CDDP), singly or in association, was assessed on two human ovarian cancer cell lines, one sensitive (A2780) and one resistant (A2780 cp8) to CDDP. Cell lines showed a similar sensitivity to TX, whereas different cytotoxicity results were obtained in the two cell lines as a function of TX and CDDP sequence. Specifically, TX followed by CDDP induced simply additive effects in both cell lines, whereas the opposite sequence produced antagonistic effects in A2780 cells and synergistic effects in A2780 cp8 cells. TX, with or without CDDP, induced oligonucleosomal DNA fragmentation typical of the apoptotic process, but the biochemical mechanisms undergoing apoptosis were different in the two cell lines. In fact, in A2780 cells, TX (with or without CDDP) treatment markedly increased p53 as well as p21waf1 protein expression. In A2780 cp8 cells, drug treatment enhanced p53 levels, whereas the expression of p21waf1 was always undetectable at mRNA and protein levels. In the latter cell line, a premature activation of p34cdc2 kinase was observed in correspondence with the drug-induced increase in the S-phase cell fraction. Such an activation was not ascribable to an increase in the overall expression of p34cdc2 or cyclin B1 proteins, but to a dephosphorylation of p34cdc2 kinase. Overall, our results indicate that TX-induced apoptosis in human ovarian cancer cells may be sustained by different events at the cell cycle-control level.


Multi-drug resistance (MDR) of cancer cells severely limits therapeutic outcomes. A proposed mechanism for MDR involves the efflux of anti-cancer drugs from cancer cells, primarily mediated by ATP-binding cassette (ABC) membrane transporters including P-glycoprotein. This article reviews the recent progress of using active ingredients, extracts and formulae from Chinese medicine (CM) in circumventing ABC transporters-mediated MDR. Among the ABC transporters, Pgp is the most extensively studied for its role in MDR reversal effects. While other MDR reversal mechanisms remain unclear, Pgp inhibition is a criterion for further mechanistic study. More mechanistic studies are needed to fully establish the pharmacological effects of potential MDR reversing agents. Multi-drug resistance (MDR) in cancer chemotherapy refers to the ability of cancer cells to survive from treatment of a wide range of drugs . In addiP-glycoprotein (Pgp) , which iABCC1 gene [The second major MDR transporter, multidrug-resistance-associated protein (MRP), was first discovered in a doxorubicin-selected lung cancer cell line . A membeCC1 gene . PhysiolCC1 gene ,16. MRP1CC1 gene . OverexpCC1 gene .Recently, ABCG2 was identified in cancer cell lines selected with mitoxantrone that do not express Pgp and MRP1. As ABCG2 was simultaneously discovered by several research groups, it was also named BCRP, ABCP and MXR -21. ABCGHow to tackle the MDR cells in chemotherapy is a pressing issue in cancer treatments. Verapamil was the first known Pgp inhibitor to increase the intracellular concentration of anticancer agents in MDR cells by binding to Pgp and inhibiting the Pgp-mediated efflux . It was This article reviews some of the recent findings on the circumvention of ABC transporters-mediated MDR by various ingredients and extracts of CM and their formulae based on whether the MDR reversal involved Pgp alteration.Stephania tetrandra (Fenfangji) [in vitro and modulated Pgp-mediated drug efflux [in vitro demonstrated synergistic anticancer effects [Tetrandrine, a calcium channel blocker, is a bisbenzylisoquinoline alkaloid isolated from the root of nfangji) . Tetrandg efflux -33. A co effects . Tetrand effects . In mice effects . A synth effects . In a re effects .Sophora alopecuroides (Kudouzi). In resistant K562/DOX cell line, matrine  increased the intracellular accumulation of doxorubicin and induced its apoptotic effects [Matrine is a quinolizidine alkaloid from  effects . Matrine effects . It was  effects .Ligusticum chuangxiong (Chuanxiong) and a calcium channel blocker [Tetramethylpyrazine is an active alkaloid from  blocker . In Pgp- blocker . Tetrame blocker . When us blocker . Tetrame blocker . However blocker .Fritillaria thunbergii (Zhebeimu) and other Fritillaria species [Peimine  is a cevanine type isosteroidal alkaloid from the bulbs of  species ,48. In r species .Mahonia fortunei (Shidagonglao). In K562/DOX cell line, berbamine inhibited cell growth by inducing apoptosis in a dose-dependent manner and reduced Pgp expression thereby increasing the intracellular concentration of rhodamine-123 and doxorubicin [Berbamine is a calcium channel blocker from orubicin ,51. In Morubicin .Panax ginseng (Renshen). Ginsenosides are mainly triterpenoid dammarane derivatives. Several ginsenosides, namely Rg1, Rg3, Re, Rc and Rd inhibited drug efflux [1, Rb2, Rc, Rd, Re and Rg1 reversed MDR whereas individual ginsenosides did not produce any effect [1 reversed MDR of harringtonolide and vincristine in K562/HHT and HL60/VCR cell lines respectively [Ginsenosides are the major active components from g efflux . A combiy effect . Ginsenoy effect . Pgp expy effect . Rb1 revectively ,58.Panax notoginseng (Sanqi) total saponins reversed MDR of doxorubicin in MCF-7/DOX and K562/VCR cell lines. The mechanism may be related to the decrease of Pgp expression [pression ,60.Sophora japonica (Huai). Quercetin inhibited the binding of heat shock factor at the MDR1 promoter, thereby decreasing MDR1 transcription and reducing Pgp expression [Quercetin is one of the most widely distributed flavonoids in natural products including Chinese medicinal herbs such as pression . Quercetpression . In HL-6pression ,64. MDR pression . Quercetpression . Althougpression .Curcuma longa (Jianghuang), inhibited the transport activity of all three major ABC transporters, i.e. Pgp, MRP1 and ABCG2 [meta-or para-position on benzamide improved MDR reversal [Curcumin, the major component in nd ABCG2 . Curcumind ABCG2 . Curcumind ABCG2 . Moreovend ABCG2 . The cytnd ABCG2 . Curcumind ABCG2 . A chlorreversal . Bisdemereversal . Tetrahyreversal . Curcumireversal . In leukreversal . A specireversal ,77.Schisandra chinensis (Wuweizi), were investigated for their MDR reversal effects. Schizandrin A was the most potent in reversing MDR by enhancing apoptosis and down-regulating Pgp and total protein kinase C expression. The crude extract of Schisandra chinensis reversed the resistance against vincristine in vivo [Schizandrins, the active constituents of  in vivo . Deoxysc in vivo . Gomisin in vivo .Panax ginseng and Ophiopogon japonicus (Maidong), down-regulated Pgp expression in peripheral blood lymphocyte membrane. When used together with oxaliplatin, 5-fluorouracil or folinic acid, the injection prolonged the survival rate of colon cancer patients ['Shengmai Injection', consisting of patients . The injpatients .Panax ginseng, Scorophularia ningpoensis (Xuanshen) and Atractylodes macrocephala (Baizhu), increased the intracellular concentration of vincristine in resistant SGC-7901/VCR cell line, possibly due to the induction of apoptosis and down-regulation of Pgp and bcl-2 expression ['Shenghe Powder', consisting of pression .Croton tiglium (Badou), Platycodon grandiflorum (Jiegeng) and Fritillaria thunbergii, induced apoptosis in SGC-7901 cell line and down-regulated the gene expressions of p53, bcl-2, rasP21CD44 and Pgp ['Modified Sanwubai Powder', consisting of herbs such as  and Pgp .in vitro and in vivo and reversed MDR in a dose-dependent manner. Tul-17 inhibited Pgp expression [Three herbal extracts used to treat diseases other than cancer, namely Ams-11, Fw-13 and Tul-17, greatly enhanced the efficacy of vincristine both pression .Brucea javanica (Yadanzi) reversed MDR when used together with other chemotherapeutic drugs such as vincristine, doxorubicin, cisplatin, mitomycin C, 5-fluorouracil or etoposide, probably due to down-regulation of Pgp expression or inhibition of TOPO II or both [Oil emulsion from  or both ,87.Reynoutria japonica (Huzhang), Actinidia arguta (Mihouligen) and Geum aleppicum (Shuiyangmeigen), reversed MDR of doxorubicin via down-regulation of Pgp expression ['Sangeng Mixture Decoction', consisting of pression .Actinidia arguta, reversed MDR in K562/DOX cell line by increasing the intracellular doxorubicin concentration [FFTLG, a formula containing ntration .Ligusticum chuanxiong, Curcuma longa and Millettia dielsiana (Jixueteng), enhanced the anticancer activities of doxorubicin in MCF-7/DOX via down-regulation of Pgp expression [R1, consisting of pression ,91.When tested with doxorubicin, 5-fluorouracil and epirubicin in HepG2/DOX cell line, Ganai-1, a commercial product, reversed MDR via down-regulation of Pgp expression . AnotherMenispermum dauricum (Bianfuge) as a calcium channel blocker. Dauricine reversed vincristine resistance in MCF-7/DOX cell line [Dauricine is a bisbenzylisoquinoline alkaloid isolated from the root of ell line . Howeverell line . Moreoveell line .Menispermum dauricum. Both dauricine and daurisoline sensitized MCF-7/DOX cell line towards doxorubicin and vincristine in a dose-dependent manner [Daurisoline, a structural analogue of dauricine, is also a calcium channel blocker isolated from the root of t manner . The MDRt manner .3, one of the active ginsenosides from Panax ginseng, restored the sensitivity of resistant KBV20 cell line to various anticancer drugs, including vincristine, doxorubicin, etoposide and colchicine in a time-and dose-dependent manner. This ginsenoside competitively inhibited the binding of substrate drugs to Pgp and its binding affinity to Pgp was remarkably higher than that of verapamil. In contrast to the dose-dependent effects in vitro, 20(S)-ginsenoside Rg3 increased animal life span in an in vivo MDR model in a dose-independent manner [20(S)-Ginsenoside Rgt manner .Paeonia suffruticosa (Mudan). In K562/DOX cell line, paeonol showed positive MDR reversal effect towards doxorubicin, daunorubicin, vincristine and vinblastine without modulating Pgp expression [Paeonol is a weak calcium channel blocker isolated from the root of pression . In parepression .Coix lacryma-jobi (Yiyi) enhanced the anticancer activities of paclitaxel and docetaxel and reversed MDR in a dose-dependent manner ['KLT Injection' consisting of the extract of t manner .Paeonia lactiflora (Shaoyao), Rehmannia glutinosa (Dihuang), Angelica sinensis (Danggui) and Ligusticum chuanxiong, reversed doxorubicin MDR without altering Pgp expression in K562/DOX cell line ['Siwu Mixture', consisting of ell line .Pseudolarix kaempferi (Jinqiansong), reversed MDR in vitro and in vivo and induced apoptosis via cell cycle arrest at G2/M phase. In either resistant cell line or nude mice model, pseudolaric acid B circumvented MDR associated with Pgp overexpression [Pseudolaric acid B, a major active component of pression .Salvia miltiorrhizae (Danshen), induced apoptosis and inhibited tubulin polymerization in various cancer cell lines, including the Pgp and MRP-overexpressing MDR cells [Salvinal, isolated from DR cells .Cephalotaxus hainanensis (Hainancufei), Artemisia annua (Qinghao) and Bufo marinus, B. viridis (Chanchu) respectively exhibited active MDR modulation [A study on 22 compounds from CM herbs found that homoharringtonine, artesunate and bufalin from dulation . Moreovedulation , arsenicdulation ,109, psodulation ,111, oridulation ,112, β-edulation ,114 alsoGanoderma species including G. lucidum (Lingzhi) were cytotoxic and induced apoptosis similar to etoposide and doxorubicin which are commonly used in chemotherapy. In etoposide-selected resistant cell line H69, G. lucidum extract increased the sensitivity to etoposide and doxorubicin significantly, possibly due to increased intracellular DNA fragmentation and caspases 3 and 9 activities [Glycyrrhiza glabra (Gancao), Hedyotis diffusa (Baihuasheshecao) and Rheum palmatum (Dahuang) reversed MDR by increasing the intracellular concentration of daunorubicin in SGC7901/VCR cell line [Nine out of 20 extracts of tivities . Moreoveell line .'Ganli Injection', consisting of matrine and tetramethylpyazine hydrochloride, reversed MDR by increasing the sensitivity of 5-fluorouracil and the intracellular concentration of doxorubicin in BEL-7402/5-FU cell line .Cinnamomum cassia (Rougui), Psoralea corylifolia (Buguzhi) and Rheum palmatum, was tested in A549/DDP cell line and S180 tumour-bearing mice. In vitro, the formula significantly increased the intracellular concentration of cisplatin at high doses and inhibited the activity of calcium channel and LRP-56 expression at both high and low doses. In vivo, the formula improved the serum concentration, reduced the inflow and the release of Ca2+ and inhibited the LRP gene expression ['Bushen Huayu Jiedu Formula', consisting of pression ,119.Glycyrrhiza glabra (GLYC), Hedyotis diffusa (OLEN), a formula consisting of 15 herbs including Cistanche deserticola (Roucongrong), Rabdosia rubescens (Donglingcao) and Zanthoxylum nitidum (Liangmianzhen) (SPES), and a formula consisting of eight herbs including Serenoa repens (Juyezhong), Scutellaria baicalensis (Huangqin), Panax ginseng and Glycyrrhiza glabra (PC-SPES) were cytotoxic to cancer cell lines in a dose-dependent manner. SPES, PC-SPES, OLEN decreased the bcl-2 gene expression and were pro-apoptotic, while GLYC was pro-necrotic without altering the over-expression of bcl-2 in MDR cells. Furthermore, OLEN, SPES and PC-SPES exhibited similar pharmacological effects to etoposide and vincristine [Four CM formulae, namely cristine .Many MDR reversing alkaloids are also calcium-channel blockers probably because of (1) their structural similarity and (2) inhibition of ABC transporters by the decrease in intracellular calcium concentration. Future research is warranted for potent MDR inhibitors without other pharmacological activities.Over-expression of ABC transporters and enhanced drug efflux are the causes for MDR. Among the ABC transporters, Pgp is the most extensively studied for its role in MDR reversal effects. While other MDR reversal mechanisms remain unclear, Pgp inhibition is a criterion for further mechanistic study. This article summarises these proposed mechanisms  inhibition on Pgp; (-) no effect on Pgp.Click here for file


Four cell lines, the mouse L1210 leukaemia, the human W1L2 lymphoblastoid and two human ovarian (CH1 and 41M) cell lines, were made resistant to ZD1694 (Tomudex) by continual exposure to incremental doses of the drug. A 500-fold increase in thymidylate synthase (TS) activity is the primary mechanism of resistance to ZD1694 in the W1L2:RD1694 cell line, which is consequently highly cross-resistant to other folate-based TS inhibitors, including BW1843U89, LY231514 and AG337, but sensitive to antifolates with other enzyme targets. The CH1:RD1694 cell line is 14-fold resistant to ZD1694, largely accounted for by the 4.2-fold increase in TS activity. Cross-resistance was observed to other TS inhibitors, including 5-fluorodeoxyuridine (FdUrd). 41M:RD1694 cells, when exposed to 0.1 microM [3H]ZD1694, accumulated approximately 20-fold less 3H-labelled material over 24 h than the parental line. Data are consistent with this being the result of impaired transport of the drug via the reduced folate/methotrexate carrier. Resistance was therefore observed to methotrexate but not to CB3717, a compound known to use this transport mechanism poorly. The mouse L1210:RD1694 cell line does not accumulate ZD1694 or Methotrexate (MTX) polyglutamates. Folylpolyglutamate synthetase substrate activity (using ZD1694 as the substrate) was decreased to approximately 13% of that observed in the parental line. Cross-resistance was found to those compounds known to be active through polyglutamation.


It has long been a practical problem for surgical pathologists to distinguish mesothelium from epithelium in order to make a positive diagnosis of mesothelioma. In this study, we developed a new monoclonal antibody, designated MS-2761 , against cultured non-neoplastic mesothelial cells. Immunohistochemistry and slot-blot analysis revealed that this monoclonal antibody reacted with 100% (12/12) of benign and malignant mesothelioma tissues and a mesothelioma cell line, but not with 99% (77/78) of epithelial tumour tissues and 97% (33/34) of epithelial tumour cell lines. A gene encoding the cell-surface antigen defined by this monoclonal antibody was isolated from a mesothelial cell cDNA library constructed with a mammalian cell expression vector through transfection of Cos-7 cells and immunoselection by panning. DNA sequencing and a database search revealed that the gene was identical to vascular cell adhesion molecule 1 . The prominent VCAM1 transcript in mesothelium was 3.2 kb in size with seven Ig-like domains, in addition to a minor transcripts with six Ig-like domains. This monoclonal antibody potentially discriminates mesothelium from epithelium and may become a tool for differential diagnosis of mesothelioma.


We selected two clones, isolated from the human colocarcinoma cell line LoVo, showing a sensitivity to doxorubicin similar to (LoVo clone 5) or three times lower than (LoVo clone 7) the parental cell line. Since vimentin was atypically expressed in a human breast carcinoma cell line made resistant to doxorubicin, we looked at vimentin expression in these two clones with spontaneously different sensitivity to the drug. For comparison we used the parental cell line LoVo WT and LoVo/DX made resistant pharmacologically. mRNA for vimentin was undetectable by Northern blot analysis in LoVo WT and in LoVo clone 5, while expression of this gene was high in LoVo clone 7 and in LoVo/DX. This increase in mRNA levels was not related to an amplification of DNA, as suggested by Southern blot analysis. Immunofluorescence and immunocytochemistry findings confirmed, at protein level, the mRNA data. In LoVo clones 5 and 7, there were respectively 8.6% and 71% vimentin-positive cells, although the two clones showed similar expression of multidrug resistance gene 1 (mdr-1) and accumulated intracellular doxorubicin at similar levels. Similarly, drug efflux was the same for both clones. Our results show for the first time that cells resistant to doxorubicin express vimentin independently of the mdr glycoprotein. However when cells from clone 5 were transfected with human vimentin cDNA, they did not become resistant, indicating that vimentin can be considered as a marker of resistance in these cells but does not give rise to a resistant phenotype by itself.


The hybrid cell line BH2 was derived by fusion between an AKR thymoma BW5147 (HGPRT-) and C57BL thymoma EL-4R (TK-). The hybrid cells showed a near-tetraploid modal number of chromosomes, in contrast to the near-diploid stem-lines of both parental cell populations; most of the BH2 hybrid cells acquired marker chromosomes from both parental cell lines. Inoculation of the parental and hybrid cells into C57BL and AKR mice revealed that the possible admixture of revertant parental cells in the hybrid cell population was less than 10(-4). Anodic electrophoretic mobilities (AEM) of the mouse thymoma lines and their hybrids were compared with each other and with normal mouse lymphoid cells. The AEM of the parental and hybrid T-cell lines was slower than that of mouse T LNC and comparable with AEM of some thymocyte subsets. The mean AEM of parental and hybrid cell lines was 0.69 micrometers/sec/V/cm for BW5147 cells, 0.96 for EL-4R cells and 0.83 for BH2 cells, the mean AEM of the hybrid cell population being identical with the mean of the parental AEM values. The mean AEM was found to be a relatively stable characteristic of each cell line.


Polyunsaturated fatty acids (PUFAs) have been implicated in tumour development and have been shown to influence cell proliferation in vitro. We report here that n-3 and n-6 PUFAs at concentration > 10 microM inhibited the proliferation of a human kidney epithelial cell line (21HKE), which has retained phenotypic characteristics of normal kidney epithelial cells. In contrast, the proliferation was stimulated by n-3 and n-6 PUFAs at concentrations < 10 microM under defined growth conditions. The stimulatory effect of n-3 and n-6 PUFAs was even more profound in the presence of EGF. In human kidney epithelial cell lines reflecting different stages of transformation (11HKE and 1THKEras), the stimulatory effect was abrogated both in the presence and absence of EGF. Saturated fatty acids did not show any stimulatory effect on cell growth. The tyrosine kinase inhibitors genistein and tyrphostin-47 inhibited EGF-induced protein tyrosine phosphorylation dose-dependently in the 21HKE cells, and abolished the growth stimulatory effect of docosahexaenoic acid (DHA). This indicates the involvement of EGF receptor tyrosine kinase activity in the observed increase in cell proliferation.


Multidrug resistance (MDR), caused by overexpression of either P-glycoprotein or the multidrug resistance protein (MRP), is characterised by a decreased cellular drug accumulation due to an enhanced drug efflux. In this study, we examined the effects of genistein and structurally related (iso)flavonoids on the transport of rhodamine 123 (Rh123) and daunorubicin in the MRP-overexpressing MDR lung cancer cell lines COR-L23/R and MOR/R. Genistein, genistin, daidzein and quercetin showed major differences in effects on Rh123 vs daunorubicin transport in the MRP-mediated MDR cell lines: the accumulation of daunorubicin was increased, whereas the accumulation of Rh123 was decreased by the flavonoids. The depolarisation of the membrane potential caused by genistein might be involved in the acceleration of the efflux of Rh123 measured in the MRP-overexpressing cell lines. These observations should be taken into account when using fluorescent dyes as probes for determination of transporter activity as a measure of MDR.


The response of multicellular tumour spheroids of the EMT6 cell line to combinations of hyperthermia and Bleomycin (BLM) or Adriamycin (ADM) has been investigated. Using this model system, we have demonstrated enhanced BLM cytotoxicity at 43 degrees C and also heat-induced drug tolerance to BLM at 43 degrees C. ADM cytotoxicity was not significantly increased after 43 degrees C x 1 h but after 6 h at 42 degrees C greatly enhanced cell-killing was evident. These results are discussed in relation to our previous data for EMT6 cells growing either as monolayer cultures in vitro or as solid tumours in mice.


The activities and the expression of 3-methyladenine glycosylase (3-meAde gly) and O6-alkylguanine-DNA-alkyltransferase (O6 ATase) were investigated in ten human cancer cell lines. Both 3-meAde gly and O6 ATase activities were variable among different cell lines. mRNA levels of the O6 ATase gene, appeared to be related to the content of O6 ATase in different cell lines, whereas no apparent correlation was found between mRNA of 3-meAde gly and the enzyme activity. No correlation was found between the activity of the two enzymes and the sensitivity to alkylating agents of different structures such as CC-1065, tallimustine, dimethylsulphate (DMSO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cis-diamminedichloroplatinum (cDDP) and melphalan (L-PAM). The most striking finding of this study is that a correlation exists between the activity of O6 ATase and 3-meAde gly in the various cell lines investigated (P<0.01), suggesting a common mechanism of regulation of two DNA repair enzymes.


Sir,In response to the lack of detection of HPV in MDA-MB-175VII and SK-Br-3 cell lines, we note that the authors have not followed the methods as set out  from DNA isolated from both MDA-MB-175VII and SK-Br-3 cell lines . The RT–Because of the difficulty in amplifying HPV in these breast cancer cells and because these cell lines have been available and passaged in many different laboratories over many years and may have changed significantly over time, we subcloned (by limiting the dilution) the MDA-MB-175VII cell line. Here we show that all six subclones of MDA-MB-175VII have the same DNA fingerprint as the parental MDA-MB-175VII cell line , as expein situ PCR as per Only three of six MDA-MB-175VII clones isolated apparently contained HPV as indicated by the successful amplification of the L1 region by nested PCR and by in situ PCR subsequent to the report in We have confirmed the presence of HPV in the nuclei of both MDA-MB-175VII and SK-Br-3 cell lines by By using a series of primers in the L1 region, it appears that the L1 sequence does not extend 5′ far beyond GP5 in the MBA-MB-175VII cell line. The region 3′ of the L1 amplicon was extended at least as far as 7244 bp. This suggests that the HPV is fragmented and/or incomplete and may indicate ‘hit-and-run’ oncogenesis as described for HPV in Syrian hamster embryo cells (We conclude that the HPV sequences are present as reported (


Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf) and Gdnf receptor alpha 1 (Gfra1) transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1) were higher than those in primary cultured normal human epithelial melanocytes (NHEM), while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT) sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma. It has recently been reported that the incidence of cutaneous malignant melanoma is increasing at a greater rate than that of any other cancer The c-RET proto-oncogene  encodes RFP-RET  is a hybThe correlation between c-RET and human malignant melanoma has been denied by a report of no c-RET transcript expression in human cultured-normal melanocytes and malignant melanoma cell lines in Northern blotting analysis We first examined dynamics of the introduced oncogenic RET RFP-RET:  transcriWe next examined expression levels of intrinsic c-Ret, Gfra1 and Gdnf transcripts in benign and malignant tumors from RET-mice . Levels We next examined the expression levels of c-RET, GFRa1 and GDNF in primary-cultured normal human epithelial melanocytes and human malignant melanoma cell lines  We next examined levels of c-RET protein expression in NHEM and human malignant melanoma cells  -carrying transgenic mice Previous studies showed that overexpression of c-RET, GFRa1 and/or GDNF indicated poor prognosis in human pancreatic In summary, we newly showed c-RET protein expression in HM3KO and MNT-1 melanoma cells in addition to its expression in the previously reported five human melanoma cell lines RET oncogene (RFP-RET) fused to the mouse MT promoter-enhancer We previously established RET-mice (line 304/B6) with C57BL/6 background by introducing the 5′-GCTGCATGAGAATGACTGGA-3′ and 5′-TGGCATfTCTCCCTCTCTCTG-3′ ; mice Gfra1, 5′-GACCTGGAAGATTGCCTGAA-3′ and 5′-CAGTGGTAGTCGTGGCAGTG-3′ ; mice Gdnf, 5′-GTCCAACTGGGGGTCTACG-3′ and 5′-AGCAACACCAGGCAGACAG-3′ ; mice Hprt, 5′-CTTTGCTGACCTGCTGGATT-3′ and 5′-TATGTCCCCCGTTGACTGAT-3′ ; human RFP-RET, 5′-TGACGGAGAGTCTAAAGCAG-3′ and 5′-GCTTTAATCCCCCGGGGC-3′ ; human c-RET, 5′-GCTCCACTTCAACGTGTC -3′ and 5′-GCAGCTTGTACTGGACGTT-3′ ; human GFRa1, 5′-CACTGCCACTACCACCACTG-3′ and 5′-GTGTATTGCCCGACACATTG-3′ ; human GDNF, 5′-CTGGGCTATGAAACCAAGGA-3′ and 5′-CAACATGCCTGCCCTACTTT-3′ ; human TBP, 5′-CACGAACCACGGCACTGATT-3′ and 5′-TTTTCTTGCTGCCAGTCTGGAC-3′ . PCR was carried out using 10 µl of power SYBR® Green PCR master mix (Applied Biosystems) containing 5 µM forward primer and 5 µM reverse primer in a final volume of 20 µl. The PCR conditions were as follows: 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min.Total RNA was prepared from frozen tumor and human cell line samples using a High Pure RNA Kit (Roche Diagnostics) according to the method previously described Polymorphism at the G691S juxtamembrane region in c-RET in melanoma cells was examined according to the method previously reported Immunoprecipitation with anti-c-RET rabbit polyclonal antibody was performed by the method previously described G361 and HM3KO cells were treated with 100 ng/ml GDNF for 15 minutes at 37°C, and then the cells were fixed in freshly prepared phosphate-buffered 2% paraformaldehyde for 15 min at 4°C. For DAB staining, the fixed cells were incubated in 3% hydrogen peroxide for 10 min at RT. All specimens were treated with a blocking reagent (phosphate-buffered 1% BSA (fraction V) solution containing 0.2% gelatin and 0.05% tween20) and then incubated with the following primary antibodies for 60 min at room temperature: anti-GFRa1 , anti-c-RET  and anti-phosphorylated tyrosine 905 in c-RET  antibodies. Then the cells were stained using a DAKO Envision-HRP/DAB system (K1390)  or labeled with Alexa fluore 488-conjugated donkey anti-rabbit IgG (A21206) . Counterstaining was performed using hematoxylin or DAPI.Cell proliferation in the presence or absence of GDNF was evaluated by counting cells with trypan blue staining Normal human normal melanocytes (NHEM) were purchased from Cell Applications Inc, and were maintained with melanocyte growth medium containing hydrocortisone and growth supplements (Cell Applications Inc.). MNT-1 and HM3KO human malignant melanoma cell lines were kindly provided by Dr. Tamio Suzuki . G361 cells were kindly provided by Cell Resource Center for Biomedical Research, Tohoku University. SK-Mel28 cells were purchased from Riken Bioresource Center Cell Bank. SK-Mel28, MNT-1 and HM3KO cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Other cell lines were maintained in RPMI 1640 supplemented with 10% FBS.


We attempted to induce anti-tumour immunity for rejecting pre-B lymphoma derived from E mu/ret transgenic mice (TGM). We established pre-B-lymphoma cell lines of C57BL/6 x Balb/c background (H-2b/d) into which H-2k alloantigen and C3H background were introduced (retL1-6 and retL6-6), and we inoculated BCF1 mice with these immunising tumour cells. After these tumours were rejected by alloantigen (H-2k/C3H background)-specific effector cells, the mice were challenged with the pre-B-lymphoma cell line derived from the original E mu/ret TGM (ret0-2). All non-immunised control mice died within 80 days, whereas half the immunised mice survived for over 300 days. The immunity was also effective against primary pre-B-lymphoma cells from E mu/ret TGM and the ret-driven melanoma cell line (MEL-ret), but not against the pre-B-lymphoma cell line from E mu/myc TGM. This immunity was at least in part mediated by cell-mediated cytotoxicity that was specific to the ret oncogene product or ret-regulated antigen. Next we immunised E mu/ret TGM by inoculating them with retL6-6 cells once every 2 weeks beginning at the age of 1 month. Interestingly, this immunisation enabled the TGM to survive longer than the non-immunised control group (P < 0.05). Moreover, 2 of 11 transgenic mice receiving such immunisation were free from both macroscopic and microscopic tumours at the time when all of the 12 non-immunised control TGM had died from their tumour. This provides a new model for oncogene-linked immunotherapy research.


Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest.We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots.Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics.Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines. About 6,500 new articles related to cancer are retrieved every 30 days in PubMed, and nearly 2,000 articles are retrieved with the key word 'cell line'.Cell lines provide indispensable tools in many aspects of laboratory research, particularly as Despite these advantages, the use of cell lines in cancer research is also disputatious . A commoin vitro applications , it is essential to know the exact differentiation state and functionality of the cells being used. This can be approached through proteomic profiling . These spots were dominant in the EM-G3 and MDA-MB-231 cell lines and almost undetectable in the MCF7 and HCC1937 cell lines. This is in accordance with the GST activity measurements. We further identified GST M3-3 (spot no. 4). This spot had a comparable intensity in each cell line. Another large spot (no. 8) was identified as carbonyl reductase [NADPH] 1. This protein was present in the EM-G3 and HCC1937 cell lines and missing in the MCF7 and MDA-MB-231 cell lines. We further identified elongation factors EF-1β (spot no. 5) and EF-1γ (spot no. 6) together with β-actin (spot no. 7) and lanC-like protein 1 .The predominant protein retained on the GSH-affinity column was GST P1-1, identified in three spots . The differences in pIs of the detected GST P1-1 isoforms might be caused by post-translational modifications or they might rose from polymorphism of the enzyme  1. All of these proteins are involved in the metabolism of xenobiotics. GSTs catalyze conjugation of glutathione with a wide variety of electrophilic compounds . CarbonySurprisingly we have not identified other typical GSH-binding proteins, such as GSH peroxidase, GSH reductase or glyoxalase. These proteins were probably retained on the column but not concentrated in sufficient quantities for identification.We also detected β-actin, which is one of the most abundant proteins in cell lysates and was likely retained on the column because it is a "sticky" protein that interacts with many partners. Actin polymerization is regulated by reversible glutathionylation, thus the interaction with glutathione might be specific to a certain extent .We also identified two subunits of elongation factor 1 (EF-1): EF1-β and EF1-γ. EF1-β contains a GST C-terminal domain and, together with EF1-δ, stimulates the exchange of GDP that is bound to EF-1-alpha for GTP. EF1-γ contains both GST C-terminal and N-terminal domains and probably plays a role in anchoring its complex to other cellular components . The rolThe final protein identified was LanCL1. LanCL1 is a mammalian ortholog of the prokaryotic enzyme lanthionin synthase and its function in eukaryotes is unknown . SimilarTo conclude, we analyzed the four breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231, and showed major differences in their phenotypes especially in GSTs activities and GSH-binding proteomes. It can be deduced that such diversity might exist among other cell lines and among activities of many other proteins. Various cell lines are often used in parallel. They are employed in determinations of bioactivities for various compounds ,41, in cThe usefulness of targeted affinity in proteome profiling has been demonstrated. However, when applying this methodology certain issues should be taken into account. The main restriction is a significant consumption of a sample, while the yields of proteins retained on affinity columns are very low. Introducing numerous steps to sample handling is a source of contamination and undefined protein losses. Affinity chromatography is not a scavenger method, and complicated affinity equilibria for various proteins towards the ligand should be anticipated . Neverth2-DE: two dimensional electrophoresis; CK: cytokeratin; GSH: glutathione; GST: glutathione S-transferase; MS: mass spectrometry; NME: normal mammary epithelialThe authors declare that they have no competing interests.JM performed affinity chromatography, GST activity measurements and prepared 2-DE gels, prepared the samples for identification of proteins and contributed to writing of the manuscript. MS performed the LC-MS/MS experiments and protein identifications. EM established and handled the cell cultures. IS designed and coordinated the study, performed the analysis of 2-DE gels and Western blots and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/449/prepub


We have examined the expression of several myeloid cell associated antigens, some of which are involved in myelomonocyte adhesion, in seven well characterized human breast cancer cell lines, since common properties of adhesiveness and migration are found in haemopoietic cells and epithelial cancer cells. Five of these cell lines were of metastatic origin and two were derived from primary breast carcinoma. Antigenic expression was evaluated by immunofluorescence (IF), flow cytometry (FCM), radioimmunoassay on live cells (RIA) and immunoperoxidase staining. None of these cell lines expressed T or B lymphoid specific antigens. Myeloid antigens My4, MO1, and MOF11  were expressed in the 7 cell lines. Leu M1, Leu M3, My9, and MO2 antigens were expressed in some of the cell lines. Leu M2 and My7 antigens were not expressed or at very low levels. The expression of these myeloid antigens was also tested by immunoperoxidase staining, and found on frozen sections of normal mammary gland, fibroadenoma of the breast, primary breast cancer, and lymph node and skin metastases of breast tumours. This common expression in epithelial breast cells and in myeloid cells might be related to common biological functions such as interaction with extracellular matrix which precedes cell migration, a normal function of macrophages and an abnormal function expressed or amplified in human cancer epithelial cells.


Patients with ovarian cancer often respond well to combination chemotherapy initially but the majority eventually relapse when, with further treatment, the initially successful regimen proves ineffectual. The cause of such failures frequently has been attributed to the development of drug resistance. Although the mechanisms of acquired resistance in situ are still poorly understood, studies in vitro have shown that cells selected for resistance to one drug often exhibit cross-resistance to other seemingly unrelated agents, suggesting a somewhat generalised mechanism of resistance. We have studied the role of glutathione (GSH) and drug transport in determining the sensitivity to adriamycin (ADR) of a panel of human ovarian cell lines established directly from biopsies of patients with diverse treatment histories. These cell lines exhibited inherent differences in sensitivity to ADR by a dose factor of up to 3; a difference that was considerably less than what has been reported when cells were selected for drug resistance in vitro. The differences in drug sensitivity reported here among the various cell lines appeared to be unrelated to drug transport, in terms of both influx and efflux. Moreover, although these cell lines have a wide range of GSH content, there was only a poor correlation between drug sensitivity and cellular GSH content per se. However, when exposed to a clinically relevant dose of ADR, the GSH content of cell lines that were 'sensitive' decreased, whereas that of cell lines that were 'resistant' increased. To take these time-dependent changes in GSH into consideration, the area under the GSH content versus time curve (AUC), with and without ADR treatment, was calculated for each cell line. When this latter factor was included in the analysis, greatly improved correlations were found between GSH kinetic parameters and responses to ADR. In particular, ADR resistance was found to be closely correlated with the positive changes in absolute GSH AUC following ADR treatment . Using 35S-labelled cysteine and methionine as tracers, it was found that the essential difference between the 'resistant' and 'sensitive' lines was that the 'resistant' lines had higher steady-state rates of GSH synthesis than the 'sensitive' lines. These results demonstrate that changes in cellular GSH concentration during treatment may be an important indicator of tumour cell response to ADR.


Cell lines are commonly used in various kinds of biomedical research in the world. However, it remains uncertain whether genomic alterations existing in primary tumor tissues are represented in cell lines and whether cell lines carry cell line-specific genomic alterations. This study was performed to answer these questions.Array-based comparative genomic hybridization (CGH) was employed with 4030 bacterial artificial chromosomes (BACs) that cover the genome at 1.0 megabase resolution to analyze DNA copy number aberrations (DCNAs) in 35 primary breast tumors and 24 breast cancer cell lines. DCNAs were compared between these two groups. A tissue microdissection technique was applied to primary tumor tissues to reduce the contamination of samples by normal tissue components.The average number of BAC clones with DCNAs was 1832 (45.3% of spotted clones) and 971 (24.9%) for cell lines and primary tumor tissues, respectively. Gains of 1q and 8q and losses of 8p, 11q, 16q and 17p were detected in >50% of primary cancer tissues. These aberrations were also frequently detected in cell lines. In addition to these alterations, the cell lines showed recurrent genomic alterations including gains of 5p14-15, 20q11 and 20q13 and losses of 4p13-p16, 18q12, 18q21, Xq21.1 and Xq26-q28 that were barely detected in tumor tissue specimens. These are considered to be cell line-specific DCNAs. The frequency of the HER2 amplification was high in both cell lines and tumor tissues, but it was statistically different between cell lines and primary tumors (P = 0.012); 41.3 ± 29.9% for the cell lines and 15.9 ± 18.6% for the tissue specimens.Established cell lines carry cell lines-specific DCNAs together with recurrent aberrations detected in primary tumor tissues. It must therefore be emphasized that cell lines do not always represent the genotypes of parental tumor tissues. Cancer cell lines are routinely used for various kinds of biomedical research under the assumption that cell lines reflect the genotypic and phenotypic characteristics of primary tumor tissues. However, such cell lines do not always faithfully represent genomic alterations and gene expression observed in tumor tissue specimens -4, and tin vitro-specific genomic aberrations. In this context, it is crucial to distinguish genomic aberrations in tumor tissues from the secondary changes with cultivation. The differentiation between these aberrations is practically difficult, because available data on difference in the genomic changes between cell lines and tumor tissue specimens are very limited at present [According to the commonly accepted model for cancer development, somatic mutations accumulate in a cell in the process of tumorigenesis. In clinically overt cancers, not only a large number of genomic aberrations are detected but also genomic instability successively yields genomic alterations in a cancer cell. This theory explains why the number of genomic aberrations is greater in advanced cancers than in early cancers -8. The e present ,14. The This study examined the DNA copy number aberrations (DCNAs) of 24 breast cancer cell lines and 35 primary breast cancer tissues using array-based comparative genomic hybridization (aCGH). The present paper showed that the breast cancer cell lines preserved genomic alterations detected in primary cancer tissue specimens and that the cell lines concurrently carried secondary genomic alterations. Some of the secondary genomic alterations were recurrent and cell line-specific.This study used 24 cell lines established from human breast cancer as follows: AU565, HCC2218, T-47D, HCC1954, MDAMB361, UACC812, UACC893, BT474, SKBR3, HCC38, HCC1008, ZR-75-30, HCC1937, MDAMB468, HCC1428, ZR-75-1, MCF7, MDAMB231, MDAMB435S (possibly derived from melanoma), BT483, HCC1806, Hs578T, MDAMB175VII and MDAMB415. These cell lines were purchased from American type Culture Collection . The original histology of these cell lines was as follows: 6 adenocarcinomas, 10 invasive ductal carcinomas, and 8 unknown tumors [Thirty-five primary breast cancers that were histologically classified as invasive duct carcinoma were used. All tumors were considered to be sporadic. The average age of patients was 57.6 years, ranging from 31 to 75 years old. In this series, the expression of estrogen and progesterone receptors was positive for 27 (77.1% of tumors) and 19 (54.3%) tumors, respectively. The Institutional Review Board for Human Use Yamaguchi University Graduate School of Medicine approved the study protocol and informed consent for this study was obtained from all patients. A tissue microdissection technique was used to reduce the contamination of samples by normal tissue components for array CGH analyses, as previously described . As a reHigh-molecular-weight DNA was extracted from each tumor specimen with a DNA extraction kit  according to the manufacturer's instructions as previously described -21.http://www.macrogen.co.kr/eng/biochip/karyo_summary.jsp. The experiments were performed as previously described [® DNA Labeling System, Invitrogen™). For hybridization, labeled DNA was mixed with Cot-1 DNA  and ethanol precipitated. The precipitated DNA was dissolved in 40 μl of hybridization mix. The probe mixture was denatured at 75°C for 5 min and incubated at 37°C for 60 min for blocking of repetitive sequences. Arrays were prehybridized with salmon sperm DNA to reduce nonspecific background staining. The probe mixture was applied to the array. The arrays were placed in a moist chamber at 37°C for 72 hr for hybridization. The array slides were washed 2 times in 2× standard saline citrate (2 × SSC), 50% formamide, pH 7.0, at 45°C. The array slides were washed in phosphate buffer with 0.1% NP-40, pH 8.0, once in 2 × SCC at room temperature.The BAC DNA array used in this study consists of 4030 human bacterial artificial chromosome (BAC) clones, including 356 cancer-related genes, which are spaced approximately 1.0 Mb across the whole genome . BAC chip information including data of end-sequenced BAC clones is available on the following websites: escribed -22. Brie2 ratio. The clones with log2 ratios that exceed least than ± 0.25 were considered gain and loss of the copy number. We defined the log2 ratio >1.0 as amplifications. A part of the cell line CGH data has been previously reported [After hybridization, the slides were scanned on a GenePix 4000A scanner  and the 16-bit TIFF images captured using GenePix Pro 5.0 software. Fluorescence images were analyzed with the MAC Viewer™ software program (Macrogen Inc.) optimized for analysis of the array as previously reported -22. Fluoreported .t-test. In this study, the Bonferroni correction was made to adjust the p-value. A difference was considered to be significant when the P-value was less than 1.25 × 10-5 (0.05/4030).The clone-by-clone comparison of the copy number was made between the cell lines and tumor tissue specimens. The differences in the prevalence of common gains and losses between cell lines and tumor tissues were determined with the chi-square test. Differences in the total number of changes and frequency were tested by Student http://cibex.nig.ac.jp/cibex2/ExperimentMiame.do?queryExperimentalDesignAccession=CBX105. The number of DCNAs was more in the cell lines than in the tissue specimens as a general trend. The average number of copy number gains and losses were 651.7 ± 148.8 (standard deviation) and 1180.7 ± 433.8, respectively in cell lines, while they were 424.2 ± 215.9 and 548.0 ± 324.7 in the tumor tissue specimens (Table -8)  and 972.2 clones 24.1%) for a cell line and tumor tissue, respectively. This result is consistent with the data reported by Naylor and colleagues .1% for a,15,24-27in vitro-specific alterations [in vitro show genomic alterations similar to those of cell lines [in vitro environments endow cells with genomic aberrations of which cell line-specific DCNAs are critical for cells to survive and proliferate in vitro. Indeed, it is known that the pattern of CGH profiles change in varying degree with the cell passage numbers [It is particularly worth noting that some of recurrent DCNAs that are frequently detected in cell lines are hardly detected in primary cancer tissues. These DCNAs correspond to cell line-specific or erations ,24,29. Ierations , lung caerations , colon cerations  and pancerations . Establierations ,34,35. Bll lines . TherefoThe difference in the CGH profiles found between the cell lines and tumor tissues is not attributed to just variations in genomic alterations between parental histological types, because DCNAs detected exclusively in cell lines are not explained by the difference in histologic types -39. AccoThe average number of amplified clones was more than double in the cell lines than the tumor tissues (41.3 vs. 15.9). In particular, the frequency of amplification for clones on 17q12-q21, encompassing many genes including ERBB2/HER2 that is the most frequent amplified gene in breast cancers ,41, was in vivo alterations. Cell line-specific DCNAs were as follows: gains of 5p14-15, 20q11 and 20q13 and losses of 4p13-p16, 18q12, 18q21, Xq21.1 and Xq26-q28. When cell lines are used as an alternative to primary tumor tissues, it is therefore important to keep in mind that cell lines do not always represent genotypes of parental tumor tissue specimens.The cell lines carry the cell lines-specific DCNAs together with CGH: comparative genomic hybridization; DCNAs: DNA copy number aberrations; BAC: bacterial artificial chromosomeThe authors declare that they have no competing interests.KT, SK and TM carried out array CGH of tumor tissues, SS and TH were invovled in array CGH of cell lines, SY and MO gathered tumor tissue specimens and clinico-pathological data, MN analyzed array CGH data. KT and SK organized the array CGH data, and KT drafted the manuscript. KS conceived of this study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/15/prepub


A novel human cell line was established from a primary botryoid rhabdomyosarcoma. Reverse transcription polymerase chain reaction investigations of this cell line, called RUCH-2, demonstrated expression of the regulatory factors PAX3, Myf3 and Myf5. After 3.5 months in culture, cells underwent a crisis after which Myf3 and Myf5 could no longer be detected, whereas PAX3 expression remained constant over the entire period. Karyotype analysis revealed breakpoints in regions similar to previously described alterations in primary rhabdomyosarcoma tumour samples. Interestingly, cells progressed to a metastatic phenotype, as observed by enhanced invasiveness in vitro and tumour growth in nude mice in vivo. On the molecular level, microarray analysis before and after progression identified extensive changes in the composition of the extracellular matrix. As expected, down-regulation of tissue inhibitors of metalloproteinases and up-regulation of matrix metalloproteinases were observed. Extensive down-regulation of several death receptors of the tumour necrosis factor family suggests that these cells might have an altered response to appropriate apoptotic stimuli. The RUCH-2 cell line represents a cellular model to study multistep tumorigenesis in human rhabdomyosarcoma, allowing molecular comparison of tumorigenic versus metastatic cancer cells. © 2000 Cancer Research Campaign