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


Colorectal cancer is (CRC) one of the commonest cancers and its therapy is still based on few drugs. Currently, no biological criteria are used to choose the most effective of the established drugs for treatment.A panel of 77 CRC cell lines was tested for sensitivity to 5-fluorouracil (5FU) using the SRB assay. The responses were grouped into three categories and correlated with genetic changes in the cell lines.DCC and mutations in TGFbIIR) are secondary to the association with replication error status.The strongest and most clearcut correlation was between 5-fluorouracil response and replication error status (mismatch repair deficiency). All the other significant correlations (loss of heterozygosity for Our findings validate previous analyses based mainly on clinical data, and indicate that replication error status could be a useful guide to 5-fluorouracil-based CRC therapy. Essentially, all previously described correlations with 5FU response are secondary to the association with replication error status. APC, KRAS, PIK3CA, BRAF, PTEN, SMAD4, MLH1, MSH2, TP53, and CTNNB1  is one of the most commonly diagnosed cancers in the western world  have been in use for many years. The most recently introduced agents are antibodies targeting EGFR (cetuximab and panitumumab) or VEGF (bevacizumab). 5-Fluorouracil, which is also used in the treatment of breast, stomach, and pancreatic cancer, remains the cornerstone of CRC treatment, although widely used in combination with several other drugs . It is aWe have accumulated in our laboratory a large collection of CRC-derived cell lines on which we have information regarding their genetic changes and variation in gene expression , KRAS (codons 12 and 13), BRAF (V600E), TP53, CDH1 (E-cadherin), CTNNB1 , MLH1, MSH2, PIK3CA (exons 9 and 20), B2M , TGFBR2 (TGFbIIR), CHK1, and SMAD4, LOH around the genes APC and CDX1 (chromosome 5), SMAD4 and DCC (chromosome 18), and promoter methylation of MLH1, MSH2, CDKN2A (p16), CDKN2B (p15), and CDX1. Most cell lines have been HLA typed to identify duplicate cell lines, and all cell lines were routinely tested negative for mycoplasma.Details of the 77 cell lines used in this study are listed in 6 cells per ml and placing them on dry ice for 90 min before transfer to liquid nitrogen. For use from frozen stocks, cells were warmed quickly to 37 °C and transferred immediately into prewarmed medium, centrifuged, and re-suspended in fresh, DMSO-free medium before use. Cell lines plated from thawed stocks were shown to have the same growth curves and drug dose-response curves as their cultured counterparts (data not shown). Lag times and doubling time (d.b.t.) for all cell lines after plating from frozen stock were established by plating 3000 cells per well and using the SRB assay as described below. The results are reported in Increased throughput was predominately achieved by plating cells directly into 96-well plates from frozen stock, thus negating the need for continuous culture of the cell lines. Optimal freezing and thawing conditions were established and the time required, after plating, for lines to begin their growth phase was determined and defined as the lag time. Standard freezing conditions involved suspending cells in 5% DMSO in FCS at a density of 4 × 10μl medium in 96-well plates and incubated for their lag time before drug treatment with a further 100 μl of drug-containing medium in duplicate wells for each condition. Final concentrations of 5FU ranged from 0.13 to 100 μM (1 : 3 titration) and the final concentration of DMSO in all wells was 0.067%. Cells were incubated with drug for 3 d.b.t. to accommodate for differences in their growth rates, before they were fixed and stained following a standard SRB protocol (μl of ice-cold trichloroacetic acid (2.6 M) was added and plates were incubated at 4 °C for 60 min. After washing the plates with 0.5 × PBSA, cells were stained with sulforhodamine B (0.04% in 1% acetic acid) for 30 min, washed with acetic acid (1%), and bound dye was dissolved in 200 μl Tris  before measuring the OD at 540 nm in a plate reader. T/Ccorr (%) (‘treated over control (corrected)’ with a correction for the OD of the number of cells at the time of treatment) is calculated as /) × 100, and GI50 values were calculated from the resulting dose-response curves using model 210 /(1+x/C)^D))^E))), A and B unlocked) in XLfit . The experiment was repeated two to four times for each cell line.For toxicity testing, 3000 cells were plated in 100 protocol . In brieμl Matrigel , no. 356231) in 96-well plates and treated with 0, 0.5, 5, and 50 μM 5FU in duplicate wells each (150 μl per well added on top of Matrigel/cells). The medium was exchanged for fresh drug-containing medium on days 3, 6, 9, and 12 before colonies were counted under a light microscope ( × 10 objective) on day 14. Three fields of vision (FOV) were counted per well, averaging counts per FOV over both wells, and the experiment was repeated three times. Four cell lines were chosen for their known different abilities to differentiate in this matrix: LS174T and HT29 (intermediate differentiation), and LS180 and SW1222 (well differentiated).A total of 700 cells were plated in 80 n=26), intermediate response (n=26), and resistant (n=25). To correlate drug response with categorical data , we used χ2 association and trend tests in 2 × 2 table and 3 × 2 tables for comparing categories of drug response with two genetic categories . For known duplicate cell lines, only one of each duplicate was counted for association studies. When drug response groupings differed between duplicate lines, both duplicates were omitted from the statistical analysis. Reported P-values were not corrected for multiple testing.The 77 cell lines were divided into terciles with respect to their sensitivity to 5FU: sensitive  to 47.5 μM (HCT15) in the 77 cell lines, giving an approximately 1600-fold maximum μM difference. 50 values for all the cell lines, including duplicates, in alphabetical order and the same data are shown in rank order in μM) to intermediate  to resistant , as shown in μM), Gp2d and Gp5d (2.54 and 3.80 μM), CACO2 and C2BBe1 (2.50 and 1.42 μM), HDC54 and HDC57 (1.76 and 1.03 μM), and LS174T and LS180 (17.87 and 20.20 μM) agree well with each other. There is a greater difference between DLD1 and HCT15 (18.67 and 47.5 μM), although these two cell lines have the fourth highest and highest GI50 values, respectively, and SW480 and SW620 (6.36 and 17.23 μM), which were established, respectively, from the primary tumour and a metastasis. For all six pairs of these duplicate cell lines, both pairs fall into the same response category. The differences are larger for VACO4A and VACO4S (6.40 and 1.85 μM), which were established from adherent and supernatant subclones of the same cell line. The generally good agreement between lines that have been separately maintained in culture for several years suggests a good measure of reproducibility in the drug response data that we have obtained, and gives confidence to the validity in the categorisation of the cell lines from sensitive to resistant.Overall, GIμM). There is no obvious relationship between the ability to form crypt-like structures and the 5FU response, suggesting that 5FU response is not related to the differentiation capacity of the lines. For all four cell lines, the concentration of 5FU that inhibits colony formation by 50% lies between 0.5 and 5 μM 5FU. This suggests a somewhat higher sensitivity of the cell lines to 5FU in this clonogenicity assay than in our conventional SRB toxicity test, for which GI50 values ranged from 5.76 (SW1222) to 20.2 μM (LS180).Because this is not a higher throughput assay, only four cell lines  were chosen for testing their sensitivity to 5FU in a 3D colony-forming assay using Matrigel. Two of these cell lines (SW1222 and LS180) readily form crypt-like structures, indicating their capacity to differentiate in Matrigel, whereas the other two (HT29 and LS174T) form crypt-like structures to a much lesser extent , with nevs RER− and for LOH around DCC, on chromosome 18. Mutations in TGFbIIR, which are nearly always found in microsatellite unstable tumours because of a coding mononucleotide repeat in the TGFbIIR gene  growing cell lines (P=0.0005). Comparing cell lines’ growth rate by RER status also gives a significant result: the average d.b.t. for RER+ lines is 35.7 h, whereas that for RER− lines is longer  and this most probably accounts for the d.b.t. association with 5FU response.We also found a significant correlation between the d.b.t. of cell lines and their sensitivity to 5FU in a vs RER− patients treated with 5FU-based chemotherapy. It has to be noted, however, that HR ratios varied between 0.48 and 1.21 in the five studies selected by them.Our key observation is that 5FU resistance in our panel of 77 CRC cell lines correlates strongly with mismatch repair deficiency, or RER+. These results are in agreement with the clinical data for the adjuvant setting  were found to carry MSH2 mutations.The well-recognised alterations in SMAD4 and DCC in RER+ CRCs and cell lines, as also shown in μM (188-fold) . We did not detect a correlation between p53 mutation status and 5FU response, which agrees with Our data agree with a much smaller study on three CRC cell lines undertaken by KRAS and a response to anti-EGFR-based chemotherapy in CRC is well established, the clinical data regarding response to 5FU are more conflicting. KRAS mutant and wild-type groups in the response of previously untreated CRC patients with liver metastasis to 5FU. Other researchers report reverse findings, with the 5FU single-agent response rate of 44% in KRAS-mutant patients vs only 32% in wild-type patients . Analysis of preliminary data on TS mRNA expression and 5FU response suggests that high TS expression may be associated with resistance to 5FU. This has been suggested previously by others (Although the link between mutations in LS174T cells originate from trypsin treatment and LS180 cells from scraping of the primary culture from a tumour in the same patient (There is now substantial evidence to support the view that cell lines are true representatives of the tumours from which they were derived. Thus, for example, both the frequency of specific mutations as well as their spectrum is similar between cell lines and tumour samples (see, e.g., We suggest that our approach of studying the response to 5FU in a large panel of well-characterised cell lines, using a relatively high throughput test procedure and an objective categorisation of response, could be a model for preclinical testing of other drugs. Our results show that our approach can be a powerful tool for the detection of predictive markers for drug treatment responses, and that the cell line studies are good predictors of the clinical situation.


Drug resistance is a major problem in cancer chemotherapy. Acquisition of chemo-resistance not only reduces the effectiveness of drugs, but also promotes side effects and markedly reduces the patient's quality of life. However, a number of resistance mechanisms have been reported and are thought to be the reason for the difficulties in solving drug-resistance problems.To investigate the mechanisms of drug resistance, a set of cell lines with different levels of sensitivity and possessing different mechanisms of resistance to 5-fluorouracil (5-FU) was established from a colorectal cancer cell line. The expression of thymidylate synthase, orotic acid phosphoribosyltransferase and dihydropyrimidine dehydrogenase, which are well known to be related to drug resistance, differed among these cell lines, indicating that these cell lines acquired different resistance mechanisms. However, swainsonine, an inhibitor of N-glycan biosynthesis, reduced 5-FU-tolerance in all resistant cells, whereas the sensitivity of the parental cells was unchanged. Further analysis of the N-glycan profiles of all cell lines showed partial inhibition of biosynthesis and no cytotoxicity at the swainsonine dosage tested.These observations suggest that N-linked oligosaccharides affect 5-FU resistance more widely than do drug-resistance related enzymes in colorectal cancer cells, and that the N-glycan could be a universal target for chemotherapy. Further, swainsonine may enhance the performance of chemotherapy by reducing tolerance. Colorectal cancer has one of the highest incidences among all forms of cancer around the world. In many cases, the cancer cannot be completely controlled by surgical intervention, so multidisciplinary treatment including chemotherapy is required. However, cancer cells often acquire drug resistance during treatment and a patient's prognosis can become very unfavorable . Acquisi5-fluorouracil (5-FU), a fluorinated pyrimidine, is a key anti-colorectal cancer drug. 5-FU affects the synthesis and repair of DNA and RNA processing in cancer cells -4. 5-FU Oligosaccharides on glycoproteins mediate a dynamic protein state, involving folding, quality control, secretion and catabolism ,10. GlycWe established various gradations of 5-FU resistant cell lines from a mouse colon cancer cell line and analyzed the expression enzymes related to resistance, the effect of swainsonine and the glycoforms present in those cells.We established various gradations of 5-FU resistant cell lines from the mouse colon cancer cell line, colon 26. The morphology of the 5-FU resistant cells was similar to that of the parental line, and their proliferation rates were similar except for L0-500, which proliferated slightly more rapidly and aggregated more easily, resulting in cell death (data not shown).Drug resistance in the parental L0 line and in the L0-200, L0-500 and L0-1000 lines was assayed using cytotoxicity assay and growth inhibition assay. Fifty percent inhibition concentration (IC-50) values of the L0, L0-200, L0-500 and L0-1000 lines against 5-FU were 0.110, 1.07, 3.87 and 4.86 μg/mL, respectively, by Mossman's 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay Fig. . To estiTS is reported to be a 5-FU catabolic enzyme, and OPRT catalyzes 5-FU to 5-fluorouracil mono-phosphate, which is finally taken into RNA where it inhibits RNA functions. Thus, we used Western-blotting to measure OPRT and TS protein levels in the resistant lines. OPRT was down-regulated and TS was up-regulated in all 5-FU resistant sublines; L0-200, L0-500 and L0-1000 Fig. .As TS, OPRT, and UP expression are correlated with 5-FU resistance, we used real-time RT-PCR to analyze the expression of these factors in the resistant lines. TS expression was up-regulated in the 5-FU resistant cell lines, though the change was only slight in the L0-500 line. OPRT expression was slightly down-regulated in the 5-FU resistant cell lines. UP expression in the L0-200 line was up-regulated 3-fold over that in the parental line; however, its expression in the L0-500 line was the same as that in the parental line, and it was down-regulated in the L0-1000 line, indicating that there was no overall linear relationship between 5-FU resistance and OPRT, TS, or UP expression Fig. . ExpressSwainsonine blocks α-mannosidase II activity, which is necessary to convert N-glycans from hybrid-type to complex-type. 5-FU resistant L0-1000 and parent cells show the same sensitivity to swainsonine. Over 90% of cells from both cell lines were killed by treatment with 30 μg/mL of swainsonine, whereas 80% of cells survived at 10 μg/mL swainsonine. Therefore, we applied 5 μg/mL swainsonine, at which concentration no cytotoxicity was observed. Swainsonine treatment significantly reduced the IC-50 against 5-FU of the resistant cells, but not that of the parental L0 line  column using high performance liquid chromatography (HPLC) Fig. , then eaHerein we describe a set of multistage 5-FU resistant cell lines: L0-200, L0-500 and L0-1000. These lines are useful in examining the mechanisms of drug resistance, as they are of the same genetic background and utilize different acquired mechanisms of drug resistance. The IC-50 values of the L0-200, L0-500 and L0-1000 lines against 5-FU were 9.7, 35 and 44 times, respectively, greater than that of the parental L0 line by MTT assay, and the 5-FU concentrations in the respective cell lines were 5.4, 7.7 and 4.9 times greater than that in the maintenance media.To analyze the mechanisms underlying 5-FU resistance, we assayed the expression of 5-FU catabolic enzymes by Western blotting and real-time RT-PCR. Western blotting showed that TS expression was up-regulated whereas that of OPRT was down-regulated in the resistant cells compared to that in the parental line. This expression profile corresponds to that in previous reports showing that TS inactivates 5-FU, and OPRT activates 5-FU function -37. OPRTIn our study, a low-dosage swainsonine treatment, which shows no cytotoxicity, increased 5-FU sensitivity and altered glycoforms in the resistant cell lines. In contrast, the 5-FU sensitivity of the parental line was unchanged following swainsonine treatment. N-glycan profiles of both the resistant and parent cells were changed by swainsonine treatment. This result shows that swainsonine rescues chemotherapeutic drugs from a range of resistance mechanisms. In all cell lines, the proportion of complex-type N-glycans was decreased from 80% to 20% Fig. , though Various gradations of 5-FU resistant cell lines acquired different mechanisms of 5-FU resistance; however, swainsonine was universally effective in increasing the sensitivity to 5-FU of all resistant cells. This result indicates that N-glycan biosynthesis is a better target for the prevention of 5-FU resistance, and points the way to the development of new chemotherapeutic strategies. To date, swainsonine has not been used as an anti-cancer drug, but this study presents a new strategy by which a low dosage of swainsonine can be used to recover the effectiveness of 5-FU or other chemotherapeutic reagents in tumors with acquired resistance.2 incubator at 37°C. To establish resistant lines, 5-FU  was added to the culture medium at an initial concentration of 25 ng/mL. Surviving cells were harvested and seeded onto another dish with 5-FU-containing media [Colon 26, a murine colorectal cancer cell line , was verng media . The con2 incubator at 37°C. A 50 μL suspension of 1.0 × 103 cells in culture medium was plated onto each well of a 96-well plate, and pre-incubated for 24 h. Then, 50 μL of medium including 5-FU was added and incubated for 70 h. Subsequently, 50 μg MTT  in PBS was added to a final concentration of 5 μg/mL and incubated for 2 h. After removal of the medium, cell membranes were permeabilized by the addition of 100 μL of dimethyl sulfoxide per well, and MTT formazan released from cells was measured by absorbance at 490 and 650 nm. Cytotoxicity was evaluated from the IC-50 value calculated using Origin 6.1J software .Drug resistance was measured using MTT assay . All cel4 cells/mL was added to 30 μL of the collagen solution, drops were hung in 6-well culture plates, and the plates were then placed in a 5% CO2 incubator at 37°C for 1 h to form a gel. Three ml of culture medium was added to each well. After preincubation for 24 h, 5-FU was added. After an additional 24 h, each well was washed twice with 4 mL of PBS by gentle shaking in the incubator for 10 min. After removal of the PBS, 4 mL of culture medium was added to each well, and cells were cultured for 6 days. Neutral red was then added to each well at 50 μg/mL. Cells were fixed 45 min later with 10% neutral-buffered formalin and washed with water for 10 minutes. Finally, collagen gel droplets were air-dried and quantified by Primage® (Nitta Gelatin) using an imaging apparatus.To verify drug resistance, we employed an alternative CD-DST method . A collaSwainsonine (Sigma) was added at 5 μg/mL to the medium when cells were harvested, and cells were incubated with the inhibitor for 5 days prior to the MTT assays, real-time RT-PCR, and glycoform analysis. Real-time RT-PCR was undertaken with the L0 and L0-1000, and the data were analyzed as the ratio of transcript levels derived from swainsonine-treated cells to those from untreated cells.6 cells of each line using CelLytic/Extraction Reagent (SIGMA), and 1.5 μg of protein, measured using the Bio-Rad DC protein assay kit , was loaded on a polyacrylamide gel  and subjected to SDS-PAGE. After blotting, PDVF membranes were blocked with a blocking buffer composed of PBS with 5% skim milk and 0.001% Tween. Membranes were then washed 3 times with a washing buffer consisting of PBS plus 0.001% Tween. OPRT and TS primary antibodies were diluted 1:500 (50 ng/mL) and 1:1000 (0.6 μg/mL) in PBS, respectively. After overnight incubation, membranes were washed 3 times with washing buffer and then incubated with anti-rabbit IgG antibody as the secondary antibody included in the ECL Plus Western blotting detection kit , according to the manufacturer's instructions. After the second antibody reaction, bands were detected using the ECL Plus Western blotting detection kit , according to the manufacturer's instructions.Antibodies against OPRT and TS were provided by Taiho Pharmaceutical Co., Ltd . Protein was extracted from 1 × 10Real time RT-PCR was performed using a LightCycler™  with a QuantiTect SYBR Green PCR Kit (Qiagen K.K.). Total RNA was isolated from each cell line with ISOGEN , following the manufacturer's directions. Five μg of total RNA was used with ReverTra Ace  for reverse transcription. The reaction mixture included 50 ng total RNA. We assayed the expression of OPRT, DPD, TS, thymidine phosphorylase (TP) and UP, the murine homologue of human PyNPase. Primer sequences were as follows: β-actin sense 5'-GCTCTTTTCCAGCCTTCCTT-3' and antisense 5'-TCTCCTTCTGCATCCTGTCA-3'; DPD sense 5'-GACTGAAAGCTGATGGCACA-3' and antisense 5'-TGAATAGCGCTGCATACCTG-3'; OPRT sense 5'-TCCCGAGTAAGCATGAAACC-3' and antisense 5'-TTAGCCGCTGCAAGTATTCC-3'; TS sense 5'-TCTGCTCACAACCAAACGAG-3' and antisense 5'-GCAGAAAATCCCAAGCTGTC-3'; UP sense 5'-TGCTCCAACATCACCATCAT-3' and antisense 5'-ACTGCACCAACTCCTGAACC-3'; and TP sense 5'-TGTTAAGTTTGGGGGAGCTG-3' and antisense 5'-TCTTCCACCTCCAGGGTATG-3'.Real-time RT-PCR cycles started with 15 min at 95°C and then 50 cycles of 15 sec at 94°C, 20 sec at 56°C and 20 sec at 72°C for β-actin, OPRT, TS and UP. Expression levels of each enzyme were expressed as ratios relative to β-actin expression and compared among cell lines using the L0 parental line as a control.6 cells) were suspended in 0.1 M Tris-HCl buffer  containing 200 μg each of trypsin and chymotrypsin , incubated for 24 h at 37°C, and then heated at 90°C for 10 min to stop enzymatic reactions. N-glycans were released from glycopeptide-containing digests by treatment with N-glycosidase F  for 24 h at 37°C in the same solution. Finally, pronase  was added to the crude mixture and incubated for 24 h at 37°C. Oligosaccharides were purified on a Bio-Gel P-4 column  with water as the eluant, and sugar-containing fractions were collected and lyophilized. Oligosaccharides were reductively aminated with 2-aminopyridine in the presence of sodium cyanoborohydride (Sigma-Aldrich Co.), according to the method of Hase et al. [Heated and lyophilized cells . Each oligosaccharide fraction separated by ODS liquid chromatography was subsequently subjected to analysis on an amide-adsorption column . Products in some peaks were digested with exo-glycosidases and further analyzed using HPLC. As a result, their structures were derived by matching the elution position with the database ,49.CD-DST- Collagen gel droplet-embedded culture drug sensitivity test;DPD- Dihydropyrimidine dehydrogenase;5-FU- 5-fluorouracil;Fuc- Fucose;Gal- Galactose;Glc- Glucose;GlcNAc- N-acetylglucosamine;HPLC- High-performance liquid chromatography;IC-50- 50% inhibition concentration;Man- Mannose; MTT- Mossman's 3--2,5-diphenyl-2H-tetrazolium bromide;ODS- Octadecylsilyl;OPRT- Orotic acid phosphoribosyltransferase;PA- Pyridylaminated;PyNPase- Pyrimidine nucleoside phosphorylase;TP- Thymidine phosphorylase;TS- Thymidylate synthase;UP- Uridine phosphorylase.


Multidrug resistance (MDR) to anti-cancer agents is frequently associated with overexpression of the drug efflux transporter P-glycoprotein (Pgp) in cancer cells, ensuing drug expulsion and maintenance of tolerable intracellular levels of certain cytotoxic drugs. Pgp may also be present in normal tissue, providing protection against toxic substances, but the physiological role of Pgp is not fully understood. Recently, it was shown that Pgp also takes part in the transport of certain growth-regulating cytokines . Therefore, we studied the effect of the highly potent Pgp inhibitor PSC 833 on proliferation of three pairs of MDR and parental human cell lines . The MDR phenotypes were characterized by Pgp overexpression, which was demonstrated by flow cytometry using the anti-Pgp antibody MRK16. Electronic cell counting of 72-96 h cultures revealed a dose-dependent antiproliferative effect of PSC 833 in the resistant KG1a/200 and K562/150 cells. The half-maximal growth inhibitory concentrations (GI50) were 0.2 microM and 0.7 microM respectively. Exposure to PSC 833 induced cell death by apoptosis in both cell types, as revealed by flow cytometry and detection of 3'-hydroxy ends of DNA (the result of DNA fragmentation associated with apoptosis), by terminal transferase-mediated dUTP-biotin nick end-labelling (TUNEL). Similar effects were not found in the hepatoma cell lines or the parental leukaemia lines. These results demonstrated a discriminating cytotoxicity of PSC 833 in two human leukaemia MDR variants, representing a possible therapeutic indication which warrants consideration during the ongoing clinical evaluation of this drug.


A panel of doxorubicin-resistant sublines of the human small-cell lung carcinoma cell line GLC4 displays decreasing DNA topoisomerase II alpha  mRNA levels with increasing resistance. In the present study we describe how this decrease may be regulated. No significant differences in TopoII alpha mRNA stability or gene arrangement were found, using mRNA slot-blotting and Southern blotting, in the most resistant cell line compared with the parental cell line. To investigate if TopoII alpha gene copy loss contributed to the mRNA decrease, fluorescence in situ hybridisation using a TopoII alpha-specific probe was performed. During doxorubicin resistance development, the composition of the population in each cell line shifted with increasing resistance, from a population in which most cells contain three TopoII alpha gene copies (GLC4) to a population in which most cells contain only two copies. A partial revertant of the most resistant cell line displayed a shift back to the original situation. We conclude that the TopoII alpha gene copy number decrease per cell line is in good agreement with the decreased TopoII alpha mRNA and protein levels, and TopoII activity levels in these cell lines which were described previously.


S1 cells displayed the highest sensitivity to cisplatin cytotoxicity, followed by S3 and distal convuluted tubule (DCT) cells. These differences in cytotoxicity did not correlate with differences in cisplatin uptake. Cytotoxic concentrations of cisplatin triggered apoptosis in all three cell lines. Although BAX and BCL-2 expression was similar among the three cell lines, the expression of the anti-apoptotic protein, BCL-XL, was significantly lower in S1 cells than in S3 and DCT cells, and this may have contributed to the heightened sensitivity of S1 cells. Cisplatin transport characteristics demonstrated a saturable component of cisplatin uptake and differences in apparent KM and Vmax values among the three cell lines. The three cell lines were 43- to 176-fold more sensitive to cisplatin than to carboplatin. This distinction between the two drugs could not be fully explained by differences in the uptake rates of carboplatin and cisplatin. We conclude that cells from different portions of the nephron display different sensitivities to cisplatin, different transport characteristics for cisplatin and different levels of expression of BCL-XL. In addition, the relative resistance of renal cells to carboplatin vs cisplatin is mostly due to the differential effects that follow internalization. © 1999 Cancer Research CampaignPathological studies suggest that cisplatin injures different portions of the nephron to different extents. To investigate this issue further, we examined the cytotoxicity and uptake of cisplatin in cell lines derived from S


Cyclooxygenase (COX)-2 has been implicated in tumour progression, angiogenesis and metastasis in non-small cell lung cancer (NSCLC). We speculated that inhibition of COX-2 activity might reduce expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in lung cancer cells.2 (PGE2) were measured by ELISA. Expression of COX-1 and COX-2 was determined by Western blotting. Inhibition or knockdown of COX-2 was achieved by treating NSCLC cells with specific COX-2 inhibitor NS-398 or COX-2 siRNA, respectively.The levels of IL-8, VEGF and prostaglandin E2 was significantly higher in NSCLC cell lines than SCLC cell lines (p < 0.001). We tested these cell lines with a potent specific COX-2 inhibitor NS-398 at concentrations of 0.02, 0.2, 2, 20 μM for 24 or 48 h. The COX-2 activity was reduced in a dose-dependent fashion as shown by reduced PGE2 production. VEGF was significantly reduced following the treatment of NS-398 in A549 (by 31%) and MOR/P (by 47%) cells lines which expressing strong COX-2, but not in H460 cell line which expressing very low COX-2. However, IL-8 was not reduced in these cell lines. To confirm these results, we knocked down COX-2 expression with COX-2 siRNA in these cell lines. VEGF was significantly decreased in A549 (by 24%) and in MOR/P (by 53%), but not in H460 whereas IL-8 was not affected in any cell line.We found that NSCLC cell lines produced more IL-8 than VEGF (p < 0.001). In contrast, small cell lung cancer (SCLC) cell lines produced more VEGF than IL-8 (p < 0.001). COX-1 was expressed in all cell lines, but COX-2 was expressed only in NSCLC cell lines. Consistent with this, PGEWe conclude that NSCLC cells produce much higher levels of IL-8 than SCLC cells whereas both NSCLC and SCLC cells produce similar levels of VEGF. COX-2 is only expressed in NSCLC cells, but not in SCLC cells. VEGF is produced in both NSCLC and SCLC cells regardless of COX-2 expression. However, VEGF production is, at least partly, COX-2 dependent in NSCLC cells expressing COX-2. In contrast, IL-8 production is COX-2 independent in both NSCLC and SCLC cells. We speculate that combined targeting of COX-2 and IL-8 may be useful in the treatment of patients with NSCLC and targeting VEGF may be useful in the treatment of patients with SCLC. Lung cancer remains the leading cause of cancer death in many countries worldwide. There is considerable interest in anti-angiogenic drugs as therapeutic agents for lung cancer. Angiogenesis in tumours is promoted through the secretion of a variety of pro-angiogenic factors. Among these, vascular endothelial growth factor (VEGF) is important in many tumour types due to both its potent activity and markedly elevated expression level. Several studies have shown that high levels of VEGF are associated with increased tumour vascularity, advanced stage and poor prognosis in patients with non-small cell (NSCLC) and small cell lung cancer (SCLC) -5. In ad+ CXC family of chemokines, is another potent pro-angiogenic factor and its expression is associated with angiogenesis, tumour progression and survival in patients with NSCLC  were GGACUUAUGGGUAAUGUUAUU (duplex 6); GAUAAUUGAUGGAGAGAUGUU (duplex 7); GUGAAACUCUGGCUAGACAUU (duplex 8) and CGAAAUGCAAUUAUGAGUUUU (duplex 9). The siRNAs were transfected into the cells in 12-well plate using DharmaFECT1 transfection reagent according to manufacturer's instructions. Four hours after transfection, the media was replaced by fresh media with 10% FCS. After further 48 h, the media were collected for ELISA and cells were collected for protein extraction.On-target COX-2 plus SMARTpool (COX-2 siRNA) and SiCONTROL Non-Targeting siRNA#1 (control siRNA) were purchased from Dharmacon. The sequences of On-target COX-2 plus SMARTpool (COX-2 siRNA) [GenBank: The results were expressed as mean ± standard error of the mean (SEM). Statistical significance was obtained using unpaired student's t-test. Each experiment was repeated at least 3 times. Values with p < 0.05 were considered significant.6cells) than SCLC cell lines (under 0.9 ng/ml/106cells). However, there was no significant difference in VEGF levels between NSCLC (ranging from 3.7 to 6.3 ng/ml/106cells) and SCLC (ranging from 4.0 to 11.2 ng/ml/106cells) cell lines.IL-8 and VEGF were detected by ELISA in conditioned medium after 48 h incubation in a panel of lung cancer cell lines Figure . In all 2 production was measured by competitive ELISA. NSCLC cell lines produced significantly higher levels of PGE2(mean 1001 pg/ml/106cells) than SCLC cell lines   Figure .2 production by ELISA and found that it was reduced in a dose-dependent fashion by NS-398 at 48 h treatment in A549. PGE2 was reduced to 390 pg/ml (p < 0.05) by NS-398 at 2 μM and to 105 pg/ml (p < 0.01) by NS-398 at 20 μM from 937 pg/ml, respectively  reduced by NS-398 (20 μM) at 24 h in all three tested cell lines  of NS-398 for 24 or 48 h. As expected, NS398 had no effect in SCLC cell lines, which do not express COX-2 (data not shown). However, VEGF production was significantly reduced by NS-398 at 20 μM in A549  and MOR/P  cells although VEGF expression was not changed in H460 cells. VEGF was slightly reduced by NS-398 at 2 μM in A549  and MOR/P . In contrast, IL-8 production was not reduced by NS-398 in these NSCLC cell lines Figure  and 4C.To test whether sublethal concentrations of NS-398 used in this study inhibited cell proliferation, we counted cell number using Trypan Blue stain and the Hemocytometer after lung cancer cells were treated with NS-398 for 24 h. There was no significant inhibition of cell growth in any of the 3 NSCLC cell lines at concentrations up to 20 μM of NS-398 Figure .To further confirm the direct involvement of COX-2 on expression of these factors, we treated NSCLC cell lines with COX-2 siRNA for 48 h. The endogenous COX-2 was markedly knocked down by COX-2 siRNA in all tested cell lines Figure . ConsistSeveral studies have shown that COX-2, VEGF and IL-8 are overexpressed in lung cancer compared to normal bronchial epithelium. Most studies have focused on NSCLC since clinical specimens of SCLC are difficult to obtain for research because SCLC patients are rarely operated on. Tas et al ,24 recen2 was determined in three NSCLC and five SCLC cell lines. VEGF was produced at similar levels in all cell lines. NSCLC cell lines produced much more IL-8 than SCLC cell lines. COX-1 was expressed at similar levels in all cell lines, whereas COX-2 was only expressed in NSCLC cell lines, albeit at low levels in H460. Interestingly, high levels of PGE2 were not only produced in MOR/P and A549 cell lines as a result of strong COX-2 expression, but also in H460 cell line in which COX-2 expression was weak. This could result from reduced catabolism of PGE2 due to low levels of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a primary enzyme responsible for PGE2 metabolism [Here, we have compared and contrasted SCLC and NSCLC using a panel of cell lines. Basal expression of VEGF, IL-8, COX-1, COX-2 and PGEtabolism . Previoutabolism ,26. Howetabolism . These dtabolism , appear 2 in these cell lines. We decided to use a maximum concentration of 20 μM in this study because COX-2 inhibition had been demonstrated and cell growth was not significantly inhibited in NSCLC cell lines as shown in Figure COX-2 expression has previously been associated with VEGF in patients with NSCLC ,20. Two Our results suggest that NSCLC cells produce much more IL-8 than SCLC cells whereas both NSCLC and SCLC cells produce similar levels of VEGF. COX-2 is only expressed in NSCLC cells, but not in SCLC cells. VEGF can be produced in lung cancer cells regardless of COX-2 expression. However, VEGF production is, at least partly, COX-2 dependent in NSCLC cells expressing COX-2. In contrast, IL-8 production is COX-2 independent in both NSCLC and SCLC cells. We speculate that combined targeting of COX-2 and IL-8 may be useful in the treatment of patients with NSCLC and targeting VEGF may be useful in the treatment of patients with SCLC.The authors declare that they have no competing interests.YMZ designed all experiments, performed some experiments and wrote the manuscript. NSMA and DC–WY performed majority of experiments. PJW contributed to experimental design and manuscript writing. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


We have investigated the effects of FGF-1 and FGF-2 on the behaviour and adhesion properties of human pancreatic adenocarcinoma cell lines  that were previously characterised for the expression of FGFRs. Here we show that exposure to FGF-1 and FGF-2 leads to significant and dose-dependent increase in E-cadherin-dependent cell-cell adhesion, tubular differentiation, and a reduced capacity to invade collagen gels. FGF stimulation produces phosphorylation of E-cadherin and β-catenin on tyrosine residues, as well as increased E-cadherin localisation to the cytoplasmic membrane and association with FGFR1 demonstrable by coimmunoprecipitation. These results demonstrate that FGF-1 and FGF-2 may be involved in the regulation of cell adhesion, differentiation and invasion of pancreatic cancer. © Cancer Research Campaign


Corresponding values for pamidronate were 40, 35 and 25 μM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research CampaignBreast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines . Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC


Metastatic testicular germ cell tumours are cured in approximately 85% of patients using cisplatin-based combination chemotherapy. Patients who fail to respond have a poor prognosis, and there is a need for more effective treatments for cisplatin-resistant disease. In this study, it is shown that two of four cell lines derived from human non-seminomatous testicular germ cell tumours are exceptionally sensitive to temozolomide, a new imidazotetrazine which can cross the blood-brain barrier in mice. In addition, three pairs of cisplatin-resistant sublines show little cross-resistance to temozolomide. These data suggest that temozolomide might have activity against non-seminomatous testicular germ cell tumours which have relapsed following cisplatin-containing chemotherapy, and could have a role in the treatment of patients with metastatic lesions in the brain.


In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour.


BCL10 gene in 81 primary prostate carcinomas, 20 squamous cell cancers of the head and neck, 15 small-cell lung cancer cell lines, 24 renal carcinoma cell lines and 13 sarcoma cell lines. We failed to find evidence of somatically acquired mutations of the BCL10 gene suggesting that BCL10 does not play a major role in the development of these malignancies. © 1999 Cancer Research CampaignWe have used single-strand conformation polymorphism (SSCP) analysis to screen for mutations in the


Br J Cancer72: 1144–1150) and with the sensitivity of all these cell types to eight unrelated cancer chemotherapeutic agents with diverse modes of action. Our results demonstrate that P450R plays a significant role in the activation of TPZ in this panel of lung lines, which is consistent with previous observations in a panel of breast cancer cell lines  Br J Cancer72: 1144–1150; Patterson et al (1997) Br J Cancer76: 1338–1347). However, in the lung lines it is likely that it is the inherent ability of these cells to respond to multiple forms of DNA damage, including that arising from P450R-dependent TPZ metabolism, that underlies the ultimate expression of toxicity. © 1999 Cancer Research CampaignThe bioreductive drug tirapazamine  is a lead compound in a series of potent cytotoxins that selectively kill hypoxic rodent and human solid tumour cells in vitro and in vivo. Phases II and III trials have demonstrated its efficacy in combination with both fractionated radiotherapy and some chemotherapy. We have evaluated the generality of an enzyme-directed approach to TPZ toxicity by examining the importance of the one-electron reducing enzyme NADPH:cytochrome P450 reductase (P450R) in the metabolism and toxicity of this lead prodrug in a panel of seven human non-small-cell lung cancer cell lines. We relate our findings on TPZ sensitivity in these lung lines with our previously published results on TPZ sensitivity in six human breast cancer cell lines


The human melanoma cell lines MM96L, A2058 and HT144 were examined for sensitivity to ionizing radiation and UVB radiation. HT144 demonstrated a significant increase in sensitivity to ionizing and UVB radiation compared with the MM96L and A2058 cells. Sensitivity to both agents was associated with susceptibility to apoptosis. Using a protein truncation assay, a mutation for the gene for ataxia telangiectasia (ATM) was identified in HT144 cells. This was confirmed to be a homozygous mutation by subsequent sequencing of the abnormal region. Protein truncation assay of the other two cell lines showed no abnormality. The results suggest that somatic mutation of the A-T gene may be important in determining tumour radiosensitivity.


Curves of growth delay (GD) or 'cure' after graded doses of radiation have been analysed for 16 lines of human and animal tumours grown as multicellular spheroids in vitro. Dose-survival curves were derived for those cellular units from which spheroids regrow after unsuccessful irradiation . For 10 sets of data from 6 spheroid lines, the Do's and extrapolation numbers of the SRU derived by GD could be compared with the response of the clonogenic cells of the spheroids. For Do, a good correlation (r = 0.910) was found between the two; this was true also for Do derived from curves of spheroid 'cure' (7 sets of data from 6 spheroid lines) and clonogenic cells (r = 0.986). Using GD, the correlation of extrapolation numbers was less good (r = 0.682), the values for SRU commonly being higher than those for clonogenic cells. This may reflect features of the growth curves of spheroids after the lower range of doses of radiation. For human and animal tumour spheroids of 250 microns or less, derived Do ranged from 0.5 to 2.5 Gy. For spheroids of 350 microns or more, derived Do for animal tumour lines ranged from 3.4 to 4.2 Gy, for human lines from 1.5 to 2.1 Gy.


Oncological results of radiotherapy for locally advanced prostate cancer (PC) are significantly improved by simultaneous application of LHRH analoga (e.g. goserelin). As 85% of PC express LHRH receptors, we investigated the interaction of goserelin incubation with radiotherapy under androgen-deprived conditions in vitro.LNCaP and PC-3 cells were stained for LHRH receptors. Downstream the LHRH receptor, changes in protein expression of c-fos, phosphorylated p38 and phosphorylated ERK1/2 were analyzed by means of Western blotting after incubation with goserelin and irradiation with 4 Gy. Both cell lines were incubated with different concentrations of goserelin in hormone-free medium. 12 h later cells were irradiated (0 – 4 Gy) and after 12 h goserelin was withdrawn. Endpoints were clonogenic survival and cell viability .Both tested cell lines expressed LHRH-receptors. Changes in protein expression demonstrated the functional activity of goserelin in the tested cell lines. Neither in LNCaP nor in PC-3 any significant effects of additional goserelin incubation on clonogenic survival or cell viability for all tested concentrations in comparison to radiation alone were seen.The clinically observed increase in tumor control after combination of goserelin with radiotherapy in PC cannot be attributed to an increase in radiosensitivity of PC cells by goserelin in vitro. Luteinising hormone releasing hormone (LHRH) analoga play an important role in the treatment of prostate carcinoma (PC). As an alternative to surgical castration to suppress testosterone levels they are used in the palliative treatment of advanced disease. Furthermore, in locally advanced disease they improve overall survival when given simultaneously to curative radiotherapy -3.The mechanism of this enhancement of survival is still obscure. Interestingly, several older trials that compared radiotherapy with radiotherapy and surgical castration or estrogen application did not show an improvement in survival -7 exceptIn vitro studies that investigated oncological relevant endpoints like clonogenic survival could not demonstrate an enhanced radiosensitivity of PC-cell lines by testosterone ablation . In LNCaP cells AD led to growth delay. This delay could be abolished by incubation with synthetic androgens. No supraadditive effects on clonogenic survival were observed, when AD was combined with irradiation. In these experimental settings androgens were withdrawn in varying protocols 3 to 5 days before irradiation. After irradiation the cells were subjected to "immediate plating" for methodological reasons. This means that the monolayer had to be trypsinized, counted and seated before colony formation took place. In contrast, we tested much shorter goserelin incubation times. This allowed us to investigate the direct interaction between goserelin and radiotherapy. We were not interested in long-term exposure of goserelin before or after irradiation. Furthermore, as we avoided "immediate plating", we could exclude possible errors caused by this methodology.Recent studies investigated the effects of AD and radiotherapy in PC cell lines in vivo. Subcutaneous tumors were grown and AD was performed by means of surgical castration in male mice. Several studies demonstrated a significant reduction of the TCD50 by the combination therapy depending on the timing of AD [Other studies investigated the interaction between AD and radiotherapy ng of AD -13.Taken together in vivo and in vitro data support the hypothesis, that androgen withdrawal during irradiation increases clonogenic cell death in an additive manner . FurtherWe chose to test goserelin concentrations between 0.01 – 10 μM. These concentrations showed significant effects on proliferation of PC-cells, receptor binding and other endpoints in vitro ,23. In pOne proposed mechanism of biological activity of LHRH in prostate carcinoma is that LHRH signalling involves MAPK-kinases. Among others, LHRH receptors trigger PLC to activate PKC. PKC activation limits EGFR tyrosine kinase activity by phophorylating EGFR at threonine 654 .We studied the effects of goserelin incubation on two main MAPK-pathways (phos. p38 and phosphor ERK1/2) and on c-fos 24 h after start of incubation, 12 h after irradiation (to reflect long term effects). In this setting, we could demonstrate biological effects of goserelin on protein expression and phosphorylation. Goserelin incubation was functionally active in our cell systems.The observation, that the incubation with goserelin induced the c-fos expression in LNCaP cells is in contrast to other reports. Dondi et al. incubated LNCaP cells with 10 μM LHRH agonist for 30 or 60 min, than added EGF to the media during the last 30 min of incubation . The expNoticeable was the minor influence of irradiation and the combination of goserelin incubation and irradiation on cell viability. One limitation of this method was the measurement of exponentially growing cultures. Minor inaccuracies in cell plating at the start of each assay aggravated over time, resulting in substantial differences in cell numbers at the time of measuring cell viability. This led to standard deviations of up to 40%. A further obstacle was, that metabolic activity assays seldom reflect cell cytotoxicity from RT in solid malignancies. We used this assay to investigate for growth arrest and apoptosis induced by the two treatment modalities. However, directly after irradiation cell numbers will not be reduced in these solid tumor cell lines. The induction of apoptosis has been investigated in LNCaP and PC-3 cells grown in standard medium by several groups. With different methodological approaches only a minor induction of apoptosis was seen 24 h after irradiation with 4 – 5 Gy (< 5% more than in sham-irradiated controls) -30. One Taken together, our cell viability data are easily explained by published results of other studies.in vitro. The clinically observed increase in tumor control after combination of goserelin with radiotherapy in PC cannot be attributed to an increase in radiosensitivity of PC cells by goserelin in vitro. It is to be explained by the synergistic effects of AD and irradiation.We could not demonstrate any significant effects of goserelin on irradiated PC cell lines antagonists (e.g. cetrurelix) and radiotherapy are still missing, we suppose no detrimental effects on tumor control, as the incubation with LHRH receptor-agonists proved not to increase radiosensitivity of PC cell lines.Although studies on the combinatory effects of LHRH receptor-Potential conflicts of interest exist: Astra-Zeneca granted 20 mg of goserelin to support our study . R.M.H. received honoraria for lecturer activities by Astra-Zeneca under 1000,- €. The authors assure that the acquisition and interpretation of laboratory data were not influenced by the donation and the honoraria.RMH designed the study, coordinated the work and drafted the manuscriptDS did the cytological work and helped with irradiation testsSF and CG designed the functional studies and performed receptor analysis and the Western blotsMRF coordinated the work, interpreted the data and helped drafting the manuscriptMN and AH did the irradiations and performed statistical analysisPT and HS participated in the study design and interpretation of the dataHC conceived the study, and participated in its design and coordination and helped to draft the manuscriptAll authors read and approved the final manuscript.


As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cell-enriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17beta-hydroxysteroid dehydrogenase type I. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines.


Invasion, the ability of an epithelial cancer cell to detach from and move through a basement membrane, is a central process in tumour metastasis. Two components of invasion are proteolysis of extracellular matrix and cellular movement through it. A potential promoter of these two processes is thrombin, the serine proteinase derived from the ubiquitous plasma protein prothrombin. Thrombin promotes the invasion of MDA-MB231 breast tumour cells (a highly aggressive cell line) in an in vitro assay. Invasion by MDA-MB436 and MCF-7 cells, less aggressive cell lines, is not promoted by thrombin. Thrombin, added to the cells, is a stimulator of cellular movement; fibroblast-conditioned medium is the chemotaxin. Thrombin-promoted invasion is inhibited by hirudin. Stimulation of invasion is a receptor-mediated process that is mimicked by a thrombin receptor-activating peptide. Thrombin has no effect on chemotaxis in vitro. Thrombin receptor is detectable on the surface of MDA-MB231 cells, but not on the other two cell lines. Introduction of oestrogen receptors into MDA-MB231 cells by transfection with pHEO had no effect on thrombin receptor expression, in the presence or absence of oestradiol. This paper demonstrates that thrombin increases invasion by the aggressive breast cancer cell line MDA-MB231 by a thrombin receptor-dependent mechanism. © 1999 Cancer Research Campaign


Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA) enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration.Seven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM), or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2.There was a high correlation of cell growth with ATP concentration (r = 0.948) in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p < 0.05).Seven human cancer cell lines grown in glucose plus acetoacetate medium showed tightly coupled reduction of growth and ATP concentration. The findings were not observed in control fibroblasts. The observed over-expression of UCP2 in cancer lines, but not in controls, provides a plausible molecular mechanism by which acetoacetate spares normal cells but suppresses growth in cancer lines. The results bear on the hypothesized potential for ketogenic diets as therapeutic strategies. One mitochondrial adaptation to increased ROS is over-expression of uncoupling protein 2 (UCP2) which has been reported in a number of human cancer cell lines . Un,9. Un8,9most colon cancer lines, the degree of over-expression correlating with the histologic grade. It was proposed that UCP2 over-expression was an adaptive mechanism that reduced ROS-induced apoptosis among the cancer cells.An increase in UCP2 has been reported to be associated with reduction of respiratory ATP production in several cancer lines  that havWe suggest that ketone bodies, if metabolized in cancers of this type, can inhibit glycoloysis and resultant ATP generation without compensatory mitochondrial respiratory ATP production, thereby inhibiting cell growth.et al. pointed out that the Randle cycle has only been persuasively demonstrated in in vitro preparations under conditions of euglycemic, hyperinsulinemic clamp, in which intracellular glucose availability remains high and fixed. Under these circumstances, in rat myocardium preparations, for example, variable fatty acid availability does indeed regulate the rate of glycolysis.The Randle cycle was proposed more than 30 years ago as a mechanism whereby products of lipolysis (fatty acids and ketone bodies) would inhibit glycolysis in normal cells to maintain stable ATP production . Accordiin vivo for most aggressive cancer cells whose membranes express high affinity insulin-independent glucose 1 transporters (GLUT1) [Interestingly, high fixed intracellular glucose availability is a likely condition  (GLUT1) . The Km et al. reported MAC16 tumor growth inhibition in mice on ketogenic diets [Evidence for such an inefficient Randle cycle is found in the seven cancer lines of our investigation which demonstrated parallel reductions in ATP concentration and cell growth, the greatest inhibition found in CaCO2 and SW48 lines, and the least in MCF7 Figure . In addiic diets  but failic diets . The latin vivo study to predict effects under physiologic conditions. Third, in vitro culture of primary cell lines is difficult for normal human cell types, and only fibroblasts were used as controls, so the response of other normal tissue types was not measured. Further, fibroblast lines capable of growth in cell culture are not normal; however, neither are they the equivalent to malignant cell lines and their difference from malignant lines was demonstrated in this investigation. Also, the range of tumor susceptibility to acetoacetate varies widely, plausibly due to increased levels of UCP2 expression, but other factors must be considered. For example SW48, with the greatest reduction of ATP and cell growth in acetoacetate medium, was a prominent outlier with the least UCP2 expression among the cancer cell lines (Figure There are limitations in the present study. The investigation does not prove metabolic inhibition of cancer growth and ATP production due to uncoupling. Proof requires direct demonstrations of acetoacetate metabolism as well as inhibition of the rate of ATP production, not just of steady state ATP concentrations. Second, glucose and acetoacetate were both incubated at supraphysiologic concentrations to maximize detectable effects. It is not possible without further s Figure . FinallyCancers are notorious for their ability to evade metabolic regulation  and therOn the other hand the initiation of a primary event transforming a normal cell into a pre-malignant phenotype is followed by a long period of subsequent adaptive cellular response to a local hypoxic, nutrient-deprived microenvironment. During this interval of abnormal cellular growth multiple adaptations eventually result in the cancer phenotype. A typicWe have shown that acetoacetate added to glucose medium causes variable but parallel reductions of ATP concentration and cell growth in seven human cancer cell lines. The effect lies along a continuum with control fibroblasts at one extreme in which neither cell growth is inhibited nor is ATP concentration reduced. The effect is consistent with an "inefficient" Randle cycle due to over-expression of uncoupling protein 2, seen only in the cancer lines, and is plausible in light of current models of a transformed cell's adaptive microscopic development and progression toward the cancer phenotype. More work must be done to prove metabolic inhibition by this mechanism. If proven, the results speak to the hypothesized potential for ketogenic diets as non-toxic therapeutic strategies, as adjuvants to standard therapies, and to multimodality therapies to improve the control of malignant disease.The authors have no conflicting or competing interests to declare in the conception, evolution, or execution of this research.EJF conceived the study, directed its primary design and coordination, performed much of the data analysis, and drafted and revised the manuscript; AM grew and counted the cell cultures, performed the ATP assays, performed substantial data analysis and participated in writing the Methods section of the manuscript; EVQ coordinated execution of the study, contributed to important aspects of the research direction, and made important revisions to the manuscript; JMS performed the UCP2 assay and wrote the methods for this aspect of the work; RDF coordinated execution of the study, contributed to important aspects of the research direction, and made important revisions to the manuscript. All authors read and approved the final manuscript.


The growth inhibitory effects of interferons, IFN-alpha and IFN-gamma on human lung cancer cell lines were studied using both a tetrazolium (MTT) colorimetric assay and direct cell counting. Significant discrepancies between the two assays were observed, the MTT assay consistently underestimating the growth inhibitory effects of the IFNs. There was no direct chemical effect of the IFNs on the tetrazolium reduction process. IFN treated cells showed increased cell size compared with control cells, although there was little or no change in cell cycle distribution. Mitochondrial activity was 30-50% greater in IFN-gamma treated cells (COR-L23) than the controls. Reduced formazan production per cell was observed in medium which had supported cell growth for several days. Differential 'medium conditioning' led to a difference in formazan production per cell between IFN and control cells and this was the major basis of the observed discrepancy. This discrepancy was not due to the differences in the glucose concentrations between these media. However, differences in pH between the media proved to be the major contributory factor of the discrepancy.


Purpose: Insulin receptor substrates (IRSs) are essential for insulin-induced mitogenic effects on several cell types but theyalso are involved in cell transformation.We investigated whether the differential constitutive expression and potential distinctdownstream signaling events of IRS-1 and IRS-2 might be related to discrete tumourigenic phenotypes of three humanuterine leiomyosarcoma cell lines, one of which was specifically isolated for the present study.            Methods and results: SK-UT-1B egressed effectively from a gellyfied Matrigel matrix and grew as did DMR cells in ananchorage-independent manner in agar and induced rapidly growing tumours in nude mice. On the contrary, SK-LMS-1cells did not emigrate from Matrigel, neither grew in agar nor were they tumourigenic. IRS-2 was highly expressed in themore malignant cell lines, whereas IRS-1 was present only in SK-LMS-1 cells. However, upon insulin stimulation both IRS-1 and IRS-2 were tyrosine phosphorylated with a similar kinetic in the respective cell lines; furthermore, after 1 min ofinsulin stimulation PI3-kinase associated with IRSs and after 2 min Shc was phosphorylated and associated with Grb2 withminor differences detectable among the various cell lines in the duration of phosphorylation and/or in their association irrespectiveof whether IRS-1 or IRS-2 were expressed.            Discussion: Our findings tend to exclude that the malignancy displayed by uterine leiomyosarcomas might be directly linkedto the activation of distinct IRS-1- or IRS-2-dependent pathways.


The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40  and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3.


Immunohistochemical studies disclosed significantly stronger staining for TF in cells from NSCLC patients with metastasis than in those without metastasis. Among the 28 patients with metastasis, ten were strongly positive, 16 were moderately positive and two were negative for TF. In contrast, among the 27 patients without metastasis, only two were strongly positive, 18 were moderately positive and seven were negative for TF. Therefore, malignant cells from patients with lung cancer produce various levels of TF, and TF may play an important role in the metastatic process. © 1999 Cancer Research CampaignTissue factor (TF) is an initiator of the extrinsic cascade of blood coagulation. Although recent studies have revealed a relationship between metastatic properties and TF expression in some neoplastic cells, the significance of TF in lung cancer, especially in non-small-cell lung cancer (NSCLC), is still unclear. In this study, TF was detected in NSCLC cell lines by functional study, Western blot analysis and immunocytochemical staining. TF levels in eight NSCLC cell lines were also quantitated by enzyme-linked immunosorbent assay (ELISA), and TF expression was evaluated in 55 specimens of surgically resected NSCLCs. NSCLC cell lines derived from metastatic lesions produced high levels of TF (48.3 ± 23.5 ng 10


The effects of combinations of dipyridamole, an effective blocker of the salvage pathway of DNA synthesis, and 8 types of anti-cancer drugs on the growth of human T, B and myeloid leukaemia/lymphoma cell lines in vitro were examined. In combinations, dipyridamole and vincristine (VCR), and dipyridamole and vindesine had synergistic inhibitory effects. Dipyridamole reduced the efflux of VCR from cells and enhanced their VCR accumulation in a dose-dependent manner at concentrations of up to 10 microM in the lymphoid cell lines, MOLT-3 and BL-TH, and of up to at least 20 microM in the myeloid cell line, ML-1. Dipyridamole also enhanced the accumulation of VCR in PHA-stimulated and un-stimulated lymphocytes of normal donors, but efflux of VCR was more rapid from normal lymphocytes than from cultured cell lines. It is proposed that combination therapy with dipyridamole plus VCR should be effective in the treatment of leukaemia and lymphoma.


INK4a. Altered expression of retinoblastoma protein (pRb) is associated with non-Hodgkin's lymphoma, particularly centroblastic and Burkitt's lymphomas. pRb is expressed in normal B-cells and its regulatory phosphorylation pathway is activated in response to a variety of stimuli. Since human B-lymphoma-derived cell lines are often used as in vitro model systems to analyse the downstream effects of signal transduction, we examined the functional status of pRb in a panel of human B-cell lines. We identified eleven cell lines which express the hyperphosphorylated forms of pRb. Furthermore, we suggest that the pRb protein appears to be functional in these cell lines. © 1999 Cancer Research CampaignHuman cancers frequently sustain genetic mutations that alter the function of their G1 cell cycle control check point. These include changes to the retinoblastoma gene and to the genes that regulate its phosphorylation, such as the cyclin-dependent kinase inhibitor p16


Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp). The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells. We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells. Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest. Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues. Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase. These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies.


Cytotoxic activity of the T-cells was assessed against autologous B lymphoblastoid cells pulsed with each peptide. Stimulation by GLQKASIFLK generated specific cytotoxic T lymphocyte (CTL) lines from HLA-A2, A3 donors, HLA-A2 donors and HLA-A3 donors. Stimulation with KASIFLKTRV generated specific CTL lines from HLA-A2 donors. No HLA-A2−, A3− CTL line showed specific cytotoxicity against these target cells. These CTL lines were also cytotoxic against HLA-A2 and HLA-A3 breast cancer cells but not against normal fibroblastoid cell lines, normal epidermal cell lines, or a melanoma cell line. RBP-1 peptide antigens may be of clinical significance as a potential peptide vaccine against human breast cancer. © 1999 Cancer Research CampaignRetinoblastoma binding protein 1 (RBP-1) is a 143-kDa nuclear phosphoprotein that promotes cell growth by inhibiting the product of retinoblastoma tumour suppressor gene (pRB). We recently found that RBP-1 contains KASIFLK, a heptameric peptide (250–256) recognized by human antibodies and overexpressed by breast cancer cells. In the present study, we demonstrate that human T-cells stimulated with RBP-1 decameric peptides containing KASIFLK can kill human breast cancer cells. These decamers, GLQKASIFLK (247–256) and KASIFLKTRV (250–259), have anchor motifs for both HLA-A2 and HLA-A3. Peripheral blood lymphocytes from 41 normal donors were stimulated by these peptides in culture media containing 15 IU ml


Loss of viability following TS inhibition occurs as a consequence of depleted dTTP pools and at least in some cell lines, accumulation of dUTP and subsequent misincorporation of uracil into DNA. The expansion in dUTP pools is largely determined by the expression of the pyrophosphatase, dUTPase. Our previous work has shown that following TS inhibition the ability to accumulate dUTP was associated with an earlier growth inhibitory effect. 3 human lung tumour cell lines and HT29 human colon tumour cells transfected with dUTPase have been used to investigate the relationship between loss of viability following TS inhibition and dUTP accumulation. Cell cycle arrest typical of TS inhibition was an early event in all cell lines and occurred irrespective of the ability to accumulate dUTP or p53 function. However, a large expansion of dUTP pools was associated with mature DNA damage (4 h) and an earlier loss of viability following TS inhibition compared to cells in which dUTP pools were not expanded. In A549 cells damage to mature DNA may have been exacerbated by significantly higher activity of the excision repair enzyme, uracil-DNA glycosylase. Consistent with results using different inhibitors of TS, transfection of dUTPase into HT29 cells significantly reduced the cytotoxicity of a 24 h but not 48 h exposure to ZD9331. Although loss of viability can be mediated through dTTP deprivation alone, the uracil misincorporation pathway resulted in an earlier commitment to cell death. The relevance of this latter pathway in the clinical response to TS inhibitors deserves further investigation. © 2001 Cancer Research Campaign


We have established a novel ascites tumour model (MDA435/LCC6) from the oestrogen receptor-negative, invasive and metastatic MDA-MB-435 human breast cancer cell line. MDA435/LCC6 cells grow as both malignant ascites and solid tumours in vivo in nude mice and nude rats, with a tumour incidence of approximately 100%. Untreated mice develop ascites following i.p. inoculation of 1 x 10(6) cells and have a reproducible life span of approximately 30 days, with all animals dying within a 48 h period. The in vivo response of MDA435/LCC6 ascites to several cytotoxic drugs, including doxorubicin, etoposide (VP-16), BCNU and mitomycin C, closely reflects the activity of these single agents in previously untreated breast cancer patients. MDA435/LCC6 cells also retain the anchorage-dependent and anchorage-independent in vitro growth properties of the parental MDA-MB-435 cells, and can be used in standard in vitro drug screening assays. The drug resistance pattern of the MDA435/LCC6 cells suggests that they may have few active endogenous drug resistance mechanisms. To generate a model for the screening of MDR1-reversing agents, MDA435/LCC6 were transduced with a retroviral vector directing the constitutive expression of the MDR1 cDNA, producing a cell line with a classical MDR1 resistance pattern (MDA435/LCC6MDR1). THese ascites models may be a viable alternative to the murine leukaemia ascites  and, in conjunction with other breast cancer cell lines, facilitate the in vitro and in vivo screening of new cytotoxic drugs and drug combinations.