The CXCR4/CXCL12 chemokine axis may play a critical role in guiding CXCR4+ circulating malignant cells to organ specific locations that actively secrete its ligand CXCL12 (SDF-1) such as bone, brain, liver, and lungs. We sought to characterize the presence of the CXCR4/CXCL12 axis in five uveal melanoma (UM) cell lines in vitro. The ability of TN14003, a synthetic peptide inhibitor that targets the CXCR4 receptor complex, to inhibit this axis was also assessed.Immunocytochemistry was performed against CXCR4 to confirm expression of this chemokine receptor in all five UM cell lines. Flow cytometry was preformed to evaluate CXCR4 cell surface expression on all five UM cell lines. A proliferation assay was also used to test effects TN14003 would have on cellular proliferation. Inhibition of cellular migration by specifically inhibiting the CXCR4/CXCL12 axis with TN14003 was also investigated. The binding efficacy of TN14003 to the CXCR4 receptor was assessed through flow cytometric methods.The CXCR4 receptor was present on all five UM cell lines. All five cell lines expressed different relative levels of surface CXCR4. TN14003 did not affect the proliferation of the five cell lines (p > 0.05). All cell lines migrated towards the chemokine CXCL12 at a level greater than the negative control (p < 0.05). All 5 cell lines pre-incubated with TN14003 prevented cellular migration towards chemokine CXCL12 (p < 0.01). TN14003 preferentially binds CXCR4 to native ligand CXCL12.Interfering with the CXCR4/CXCL12 axis, using TN14003 was shown to effectively down regulate UM cell migration in vitro. Knowing that UM expresses the CXCR4 receptor, these CXCR4+ cells may be less likely to colonize distant organs that secrete the CXCL12 ligand, if treated with an inhibitor that binds CXCR4. Further studies should be pursued in order to test TN14003 efficacy in vivo. Uveal melanoma is the most common primary intra-ocular malignant tumor in adults. Despite advances in diagnosis and local treatment of patients over the last 30 years, the mortality rate has remained constant, with 30 to 50% of patients developing metastatic disease . Due to Metastatic disease is typically characterized by initial cell transformation within the primary tumor site followed by angiogenesis, facilitating nutrient delivery to the newly transformed cells. Later events include cellular proliferation and detachment from the local extracellular matrix, followed by cell migration and intravasation into the newly formed vessels . InteracIt is now well described and understood that chemo-attractive cytokines referred to as chemokines play a number of functional roles in tumor biology -8. ChemoThese low molecular weight signaling molecules regulate and direct circulating immune system cells to specific locations in the body. It has also recently been discovered that cancer cells, themselves exploit and "hijack" the chemokine system in order to facilitate their movement, and extravasate out of the primary site into systemic circulation .It is widely believed that the CXCR4/CXCL12 axis may play a critical role in guiding circulating malignant cells (CXCR4+ cells) to organ specific locations that actively secrete CXCL12 (SDF-1) such as bone, brain, liver, and lungs . Many anWe have shown that interfering with the CXCR4/CXCL12 axis using TN14003 in vitro will effectively inhibit tumor cell migration. Further in vivo studies are warranted to test the efficacy of TN14003 to inhibit UM metastasis.2 enriched atmosphere. These cells were cultured with RPMI-1640 medium (Invitrogen), supplemented with 5% heat inactivated fetal bovine serum , 1% fungizone (Invitrogen), and 1% penicillin twice weekly, at every media change, for normal growth by phase contrast microscopy. The cultures were grown to confluence and passaged by treatment with 0.05% trypsin in EDTA (Corning) at 37°C and washed in 7 ml RPMI-1640 media before being centrifuged at 120 g for 10 minutes to form a pellet.Four human UM cell lines  and one transformed melanocyte cell line (UW-1) were incubated at 37°C in a humidified 5% COThe UM cell lines 92.1, SP6.5, MKT-BR, OCM-1, and UW-1 had been established by Dr. Jager , Dr. Pelletier , Dr. Belkhou  and Dr. Albert , respectively ,17.The CXCR4 synthetic peptide inhibitor was synthesized and purchased from the Emory University Microchemical Facility, Winship Cancer Center. The peptide itself is approximately 2.03 kDa in size and its linear amino acid sequence is as follows: NH2-RR-Nal-CY-Cit-K-dLys-PYR-Cit-CR-amide.Cytopsins of the 5 UM cell lines were made using a Cytospin3 machine (Shandon). Cells from culture were diluted to a concentration of 250,000 cells/ml, and a 300 μL solution at that concentration was placed in each spin to be evenly plated on each slide. All slides were then immunostained with primary anti-human monoclonal antibody against CXCR4  using the Ventana™ automated immunostaining machine programmed to use a standard Avidin-Biotin Complex method.3 cells per well, with twelve wells per cell line. A row of twelve wells containing only RPMI-1640 medium was used as a control. Cells were allowed to adhere overnight. The CXCR4 peptide inhibitor TN14003 was added the previous day to six of the twelve wells per cell line at a concentration of 4 ng/ml [The Sulforhodamine-B based assay kit  was performed according to the National Cancer Institute protocol . Each of 4 ng/ml . Cells w5 cells were then placed in the upper chamber and re-suspended in RPMI serum-free media. The lower chamber contained CXCL12 (SDF-1α) also re-suspended in serum free media at a pre-determined optimum concentration range 200 ng/ml [2 supplemented atmosphere. Following incubation, cells that did not migrate were removed from the top of the insert with sterile cotton swabs. Cells that migrated were then stained, eluted from the bottom of the wells, and the optical density of the stain released from the well was read at an O.D of 565 nm.The migratory and inhibitory properties of 5 UM cell lines  for the CXCR4/CXCL12 chemokine axis were investigated using a QCM™ 24-Well Colorimetric Cell Migration Assay (Chemicon). This assay is based on the Boyden chamber system that contains smaller well inserts. These well inserts have a PET membrane made of a polycarbonate material perforated with 8 μm pores. 5.0 × 106 cells/ml. 10 μL of the biotinylated cytokine reagent was added to each vial containing the cell lines , 10 μL of biotinylated negative control reagent was added to separate vials containing each of the 5 cells lines as an appropriate negative control for this experiment. All vials were incubated for 60 minutes at 5°C as per protocol instructions. Then 10 μL of the avidin-FITC reagent was added to each tube and were incubated for 30 min. at 5°C in the dark as per protocol instructions. The vials containing the cell reaction mixture were then washed twice with 2 mL of RDF1 buffer provided with the kit in order to remove unreacted avidin-fluorescein. The cells were then finally resuspended in 200 μL of RDF1 buffer for flow cytometric analysis.The CXCR4 cell surface expression was measured and quantified using a Flurokine: Cytokine Flow Cytometry Kit for human SDF-1α Biotin Conjugate (R&D Systems). Cells from culture were scrapped from the bottom of each flask in order to avoid cell surface receptor damage due to trypsinization of adherent cells. Once scrapped, cells were washed twice in 10 mM HBSS (invitrogen), centrifuged at 500 × g for 5 minutes to avoid any cross reactivity with endogenous cytokine secretions. Preceding the washes, cells were counted and seeded in 1.5 ml tubes at a final concentration of 4 × 10Results from the migration assay for each cell line (cells incubated with TN14003) were directly compared to a matched group of cells incubated solely with CXCL12 using a Student's t-test. A significant result was considered when a p-value < 0.05 was obtained. Results from the proliferation assay for each cell line (cells incubated with TN14003) were directly compared to a matched group of cells that did not contain any inhibitor using a Student's t-test. A significant result was considered when a p-value < 0.05 was obtained.All 5 UM cell line cytospins  stained positive for the CXCR4 receptor. All the slides displayed cytoplasmic staining of the receptor in the cell lines when viewed under a light microscope at a magnification of 400x  when compared back to the lines that were not pre-incubated with the peptide inhibitor Figure .All cell lines migrated towards the chemokine CXCL12 (200 ng/ml) at a level greater than the negative control (p < 0.05). All 5 cell lines pre-incubated with TN14003 (4 ng/ml) did not migrate towards the chemokine CXCL12 . A student's t-test was used in order to ascertain a significant change between groups that contained the inhibitor and the groups that did not contain the inhibitor. The 10% FBS (positive control) gave the highest levels of migration for each cell line. The serum-free medium (negative control) caused little to no migration in all cases . All 5 cell lines expressed different relative levels of the cell surface receptor CXCR4  would bind to cell surface CXCR4 receptors and shift the binding of the biotinylated SDF-1α cytokine, in turn causing a shift in the level of florecesence when being read by the flow cytometer. A negative shift in overall fluorescence was observed for all 5 cell lines when pre-incubated with TN14003 (4 ng/ml) for 5 minutes prior to treatment with SDF-1α biotinylated cytokine  in all 5 cell lines used. This proves active binding of the inhibitor which adversely shifted the mean fluorescent values of all 5 UM cell lines pre-incubated with the drug at a very low concentration (4 ng/ml) (Figure There has recently been an overwhelming amount of evidence suggesting that the CXCR4/CXCL12 chemokine signaling axis may play an integral role in the metastatic progression of many tumor types. It has been shown that CXCR4 may have a prognostic value when expressed in certain tumors such as esophageal cancer . These aThe inhibitory potential of TN14003 a CXCR4 peptide antagonist has been previously studied in inhibiting breast cancer metastasis  and inhiFITC-avidin kit for biotinylated SDF-1α displayed different relative surface expression of the CXCR4 receptor in the UM cell lines. Cells lines previously described as having a higher invasive and proliferative potential in vitro correlated with higher CXCR4 expression. For example, the 92.1 cell line has been previously characterized in vitro  and in vOur study has shown that 92.1 does indeed have higher expression of surface CXCR4 relative to the other 4 UM cell lines used this experiment. This may implicate that CXCR4 may be necessary in higher concentration in order to achieve a greater metastatic potential. Knowing this, further in vivo studies should be preformed in order to substantiate the previously mentioned hypothesis.The characterization of the CXCR4/CXCL12 chemokine axis in five UM cell lines have shown significant reduction of cancer cell migration in vitro. We have also displayed that the TN14003 inhibitor binds CXCR4 with high affinity shifting the binding of its native ligand CXCL12. Cellular proliferation of the UM cell lines tested were not significantly inhibited by TN14003. This data indicates that further in vivo studies should be pursued in order to test TN14003 ability to inhibit UM metastasis.SD carried out the cellular migration assays, flow cytometry experiments, and wrote the manuscript. JCM and VBF preformed all proliferation assays and statistical analysis. EA and PL prepared all the cytospins and preformed all immunocytochemistry. BFF and MNB evaluated all immunocytological stainings, and revised the entire manuscript.


In all the cell lines expressing measurable amounts of active enzymes, genistein induced a shift towards antiproteolysis in both matrix metalloproteinase/tissue inhibitor of metalloproteinase and urokinase/plasminogen activator inhibitor proteolytic balances. On the other hand, 2-methoxyestradiol did not produce any clear net shift of the proteolytic balance, with the significant exception of the matrix metalloproteinase/tissue inhibitor of metalloproteinase balance in WAC-2 cells, a neuroblastoma cell line with enhanced expression of the N-myc oncogene. © 1999 Cancer Research CampaignThe cytotoxicity of two compounds described as anti-angiogenic, the isoflavone genistein and the oestrogen metabolite 2-methoxyestradiol, has been studied in different human tumour cell lines. Since the degradation of the extracellular matrix is one of the essential steps in angiogenesis, the potential modulatory effects of both compounds on the proteolytic balance in media conditioned by different human tumour cells have been also investigated. The IC


The characteristics of 7 human melanoma cell lines were compared with those of the xenografts from which they were established. The ultrastructure, melanin content, isozyme pattern and chromosome numbers of the cell lines were closely similar to those of the corresponding xenografts. The different cell lines gave rise to colonies in soft agar of size and morphology similar to the parent xenografts, and the plating efficiencies were clearly correlated. However, no correlation was found between the growth rates in vivo and either the doubling times and saturation densities in monolayer cultures or the plating efficiencies in soft agar. Moreover, one of the cell lines lost its tumorigenic ability upon establishment in culture. Thus, although the properties of the cell lines by and large reflected those of the parent xenografts, important inconsistencies were seen. The data emphasize that extrapolations from continuous cell lines in vitro to tumour cells in vivo are not necessarily valid. A high content of cellular fibronectin was correlated with a compact colony morphology in soft agar and rapid attachment and spreading on plastic. The growth rates and cellular morphology of the cell lines were strongly influenced by TPA, DMSO, retinoic acid and theophylline, but not by alpha-melanocyte-stimulating hormone. A murine cell line established from one of the xenografts grew in soft agar and produced sarcoma in mice. The malignant murine cells had arisen by transformation of murine stromal cells during the first subcultures in vitro, possibly caused by a factor produced by the human melanoma cells.


The anti-mitotic drug vindesine was coupled chemically to a monoclonal antibody raised originally against the human osteogenic sarcoma cell line, 791T. The cytotoxicity of the conjugate in vitro was tested, in comparison with free vindesine, against sarcoma 791T and other antigenically cross-reactive osteogenic sarcoma-cell lines, and also against tumour cell lines which have no detectable reaction with the monoclonal antibody. Continuous exposure of cultured 791T cells indicated that the vindesine was partially inactivated following conjugation since the conjugate was less toxic than the free drug. However, antibody-binding activity was essentially preserved following conjugation. Despite diminished drug activity in the conjugate, assays designed to mimic antibody binding to tumour in which target cells were treated with conjugate and washed before culture, showed selective cytotoxicity for osteogenic sarcoma lines with little or no effect on non-cross reactive control cells. In comparison, free vindesine was toxic equally for all cell lines and free antibody was non-toxic. These studies indicate that conjugation of a cytotoxic agent to a monoclonal antibody can confer on that agent selectivity for a particular target cell type which is recognised by the antibody.


The primary chemosensitivity of 16 highly malignant xenografted human soft-tissue sarcomas to ifosfamide, dacarbazine, adriamycin and cisplatin and the development of secondary drug resistance in two chemosensitive sarcoma cell lines was tested in the xenograft system. Single-dose, single-agent treatments with 350 mg kg-1 ifosfamide, 200 mg kg-1 dacarbazine, 10 mg kg-1 doxorubicin and 6.6 mg kg-1 cisplatin were administered and response measured as specific growth delay. Since ifosfamide induced unexpectedly higher toxicity, response was corrected based on the shape of the dose-response curve for ifosfamide. Taking a specific growth delay > 3 as the cut-off point for chemosensitivity, ifosfamide, dacarbazine, doxorubicin and cisplatin were effective in 10/16, 4/16, 2/16 and 1/16 sarcoma cell lines respectively. Five out of 16 sarcoma cell lines were resistant to all tested drugs. Ifosfamide-resistant sarcoma lines were also resistant to doxorubicin and cisplatin, indicating a high degree of cross-resistance. Dacarbazine was still effective in 1/6 ifosfamide-resistant sarcoma cell lines. Secondary drug resistance developed slowly after doxorubicin and ifosfamide pretreatments at moderate selection pressure and developed rapidly after dacarbazine pretreatment at high selection pressure.


Novel imidazoacridinone derivatives, C1310 and C1311, have been evaluated for their potential to inhibit tumour cell growth in vitro and in vivo. A cell line panel, including seven human and murine colon carcinoma cell lines and three in vivo models, was used. The compounds were found to be potent inhibitors of tumour cell growth with IC50 values ranging between 10 nM and 2 microM in human colon cancer cell lines. Statistically significant tumour growth delay (P < 0.01) was observed after a single intraperitoneal (i.p.) dose of C1311 (100 mg kg-1 body weight) in MAC15A, MAC29 murine and HT29 human adenocarcinomas of the colon. Rapid accumulation of fluorescence of both C1310 and C1311 was seen in the nuclei of HT29 human colon tumour cells in culture. C1311 was also found to bind into calf thymus DNA as shown by spectrophotometric titration and thermal denaturation and to cause early inhibition of thymidine incorporation in HT29 cells in vitro. The results of this study suggest that C1311 should be considered as a candidate for clinical development.


We have previously shown that the metastatic breast cancer cell line MDA-MB-231 over-expresses urokinase and binds and efficiently activates plasminogen at the cell surface compared to non-metastatic cells. The aim of this study was to further characterise plasminogen binding and determine the topology of cell surface-bound plasminogen in terms of its potential for activation. The lysine-dependent binding of plasminogen at 4°C to MDA-MB-231 cells was stable and resulted in an activation-susceptible conformation of plasminogen. Topologically, a fraction of bound plasminogen was co-localised with urokinase on the surfaces of MDA-MB-231 cells where it could be activated to plasmin. At 37°C plasmin was rapidly lost from the cell surface. Apart from actin, other candidate plasminogen receptors were either not expressed or did not co-localise with plasminogen at the cell surface. Thus, based on co-localisation with urokinase, plasminogen binding is partitioned into two functional pools on the surface of MDA-MB-231 cells. In conclusion, these results shed further light on the functional organisation of the plasminogen activation cascade on the surface of a metastatic cancer cell. © 2001 Cancer Research Campaign


Previous studies have shown that normal as well as neoplastic B-cell lines vary substantially in their response to the antiproliferative effects of human interferon (HIF). In this study we took advantage of a recent method to generate long-term continuous normal T-cell cultures (CTC) to investigate the effects of HIF on proliferating lymphoid cells. Normal CTC proved to be resistant to inhibition of proliferation; up to 1000 u HIF had little effect on [3H] TdR uptake, and up to 2000 u HIF had little effect on cell-cycle progression, measured by flow cytometry. Proliferating normal B cells were also resistant to the antiproliferative effect. Nor did up to 500 m HIF inhibit RNA synthesis or immunoglobulin biosynthesis of normal B cells. In contrast, a neoplastic myeloma B cell, a Burkitt's lymphoma cell and a neoplastic leukaemic T cell showed marked inhibition of [3H] TdR uptake and cell cycle progression with as little as 5 u HIF. These results suggest that amounts of HIF sufficient to inhibit proliferation of some neoplastic lymphoid cells have little effect on T- and B-cell proliferation and differentiation of normal B lymphocytes.


Incubation in vitro of lymphnode cells (LNC) from rats bearing a transplanted syngeneic methylcholanthrene-induced sarcoma (Mc7) resulted in the generation of a potent cytotoxic activity. Four to seven days' culture was required for development of cytotoxic activity, which was shown to be mediated by a heat-stable soluble factor. The cytotoxicity was not detectable in a 3 h or 15 h 51Cr-release assay, but was demonstrated in a 48 h microcytotoxicity assay, where post-labeling with isotopically labelled cell precursors was used to quantitate cell survival. The cytotoxicity of the cultured tumour-bearer LNC and their supernatant factor was shown to be cross-reactive for tumour cell lines other than sarcoma Mc7, and was also expressed against adult or embryonic fibroblasts.


There was no effect of an absence of OPN on the ability of the cells to undergo immortalization or to form morphologically transformed foci following ras transfection. Wildtype and OPN-deficient cell lines were established from such foci, and lines with similar ras mRNA levels selected for further analysis. Ras -transformed cell lines from both wildtype and OPN-deficient mice could form colonies in soft agar indicating that this process can occur in the absence of OPN. However, the ability of the OPN-deficient cell lines to form colonies was reduced as compared to wildtype cell lines. Tumorigenesis in syngeneic and nude mice was assessed for a subset of cell lines that formed colonies efficiently in soft agar. Cell lines unable to make OPN formed tumors in these mice much more slowly than wildtype cells, despite similar growth of the cells on plastic and in soft agar. Taken together, these results indicate that maximal transformation by ras requires OPN expression, and implicate increased OPN expression as an important effector of the transforming activity of the ras oncogene. © 2000 Cancer Research CampaignThe secreted phosphoprotein osteopontin (OPN) is strongly associated with the process of neoplastic transformation, based both on its pattern of expression in vivo and in vitro and on functional analyses. We have used 3T3 cells derived from wildtype and OPN-deficient mice and transformed by transfection with oncogenic


We recently established a new human breast cell line, designated KPL-1, which was derived from the malignant effusion of a patient with breast cancer. This cell line is highly tumorigenic and grows rapidly in female nude mice. Cytogenetic analysis indicated its human origin and revealed a hypertriploid modal number of chromosomes. Electron microscopic examination suggested that the KPL-1 cells are of epithelial origin. Immunohistochemical studies revealed that the cells express cytokeratin, carcinoembryonic antigen and CA 15-3. They also possess a large number of oestrogen receptors but not progesterone receptors. Interestingly, KPL-1 cells seem to grow oestrogen independently in vitro. No amplification of c-erbB-2, c-myc, H-ras and N-ras genes was detected. KPL-1 cells secrete a large amount of tissue polypeptide antigen (TPA). Although the secretion of CA 15-3 seemed to be constant throughout all cell growth phases, TPA secretion increased during the exponential growth phase and decreased during the plateau phase. Serum TPA levels significantly correlated with the volume of KPL-1 tumours transplanted into nude mice. These data suggest that this KPL-1 cell line may be useful for studying oestrogen-independent growth and the kinetics of tumour-associated antigens in vivo as well as in vitro.


Using rat glioma C6 cells as a model, we have found a bimodal effect of alpha-thrombin on cell growth. In C6 cells treated with alpha-thrombin at concentrations from 0.02 nM to 1.0 nM, inhibition of cell proliferation was noted. Because the thrombin receptor agonist peptide TRAP-6 also induced inhibition of cell proliferation and the thrombin receptor antagonist peptide T1 prevented the inhibitory effect of alpha-thrombin on C6 glioma cell growth, thrombin receptor involvement in antiproliferative action of alpha-thrombin in C6 glioma cells is highly likely. However, stimulation of cell proliferation observed when C6 cells were treated with alpha-thrombin at higher doses (> 1.0 nM) seems to be mediated by as yet undefined thrombin receptor-independent biochemical mechanisms.


Successful imaging or treatment of neuroblastoma with 131I-meta-iodobenzylguanidine (131I-mIBG) depends on the selectivity of active (type 1) uptake of mIBG in neuroblastoma cells relative to passive (type 2) uptake present in most normal tissues. This study investigates the effects of moderately elevated temperature (39-41 degrees C) on the cellular uptake of 131I-mIBG in two neuroblastoma cell lines [SK-N-BE(2c) and IMR-32] and in a non-neuronal (ovarian carcinoma) cell line (A2780). In SK-N-BE(2c), a cell line with high active uptake capacity, the specific (type 1) uptake was reduced by 75% (P < 0.001) at 39 degrees C. Both IMR-32 and A2780 have a low capacity for accumulation of mIBG by active uptake. These cell lines demonstrated a statistically significant increase in accumulation at 39 degrees C, mainly as a result of increased non-specific transport. At 41 degrees C uptake of 131I-mIBG was reduced in all cell lines. Thus, the active component of mIBG uptake is more vulnerable to increased temperature than the passive component. It seems probable that moderately increased temperature will have an unfavourable effect on the therapeutic differential for targeted radiotherapy of neuroblastoma using radiolabelled mIBG.


Ten human ovarian carcinoma cell lines have been studied as a potential in vitro screen for the development of novel anticancer platinum complexes. Lines have been established and developed both from solid and ascitic tumours, from pretreated and untreated patients, and are available at a range of in vitro passage numbers. The biological properties of the lines were consistent with them being human, epithelial and of ovarian carcinoma origin. Using a tritiated thymidine or leucine uptake method, and a 96 hour continuous drug exposure, the lines have been calibrated against four platinum-containing chemotherapeutic agents: cisplatin, iproplatin, carboplatin and tetraplatin. Striking differences in cytotoxicity were observed across the lines for each agent. Some lines were consistently resistant, others generally sensitive, whereas some showed clear evidence of differential sensitivity to a particular agent. Statistical analysis (Spearman rank correlation) involving the six possible pairings of drugs showed that cisplatin, iproplatin and carboplatin elicit a very similar pattern of response in these lines whereas tetraplatin elicits a completely different response pattern. Similar cytotoxicity values were obtained using a soft agar cloning assay. Results using a tetrazolium dye reduction assay, however, gave somewhat higher and more variable values, particularly with tetraplatin. The thymidine uptake assay will be adopted in further studies on a selected panel of six lines. This panel encompasses the spectra of sensitivities identified for each of the four agents against the original ten lines and may provide a useful screening facility for the development of novel platinum drugs, in that it detects both cell line-determined and structure-determined differences in cytotoxicity.


We have previously shown that epidermal growth factor (EGF) enhances the in vitro and in vivo sensitivity of human ovarian carcinoma 2008 cells to cisplatin. EGF was found to enhance selectively the in vivo toxicity of cisplatin to 2008 cell xenografts without altering the toxicity of cisplatin to non-malignant target tissues such as the kidney or bone marrow. We now show that recombinant human EGF (rhEGF) enhances the cisplatin sensitivity of cell lines representative of many other types of malignancies in addition to ovarian carcinoma, including cancers of the head and neck, cervix, colon, pancreas and prostate, as well as non-small-cell carcinoma of the lung. In addition, rhEGF was found to sensitise cells to other platinum-containing drugs and several other classes of chemotherapeutic agents. rhEGF sensitised 2008 cells not only to cisplatin, but also to carboplatin and tetraplatin, as well as taxol, melphalan and 5-fluorouracil. We conclude that modulation of drug sensitivity by rhEGF is observed in cell lines representative of many human malignancies and for multiple classes of chemotherapeutic agents, indicating that it alters one or more components of the cellular damage response that are both common between cell lines and classes of drugs and fundamental to survival.


Microtitre plate proliferation assays of cell density and DNA synthesis were devised to study the effects of TNFα on confluent monolayers of the human foetal jejunal cell line I407 and the mouse fibrosarcoma cell line L929. Both cell lines showed a similar response to TNFα. Exposure to TNFα alone did not reduce cell numbers but did cause a significant increase in DNA synthesis (p < 0.05). When cycloheximtde was added in tandem with TNFα there was a significant reduction in cell number (p < 0.001) and level of DNA synthesis (p < 0.01) indicative of cell death. The DNA of cells exposed to TNFα and cycloheximide was fragmented when viewed on an electrophoresis gel. The results show that BrdU incorporation might be a good indicator of damage to the DNA of cells after cytotoxic insult. TNFα may be responsible for villus enterocyte damage in enteropathies such as coeliac disease and GVHR of the small bowel.Bromodeoxyuridine incorporation is a useful method for studying the pattern of DNA synthesis in proliferating cells. The distribution pattern of incorporated BrdU in villus enterocytes of duodenal explants was analysed after exposure to TNFα in organ culture. TNFα caused a consistent, low level uptake of BrdU in the portion of the nucleus close to the nuclear membrane, this pattern was absent from the control cultures. As these epithelial cells are terminally arrested in G


Pyrrolidino-4-iodotamoxifen (idoxifene) is a new non-steroidal antioestrogen currently undergoing phase I clinical evaluation. Using idoxifene and tamoxifen and two additional analogues of tamoxifen (3-hydroxytamoxifen and 4-iodotamoxifen) and the imidazole-based calmodulin inhibitor, calmidazolium, a strong positive correlation (r2 > 0.95) was observed between cytotoxicity and inhibition of calmodulin-dependent cyclic AMP phosphodiesterase (e.g. mean IC50 across four human ovarian carcinoma cell lines of 4.5 microM for idoxifene and 6.3 microM for tamoxifen). Using two parent human ovarian carcinoma cell lines  in which acquired resistance to doxorubicin or cisplatin has been generated, we have determined the ability of idoxifene to overcome resistance in these lines. At a non-toxic concentration of 2 microM, idoxifene appeared at least as effective as the clinically used multidrug resistance modifiers verapamil and tamoxifen in overcoming doxorubicin resistance in two acquired resistant cell lines shown to overexpress the P-170 efflux glycoprotein. Non-cross-resistance between cisplatin and idoxifene was observed in two acquired resistant cell lines possessing contrasting mechanisms of resistance to cisplatin (41McisR6 reduced drug transport and CH1cisR6 resistance mediated at the level of DNA). In one of four cell lines (CH1), synergism between idoxifene and cisplatin was observed by median effect analysis. However, with the 41M and its 6-fold cisplatin-resistant variant, antagonism was observed. These observations made by median effect analysis appeared to be unrelated to platinum uptake or removal of platinum-induced DNA interstrand cross-links. These in vitro data suggest that idoxifene may be usefully combined with doxorubicin in the clinical setting, but caution should be exercised in combining it with cisplatin in the treatment of certain tumours.


In vitro interactions of thymocytes and thymocyte hybridomas with cortical (R-TNC.1) and medullary (TE-R 2.5) rat thymic epithelial-cell (TEC) lines were studied. It was found that the cortical line had better adhesion capability. It bound exclusively immature CD4+CD8+            αβTCR10 thymocytes, induced apoptosis of a subset of these cells, and stimulhted proliferation of the BWRT (CD4-CD8-            αβTCR-) hybridoma. The medullary line bound both immature and mature thymocytes, decreased their apoptosis, and induced apoptosis of the BWRT 8 (CD4+CD810            αβTCRhi) hybridoma. Thymocyte differently modulated cytokine production by TEC lines, upregulating the secretion of IL-1 by R-TNC.1 and IL-6 by TE-R 2.5 cells. Finally, coculture of thymocytes with TEC lines resulted in different patterns of protein-tyrosine phosphorylation in thymocytes. These results show the existence of mutual bidirectional interactions between thymocytes and TEC lines in vitro, but these processes differed depending on phenotypic characteristics and origin of TEC lines used.


The potential of radiolabelled phenylpiperazines as agents for the detection and therapy of tumours of neural crest origin was evaluated by in vitro pharmacological studies with human neuroblastoma cell lines [SK-N-SH and SK-N-BE(2C)], and in vivo by biodistribution measurements. The ability of phenylpiperazines: 4-phenyl-piperazine (PP), 1-carboxamidino-4-phenyl-piperazine (CAPP), , to inhibit MIBG uptake by neuroblastoma cells was determined by incubation with [125I]MIBG (0.1 microM) for 2 h in the presence of varying concentrations (10(-8)-10(-3) M) of ligand. For measuring uptake, cells were incubated with [125I]IPP (0.1 microM) and cell-associated radioactivity was measured at various times. Retention was studied by incubating cells in the presence of [125I]IPP (0.1 microM) for 2 h, followed by replacement with drug-free medium and determination of cell-bound radioactivity. Selectivity of [125I]IPP uptake was studied by inhibition studies with MIBG, DMI, 5HT and phenylpiperazines. The biodistribution of [125I]IPP was measured in normal rats at 0.083, 0.5, 1, 2 and 24 h . The IC50S (microM) for inhibition of [125I]MIBG uptake were: PP, 1.5; CPP, 2.5; CAPP, 2.5; DMPP, 5; CP(OH)P, 30 and TFMPP, 65. The rate of cellular uptake of [125I]IPP was greatest between 0 and 60 min and decreased after 60 min, similar to MIBG. After an initial rapid washout of approximately 50% of the radioactivity, retention remained constant for 3 h. The IC50S (microM) for inhibition of [125I]IPP uptake were: MIBG, 18-25; DMI, 0.6-1.5; 5HT, > 100; IPP, 1.8-2.5; CPP, 7.0-9.0 and TFMPP, > or = 20. The in vivo studies demonstrated a pattern of distribution similar to MIBG. The results demonstrate that phenylpiperazines display significant affinity for neuroblastoma with uptake and retention characteristics similar to MIBG.


Although antagonist G blocks SCLC cell growth (IC50 = 24.5 ± 1.5 and 38.5 ± 1.5 μM for the H69 and H510 cell lines respectively), its exact mechanism of action is unclear. This study shows that antagonist G stimulates apoptosis as assessed by morphology (EC50 = 5.9 ± 0.1 and 15.2 ± 2.7 μM for the H69 and H510 cell lines respectively) and stimulates c-jun-N-terminal kinase (JNK) activity in SCLC cells (EC50 = 3.2 ± 0.1 and 15.2 ± 2.7 μM). This activity is neuropeptide-independent, but dependent on the generation of reactive oxygen species (ROS) and is inhibited by the free radical scavenger n-acetyl cysteine. Furthermore, antagonist G itself induces inflammation (59% increase in oedema volume compared to control) and potentiates (by 35–40%) bradykinin-induced oedema formation in vivo. In view of these results we show that, as well as acting as a ‘broad-spectrum’ neuropeptide antagonist, antagonist G stimulates basal G-protein activity in SCLC cell membranes (81 ± 12% stimulation at 10 μM), thereby displaying a unique ability to stimulate certain signal transduction pathways by activating G-proteins. This novel activity may be instrumental for full anti-cancer activity in SCLC cells and may also account for antagonist G activity in non-neuropeptide-dependent cancers. © 1999 Cancer Research Campaign[Arg


We examined the importance of dosing interval between leucovorin (LCV) and 5-fluorouracil (5-FU) on intracellular thymidylate synthase (TS) ternary complex, free TS and total TS protein levels in human MCF-7 breast and NCI H630 colon cancer cell lines. A 2- to 3-fold increase in total TS was noted when either cell line was exposed to 5-FU 10 microM plus LCV (0.01-10 microM) compared with a 1.4- to 1.6-fold increase in total TS due to 5-FU 10 microM alone. The amount of TS ternary complex formed was 2- to 3-fold higher in both cell lines treated with the combination of 5-FU and LCV compared with 5-FU alone. TS complex formation and total TS protein increased with LCV dose (0.1-10 microM). In MCF-7 cells, the maximal increase in total TS protein and TS ternary complex formation was observed when 5-FU was delayed for 4 h after the start of LCV exposure. In NCI H630 cells, maximal total TS protein and ternary complex formation occurred when 5-FU was delayed for 18 h after the start of LCV exposure. The amount of free TS did not change in either cell line whether 5-FU was given concurrently with LCV or delayed for up to 24 h. The accumulation rate of intracellular folates in the form of higher glutamates Glu3-Glu5 was rapid in MCF-7 cells , whereas in H630 cells accumulation of higher polyglutamates continued to increase up to 18 h. The time of peak folate polyglutamate (Glu3-Glu5) formation coincided with the time of peak TS complex formation and total TS protein in each cell line. In these human carcinoma cell lines, the LCV dose and interval between 5-FU and LCV play a role in increased TS total protein and TS ternary complex; however, the amount of free TS is independent of the interval between 5-FU and LCV. The time-and dose-dependent increases in TS ternary complex and TS total protein are associated with differences in the accumulation of folate polyglutamates in these cell lines.


The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC4) and -resistant (GLC4-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the platinum and radiation toxicity with the clinically relevant dose ranges applied, this assay was used to obtain information on cell survival after the treatments. Apart from effects on cell survival effects on DNA were also investigated. Configurational DNA changes could be induced by platinum drugs and thereby these drugs might change the frequency of DNA double-strand breaks (dsbs). DNA fragmentation assayed with the clamped homogeneous electric field (CHEF) technique was used as a measure for dsbs in DNA. The radiosensitising effect of the platinum drugs was expressed as enhancement ratio (ER) calculated directly from survival levels of the initial slope of the curve. The highest ER for cisplatin in GLC4 was 1.39 and in GLC4-CDDP 1.38. These were all at 75% cell survival. Carboplatin showed increased enhancement with prolonged incubation up to 1.21 in GLC4 and was equally effective as cisplatin in GLC4-CDDP. According to isobologram analysis, prolonged incubation with both platinum drugs showed at least additivity with radiation for both cell lines at clinically achievable doses. GLC4-CDDP showed cross-resistance to radiation. The radiosensitising capacity of both lung cancer cell lines was not dependent on their platinum sensitivity. The formation of dsbs in DNA directly after radiation was not influenced by pretreatment of either drug in the sensitive or in the resistant cell line. Drug treatment resulted in decreased DNA extractability in control as well as in irradiated cells. Modest enhancement ratio for radiosensitisation by platinum drugs cannot be explained on the level of dsb formation in DNA in both cell lines. Interaction of radiation with the clinically less toxic carboplatin can be improved by prolonged low-dose carboplatin exposure before irradiation and is as potent as cisplatin in the resistant lung cancer cell line. This suggests an advantage in combining radiation and carboplatin in lung cancer patients.


Signal transduction pathways activated by injury play a central role in coordinating the cellular responses that determine whether a cell survives or dies. GADD153 expression increases markedly in response to some types of cellular injury and the product of this gene causes cell cycle arrest. Using induction of GADD153 as a model, we have investigated the activation of the cellular injury response after treatment with taxol and cisplatin (cDDP). Activation of the GADD153 promoter coupled to the luciferase gene and transfected into human ovarian carcinoma 2008 cells correlated well with the increase in endogenous GADD153 mRNA after treatment with taxol but not after treatment with cDDP. Following treatment with cDDP, the increase in endogenous GADD153 mRNA was 10-fold greater than the increase in GADD153 promoter activity. Likewise, at equitoxic levels of exposure (IC80), cDDP produced a 5-fold greater increase in endogenous GADD153 mRNA than taxol. The tyrosine kinase inhibitor tyrophostin B46 had no significant effect on the ability of taxol to activate the GADD153 promoter, but inhibited activation of the GADD153 promoter by cDDP in a concentration-dependent manner. Tyrphostin B46 synergistically enhanced the cytotoxicity of cisplatin; however, the same exposure had no significant effect on the cytotoxicity of taxol. We conclude that (1) taxol and cDDP activate GADD153 promoter activity through different mechanisms; (2) the signal transduction pathway mediating induction by cDDP involves a tyrosine kinase inhibitable by tyrphostin B46; and (3) that inhibition of this signal transduction pathway by tyrphostin synergistically enhances cDDP toxicity.


The efficacy of cisplatin  is hampered by acquired or de novo resistance of malignant cells to its cytotoxic effects. We have previously reported that cisplatin resistance parallels glutathione S-transferase (GST) activity in several human small-cell lung cancer cell lines. In the presently described studies, we used sulphasalazine, an inhibitor of GSTs, to evaluate the relative role of GSTs in mediating cisplatin resistance in two human small-cell lung cancer cell lines, NCI H-69 and H-2496. The H-69 cell line, which contained relatively higher GST activity than the H-2496 cell line (317 +/- 7 vs 9 +/- 1 mU mg-1 protein respectively), also displayed a greater degree of cisplatin resistance . Western blot and Northern blot analyses of purified GSTs revealed the expression of only the pi-class GST in both cell lines. Sulphasalazine inhibited the purified GSTs (IC50 of 10 microM for H-69 and 12 microM for H-2496) from both lines in a competitive manner with similar Ki values (6.5 and 7.9 microM for the H-69 and H-2496 cell lines respectively). Cytotoxicity studies revealed that sulphasalazine increased the cytotoxicity of cisplatin towards both cell lines. Isobologram analysis showed that sulphasalazine synergistically enhanced the cytotoxicity of cisplatin towards both cell lines, the magnitude of synergy being remarkably higher in H-69 cells than in H-2496 cells. Our studies indicate that clinically achievable concentrations of sulphasalazine may be useful in modulating cisplatin resistance in malignancies with increased GST-pi content.


The effects of transforming growth factor beta 1 (TGF-beta1) and sodium butyrate on cell proliferation and the urokinase plasminogen activator (uPA) system were examined in normal human breast epithelial cells (HBECs) and in a breast cancer cell line, MDA-MB-231. In HBECs, TGF-beta1 inhibited cell proliferation and uPA activity, while it augmented plasminogen activator inhibitor-1 (PAI-1) antigen level. Sodium butyrate inhibited both cell proliferation and uPA activity but did not affect the level of PAI-1. In MDA-MB-231, TGF-beta1 had no effect on cell proliferation but increased uPA activity and PAI-1 antigen level; sodium butyrate inhibited both cell proliferation and uPA activity but had no effect on PAI-1 level. Moreover, in the presence of plasminogen, cell detachment could be modulated by the level of cell-associated uPA. Our results indicate (1) that the effects of TGF-beta1 on cell growth can be dissociated from its effects on the uPA/PAI system; (2) that, while TGF-beta1 is a potent inhibitor of cell proliferation and uPA activity in normal cells, it may promote invasion and metastasis of tumour cells by increasing uPA activity and PAI-1 levels; and (3) that sodium butyrate offers a potential approach to preventing tumour development by inhibiting both cell proliferation and invasion.


The effects of expressing neu-T, a mutated constitutively activated form of c-neu, have been examined in the non-transformed conditionally immortalised human mammary luminal epithelial cell line, HB4a. A variant cell line, N4.1, which expressed neu-T, showed evidence of transformation, including partial loss of growth factor dependence and acquisition of anchorage-independent growth, but failed to give rise to tumours in nude mice, indicating that expression of neu-T alone was probably insufficient to cause tumorigenic progression to a full malignant phenotype. During characterisation of the N4.1 cell line, it was observed that under conditions of serum deprivation, it underwent apoptotic cell death, as demonstrated by light microscopy, flow cytometry and DNA gel electrophoresis. The induction of apoptotic cell death in the N4.1 cell line by serum deprivation was abrogated specifically by the addition of steroids with glucocorticoid activity but not any peptide growth factors studied. This study shows the induction of apoptosis by serum deprivation, and its abrogation by glucocorticoids occurring in human mammary luminal epithelial cells transformed by expression of neu-T, and implicates the involvement of receptor protein tyrosine kinases in an apoptotic signalling pathway in this cell type.


Development of resistance to cytotoxic agents is a major limitation to their clinical use. Novel compounds are synthesized with a view to develop non-cross-resistant, less toxic and more potent activity. The detection of the anti-tumour properties of the inorganic compound cisplatin stimulated a broad search for other metal-containing complexes. Titanocene dichloride was synthesized on this basis and has shown potent anti-neoplastic activity in experimental animals. We have examined the in vitro activity of titanocene dichloride in two pairs of platinum-sensitive and resistant human ovarian carcinoma cell lines, A2780/2780CP and CH1/CH1cisR, and in mutated p53- and bcl-2-transfected clones of A2780 cells. A time- and concentration-dependent anti-proliferative effect was observed in all cell lines treated with titanocene dichloride. The drug was found to significantly overcome platinum resistance in the 2780CP and the CH1 cisR cell lines and in the bcl-2 and the mutant p53 transfectants of A2780 cells. Titanocene dichloride induced a block in late S/early G2 phase of the cell cycle; however apoptotic cell death occurred from any phase of cycle. Titanium-DNA adducts were detected in A2780 cells treated with titanocene dichloride using atomic absorption spectrometry, suggesting that DNA may be a target for this drug. In agreement with this finding, p53 accumulated rapidly in drug-treated A2780 cells, indicative of a role for titanocene dichloride as a DNA-damaging agent. We have also performed studies to determine whether titanocene dichloride could demonstrate synergy with other cytotoxic agents in vitro. Isobologram analysis of cytotoxicity data obtained suggests that the combination of titanocene dichloride and 5-fluorouracil (5-FU) is synergistic. The potent in vivo anti-tumour activity of this compound, supported by the encouraging results from two phase I clinical trials, suggests that titanocene dichloride could be a promising novel chemotherapeutic agent.


The establishment and characterisation  of six cell lines from primary human colorectal adenocarcinomas is described. These lines were established from surgical specimens, from 49 unselected patients, without the use of 'feeder' cells, 'conditioned' medium or passage of cells in nude mice. The six cell lines exhibit considerable variation in morphology, CEA secretion and tumourigenicity in nude mice. At least two of the lines retain some of the differentiated characteristics of colorectal epithelium.


Expression of these genes had at most small effects to influence cell survival. We conclude that apoptotic mechanisms had at most a minor role in leading to reproductive death of MGH-U1 and CHO cells after chemotherapy. When apoptosis is observed following treatment with anti-cancer drugs it may be a secondary event which occurs in lethally-damaged cells, leading to their lysis, rather than a primary event that leads to loss of reproductive integrity. © 2001 Cancer Research Campaign http://www.bjcancer.comAn increase in apoptotic cells may be observed after treatment with chemotherapy, and many authors have assumed that anti-cancer drugs kill cells by inducing apoptosis. The most relevant endpoint of cell death following treatment of tumour cells is loss of reproductive ability as measured by a colony-forming assay, since cells with limited reproductive potential cannot regenerate a tumour. We have therefore investigated the relationship between apoptosis and reproductive cell death following in vitro treatment of mammalian cell lines with anti-cancer drugs. Markers of apoptosis  were evaluated at various times after treatment of Chinese Hamster Ovary (CHO) cells, human bladder cancer MGH-U1 cells, and a murine T-lymphocytic cell line (CTLL-2) with several anti-cancer drugs. These markers were found infrequently, despite the use of doses that cause loss of colony-forming ability, except in CTLL-2 cells. We also transfected and expressed the human pro-apoptotic gene


The effects of acute hypoxia on integrin expression and adhesion to extracellular matrix proteins were investigated in two human melanoma cell lines, HMB-2 and DX3, and a human adenocarcinoma cell line, HT29. Exposure to hypoxia caused a significant down-regulation of cell surface integrins and an associated decrease in cell adhesion. Loss of cell adhesion and integrin expression were transient and levels returned to normal within 24 h of reoxygenation. Other cell adhesion molecules, such as CD44 and N-CAM, were also down-regulated after exposure of cells to hypoxia. Acute exposure to hypoxia of cells at confluence caused rapid cell detachment. Cell detachment preceded loss of viability. Detached HMB-2 and DX3 cells completely recovered upon reoxygenation, and floating cells re-attached and continued to grow irrespective of whether they were left in the original glass dishes or transferred to new culture vessels, while detached HT29 cells partly recovered upon reoxygenation. Cell detachment after decreased adhesion appears to be a stress response, which may be a factor enabling malignant cells to escape hypoxia in vivo, with the potential to form new foci of tumour growth.


A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line.


Chromophilic renal cell carcinoma is a distinct type of human renal cancer, only recently recognised and defined by its characteristic histomorphological aspect and cytogenetic aberrations. We are the first to report on the establishment and cytogenetic characterisation of a panel of four permanent cell lines, i.e. chromphi-1, -2, -3 and -4, derived from strictly defined renal cell carcinomas (RCCs) of the chromophilic type and kept in continuous culture for up to 5 years. Immunohistochemistry revealed coexpression of vimentin and cytokeratins in all cell lines the cytokeratin polypeptide patterns, however, varying between the different cell lines. By light and transmission electron microscopy, various amounts of cytoplasmatic glycogen deposition were observed, being most pronounced in chromphi-3 and -4. The mean population doubling time ranged from 24 h (chromphi-1) to 51 h (chromphi-4). Chromphi-1 tumour cells produced slowly growing tumours in nude mice using the subrenal capsule assay. In all cell lines, cytogenetic analysis revealed numerical chromosomal aberrations known to be characteristic for chromophilic RCCs, i.e. loss of the Y chromosome, tri- or tetrasomy of chromosomes 7 and 17 as well as various combinations of additional structural and numerical chromosomal aberrations. Karyological aberrations were least pronounced in chromphi-2 and most complex in chromphi-1. Chromosomal aberrations typically affecting the short arm of chromosome 3 in clear cell RCCs were not observed in any of our cell lines.


Previous work has shown that the lethal effects of glucocorticoids on the human lymphoblastoid cell line, CEM-C7, are antagonized by the simultaneous presence of 1-beta-D-arabinofuranosylcytosine (Ara-C). A possible cell cycle mechanism prompted further studies using flow microfluorimetry. We now report that (1) Ara-C (10-100 nM) blocks cells in S-phase and (2) the block is reversible after the drug is removed. A second treatment protocol, in which glucocorticoid is added to cells recovering from the effects of 24 h exposure to Ara-C, results in a clear synergism between the 2 drugs. This synergism is observed over a range of concentrations (5-100 nM), but is most significant at low doses, where inhibition of cell growth by Ara-C occurs but cell killing is minimal. Prior treatment with Ara-C increases the number of cells killed in the presence of steroid during the period 12-24 h after removal of the S-phase block. Combinations of Ara-C and steroid can thus be either synergistic or antagonistic, depending on the drug scheduling.


The occurrence of organ-specific antigens on the surface of cells freshly isolated from normal adult rat liver and from liver cell lines cultured as monolayers for up to 6 months is demonstrated. Enzyme treatment used to disaggregate parenchymal cells from liver tissue did not result in appreciable changes in the antigen profile of the cell surface membrane.


Two naturally occurring cytotoxic cell populations have been identified in the peritoneal cavity of mice inoculated with C. parvum (CP), and are distinguishable on the basis of target-cell reactivity and intrinsic properties. The first effector cell was non-adherent to nylon wool and glass and non-phagocytic. These cells were selectively cytotoxic to the NK-sensitive target cell line K562, and present in the peritoneal cavity of mice 2 days after treatment with 700 micrograms of CP. The second cytotoxic effector cell was adherent to nylon wool and glass, and killed EL4 lymphoma cells derived from in vivo tumour transplants; these target cells are susceptible to phagocytic cell killing, but not NK-cell cytotoxicity in short-term (4h) assays. The adherent cytotoxic population of effector cells was present 4 days after inoculation of CP. In vivo studies showed that CP injected i.p. induced resistance to i.p. challenge with lymphoma EL4 cells, but no resistance was evident when the challenge dose was administered s.c. Adoptive-transfer studies showed that the effector cell type responsible for inhibiting tumour growth was nylon-wool adherent, probably CP-activated macrophages.


Platelet-derived endothelial cell growth factor (PD-ECGF) is identical to human thymidine phosphorylase (dThdPase). The human MCF-7 breast cancer cell line was transfected with the dThdPase cDNA and expressed a 45 kDa protein that was detected with anti-dThdPase antibody. Cell lysates possessed elevated dThdPase activity and cells had up to 165-fold increased sensitivity to the prodrug 5'-deoxy-5-fluorouridine (5'-DFUR) in vitro. Sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (5-FUdR) was unchanged. Recombinant dThdPase was shown to catalyse directly the phosphorolytic cleavage of 5'-DFUR to 5-FU. Exogenous thymidine (dThd) reversed the toxicity of 5-FUdR on the parental line , but the dThd rescue was substantially modulated in the dThdPase-expressing clone 4 . We observed a substantial 'bystander' killing effect when small proportions of dThdPase-expressing cells were mixed with parental MCF-7 cells. dThdPase activity was on average 27-fold higher in breast tumours than in normal breast. The levels of wild-type MCF-7 are similar to the low end of the tumour expression. Thus, in some tumours resistance to 5'-DFUR therapy could be due to low dThdPase activity, and transfection to raise the dThdPase levels within the broad tumour range or above it should markedly enhance sensitivity to the prodrug. These results confirm that dThdPase is a major pathway in the metabolic activation of 5'-DFUR, and the bystander effect suggests that this may be a suitable enzyme for gene therapy-directed enzyme/prodrug activation therapy.


The human promyeloid cell line H60 can be induced to differentiate towards either neutrophils or monocytes. Variant cell lines, derived from HL60, which show reduced capacities for neutrophil and monocyte differentiation can be arranged in a developmental sequence which suggests that the potentials for neutrophil and monocyte differentiation are expressed sequentially by HL60 cells in this order. Analysis of the patterns of total cellular phosphoproteins within HL60 and 5 variant cell lines, by two-dimensional gel electrophoresis, has identified 6 distinct phosphoproteins which show progressive differences in the intensity of spots between the variant lines. The changes in these phosphoproteins relate to the position of the lines within the proposed development sequence. Similarly, lines placed close together in the sequence are more similar, as regards phosphoprotein profiles, than lines placed far apart. These studies provide direct evidence in favour of the hypothesis that the potentials for neutrophil and monocyte differentiation are expressed sequentially during myelopoiesis. Furthermore, two phosphoprotein spots were found to be restricted to lines able to differentiate towards monocytes. These proteins may play important roles during commitment to monocyte differentiation.


Treatments shorter than 72 h with FUdR-dP in immunoliposomes resulted in anti-tumour activities comparable to, or even higher than, that of free FUdR. The shorter treatments reflect more closely the in vivo situation and illustrate the potential advantage of the use of immunoliposomes over non-targeted liposomal FUdR-dP or free FUdR. Association of tumour cell-specific immunoliposomes with CC531 cells was up to tenfold higher than that of liposomes without antibody or with irrelevant IgG coupled, demonstrating a specific interaction between liposomes and target cells which causes an efficient intracellular delivery of the drug. Since biochemical evidence indicates a lack of internalization or degradation of the liposomes as such, we postulate that entry of the drug most likely involves the direct transfer of the prodrug from the immunoliposome to the cell membrane during its antigen-specific interaction with the cells, followed by hydrolysis of FUdR-dP leading to relatively high intracellular FUdR-levels. In conclusion, we describe a targeted liposomal formulation for the anticancer drug FUdR, which is able to deliver the active drug to colon carcinoma cells with high efficiency, without the need for the cells to internalize the liposomes as such. © 1999 Cancer Research CampaignWe have investigated the antiproliferative action towards CC531 colon adenocarcinoma cells of target cell-specific immunoliposomes containing the amphiphilic dipalmitoyl derivative of 5-fluorodeoxyuridine (FUdR-dP). FUdR-dP incorporated in immunoliposomes caused a 13-fold stronger inhibition of CC531 cell growth in vitro, during a 72-h treatment, than FUdR-dP in liposomes without antibody, demonstrating that the prodrug is efficiently hydrolysed to yield the active drug, FUdR, intracellularly. The intracellular release of active FUdR was confirmed by determining the fate of


Our studies of the metastatic behaviour of FSA1231 and FSA1233 show that the role of selection processes of metastatic variants at the secondary sites is minimal in the spontaneous metastasis of these systems. In contrast, tumour-cell release efficiency (number of blood-borne clonogenic tumour-cells) correlates well with the difference in spontaneous lung metastasis efficiencies of the cell clones FSA1231 and FSA1233. Also, the different tumour-cell release efficiencies could explain the discrepancy between artificial and spontaneous metastasis of these cell clones.


The paradigm that human malignancies are monoclonal has been questioned during recent years by the finding of unrelated, cytogenetically aberrant clones in short-term cultures from certain tumour types, notably carcinomas of the breast, skin and upper aerodigestive tract. In order to analyse whether cytogenetically unrelated clones are also unrelated at the molecular level, we analysed the X-chromosome inactivation status in cell cultures from a cytogenetically highly polyclonal acinic cell carcinoma of the parotid gland. By using cell cultures dominated by a single abnormal clone, obtained through in vitro culturing for 3-5 passages, we showed that the different clones must indeed have originated from different cells.


Recently we reported the variable presence of hypoxia adjacent to necrosis in human glioma lines grown as subcutaneous tumours in severe combined immunodeficient (SCID) mice. To assess the basis for this observation, we examined the pattern of oxygenation in M006 and M006XLo glioma spheroids. We found a wide range of binding of [3H]misonidazole to cells adjacent to the necrotic core, analogous to the patterns seen in xenografts, indicating substantial differences in the central oxygen tension of the spheroids. Clonal selection was used to isolate single cell-derived sublines of the M006XLo line. Some sublines gave spheroids that showed narrow distributions of [3H]misonidazole binding to the cells adjacent to necrosis, whereas other sublines showed a range of binding similar to that seen in spheroids of the parent line. After additional passages in monolayer culture, clonal sublines occasionally gave rise to spheroids in which the mean oxygen tension of cells adjacent to necrosis differed substantially from that of the initial spheroids. No relationship was evident between the thickness of the rim of viable cells and the presence or absence of central hypoxia, over a wide range of rim thickness. These results indicate that different oxygenation characteristics of glioma spheroids and tumour microregions are unlikely to arise from stable genetic variants coexisting in the parent line.


The HT29R human colonic adenocarcinoma cell line grows as locally invasive, mucin-secreting tumours in immunosuppressed mice with a doubling time of 6 days. These tumours may be disaggregated to give single-cell suspensions with plating efficiencies of 25-45% in technically simple in vivo-in vitro cell-survival assays. The effect of maximum tolerated doses of 5-fluorouracil, melphalan and cyclo-phosphamide on in situ growth is only slight. In vivo-in vitro cell-survival assays phosphamide on in situ growth is only slight. In vivo-in vitro cell-survival assays are consistent with these in situ results. The relative ease of experimental manipulation and the high clonogenic efficiency of this tumour make it a useful addition to human tumour xenograft models.


The ultrastructure of 16 tumours derived from spontaneously transformed cell lines established from young and old C57 and C3H mouse organs is described. Three types of tumour were found: myxoid (fibrosarcomatous), consisting of cells with long processes and much interstitial material; leiomyomatous, consisting of a bundle of smooth muscle-like cells with less interstitial material; or epithelial-like consisting of closely packed round cells with little interstitial material. The cell types in the tumours were similar to those found in the tissue culture cell lines from which they were derived.


Monolayer cultures have been established from a poorly differentiated carcinoma of the lung. Homogeneous cell growth and morphology have been maintained for over 18 months through more than 80 subculture passages, and the cells have been found to produce both immunoreactive calcitonin and an immunoreactive carcinoembryonic antigen-like material.


Treatment of non-small cell lung cancer (NSCLC) remains a difficult task in oncology. Targeted inhibition of oncogenic proteins is promising. In this study, we evaluate the expression of MET and PKCß and in vitro effects of their inhibition using SU11274 and enzastaurin (LY317615.HCl) respectively.Patient samples were analyzed by immunohistochemistry for expression of PKCß and MET, utilizing tissue microarrays under an IRB-approved protocol. Expression of PKCß and MET was evaluated in cell lines by immunoblotting. Treatment with SU1174 against MET and enzastaurin against PKCß was performed in H1993 and H358 cell lines, and cell proliferation and downstream signaling  were evaluated by immunoblotting. Statistical analysis was performed using SPSS 16.0.Expression of MET positively correlated with lymph node metastases (p=.0004), whereas PKCß showed no correlation (p=0.204). MET and PKCß expression were also strongly correlated (p<0.001). Expression of MET was observed in 5/8 cell lines , whereas PKCß expression was observed in 8/8 cell lines. Cell proliferation was significantly impaired by treatment with SU11274 and enzastaurin, and their effects were synergistic in combination (CI=0.32 and 0.09). Phosphorylation of MET, FAK, AKT, and GSK3ß were strongly inhibited with both agents in combination.Concomitant inhibition of MET and PKCß significantly increased cytotoxicity in vitro against NSCLC, disrupting important downstream signaling pathways. Further evaluation in animal models is warranted. Lung cancer is the most common cause of cancer mortality in the United States and worldwide.2 The disThe protein kinase C (PKC) family of serine-threonine protein kinases has been implicated in several important cellular functions including proliferation, motility, invasion, and apoptosis. Of the v12The c-MET receptor tyrosine kinase (MET) was originally identified as the cellular homologue of the TPR-MET oncoprotein. MET is oin vitro. Intracellular signaling networks were also evaluated for effects of the combination therapies.Combination therapies, especially those involving MET inhibition, have been increasingly studied as a treatment strategy in lung cancer.–24 GivenParaffin-embedded, formalin-fixed tumor tissues from patients with NSCLC with associated clinical information  were processed into a tissue microarray (TMA) under an Institutional Review Board-approved protocol (UCMC #9571 and #13473). Immunohistochemistry (IHC) was performed using biotin-free HRP-labeled polymer complex bound to secondary antibody , and performed according to previously published procedures. NegativeNCI-H522 (adenocarcinoma), NCI-H1703 (adenocarcinoma), A549 (adenosquamous carcinoma), NCI-H1993 (adenocarcinoma), NCI-H2170 (squamous cell carcinoma), NCI-H661 (large cell carcinoma), SW1573  and NCI-H358  cell lines were obtained from the American Type Culture Collection  All cell lines were cultured as per our established protocols.Enzastaurin was provided by Eli Lilly . Fetal bovine serum (FBS) was obtained from Gemini Bioproducts . SU11274 was obtained from EMD Biosciences . Cell culture media, penicillin, and streptomycin were obtained from Cellgro . Antibodies used included: anti-PKCß2, anti-Met, anti-phospho-FAK (Tyr925), anti-FAK, anti-phospho-AKT (Ser473) and anti-phospho-GSK3ß (Ser9) ; anti-phospho-Met (Tyr130/1234/1235) ; ß-actin monoclonal antibody . All other chemicals were purchased from Sigma .μM enzastaurin, 2.5 μM SU11274, or enzastaurin and SU11274 in combination (or DMSO as a negative control) for different durations as described, in serum-free media. Whole cell lysates were collected and immunoblotting was performed following routine protocols.[To examine protein expression in NSCLC cell lines under basal conditions, subconfluent cells were cultured in medium supplemented with 10% FBS. To detect the inhibition of cell transduction pathways, cells grown on 10-cm culture dishes for 24 hours were washed twice with PBS and incubated at 37°C with 2.5 rotocols. The sameCells were plated in 96-well plates at 5×10^3 cells per well in serum-containing media and grown for 24 hours. Drugs (or drug carrier) were added in serum-free media, and cells were incubated for 72 hours. Cell growth was estimated utilizing fluorometric readings after the addition of Alamar Blue , a non-radioactive, non-toxic dye that is reduced and whose fluorescence is proportional to cellular metabolic activity. A HT Synergy Plus microplate reader  was used to measure fluorescence (530-560 nm excitation wavelength and 590 nm emission). Drug synergism was estimated by the median-effect analysis using thComparisons between average means of protein expression were made by utilizing the Student's t-test for independent samples. For evaluation of differences in frequencies among different categories, the Chi-square test was used. The statistical software used was SPSS, version 16.0 .Evaluation of protein expression of PKCß and MET in the TMAs was performed. In order to further investigate the potential therapeutic implications of concomitant PKCß and MET inhibition, eight NSCLC cell lines were evaluated for protein expression by immunoblotting. MET expression was robustly observed in 5/8 cell lines , while PKCß expression was seen in all 8 cell lines .Cell growth inhibition with various drugs was evaluated in H1993 and H358 cell lines. After treating these cells for 72 hours with SU11274, enzastaurin, or both drugs in combination in increasing concentrations, it was observed that the combination was synergistic in causing significant effects on cell proliferation in both cell lines. Combinatorial indices for H358 cells at ED50 was 0.32, and for H1993 was 0.09, therefore proving significant synergistic effect .In order to investigate the molecular effects of treatment with SU11274 and/or enzastaurin, immunoblotting with phospho-specific antibodies was carried out in H1993 cells treated with these drugs for the time periods shown . TreatmeThere have been multiple examples of the utilization of a combination of targeted agents in lung cancer. Unfortunately, the accumulated experience has been largely unsuccessful in the treatment of this disease. Our work presents a combination that might prove to be biologically relevant, and perhaps have a reasonable toxicity profile to be investigated in the clinical setting. Herein, we have identified that MET and PKCß are expressed in lung cancer, and their inhibition can be synergistic. Interestingly, also, metastasis is common in lung cancer, and lymph node metastasis correlated with both MET and PKCß expression.For nearly two decades, MET has been known to be commonly aberrant in lung cancer, whether overexpressed,2829 ampl2834MET activation can lead to autophosphorylation or phosphorylation of downstream intermediates and activation of signaling pathways. As an example, in small cell lung cancer (SCLC), activation of MET with HGF leads to phosphorylation/activation of several pathways involving cell proliferation/survival , cell cycle (RB), and cytoskeletal proteins .in vitro and in vivo settings.[MET has also been shown to signal synergistically with EGFR as part of a broad signaling network that cooperatively drives activation of these downstream pathways.40 Indeedsettings.in vitro effect of enzastaurin against malignant pleural mesothelioma,[in vitro activity of enzastaurin and pemetrexed, a commonly used antifolate compound, has also revealed synergistic activity against SW1573 and A549 cell lines. Multiple biochemical pathways were shown to be affected, such as cell cycle control, apoptosis, and angiogenesis.[Inhibition of PKCß with enzastaurin has been recently studied in thoracic malignancies. Our previous work has demonstrated the thelioma, and its ogenesis. In anothogenesis. The effiogenesis. A phase ogenesis.in vitro assays. This finding was associated with decrease in activation of downstream effectors such as GSK3ß, AKT and FAK. These data suggest that concomitant inhibition of MET and PKCß may be an effective treatment strategy for NSCLC, especially for those patients whose tumors have developed prior tyrosine kinase resistance. These findings warrant further investigation in vivo to determine whether such a dual inhibition strategy is effective in reducing tumor progression.We have shown here that MET and PKCß tend to be coexpressed in NSCLC cell lines and tissues and that simultaneous inhibition of MET and PKCß significantly decreased cell proliferation in


Retinoids and vitamin D are important factors that regulate cellular growth and differentiation. An additive growth-inhibitory effect of retinoids and vitamin D analogues has been demonstrated for human myeloma, leukaemic and breast cancer cells. We set out to study the effects of the vitamin D analogue EB1089 and the retinoids all-trans- and 9-cis-retinoic acid on the human pancreatic adenocarcinoma cell lines Capan 1 and Capan 2 and the undifferentiated pancreatic carcinoma cell line Hs766T. The cell lines investigated expressed vitamin D receptor, retinoic acid receptor (RAR)-alpha and gamma as determined by polymerase chain reaction after reverse transcription. RAR-beta was expressed only in Hs766T cells. Addition of all-trans-retinoic acid increased the amount of RAR-alpha mRNA in the three cell lines and induced RAR-beta mRNA in Capan 1 and Capan 2 cells. All-trans-retinoic acid at a concentration of 10 nM inhibited the growth of Capan 1 and Capan 2 cells by 40% relative to controls. 9-cis-Retinoic acid was less effective. Neither all-trans-retinoic acid nor 9-cis-retinoic acid affected the growth of Hs766T cells. EB1089, if added alone to the cells, did not significantly inhibit growth. However, the combination of 1 nM EB1089 with 10 nM all-trans-retinoic acid exerted a growth-inhibitory effect of 90% in Capan 1 cells and of 70% in Capan 2 cells. Our data suggest that vitamin D analogues together with retinoids inhibit the growth of human pancreatic cancer cells. However, in vivo studies are necessary to examine the potential use of retinoids and vitamin D analogues on pancreatic cancer.


Verapamil, the prototype calcium channel blocker, reversibly inhibits cell proliferation in many normal and tumour cell lines . We have found that two closely related cell lines - B16 murine melanoma cells and B10.BR normal murine melanocytes growing in culture - behave differently in the presence of verapamil, and we are now utilising these two related cell lines to help elucidate the molecular basis of verapamil's antiproliferative effect. In this study, we studied cell cycle phase distribution and c-myc gene expression in both cell lines in the absence of verapamil, during incubation with verapamil and after the cells were washed free of verapamil. Our studies show that 100 microM verapamil rapidly blocks DNA synthesis in melanocytes but not in B16 cells. Similarly, incubation with verapamil for 6-24 h results in a decreased c-myc signal in melanocytes, but a transient increase in c-myc expression in B16 cells. After verapamil is washed from the cells following a 24-h incubation with drug, c-myc expression increases in melanocytes as they begin again to proliferate, but decreases in B16 cells as they begin to die. Our disparate results with these cell lines suggest that c-myc gene expression, regardless of its known involvement in growth control, is not the immediate target for verapamil's inhibitory action.


This study investigates the response to pemetrexed in a panel of eight mesothelioma cell lines and the clinical outcome for patients treated with pemetrexed in relation to folate receptor alpha (FR50). FRα expression was determined by western blotting and that of FRα, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) by real-time quantitative RT–PCR. Immunohistochemistry for FRα was carried out on 62 paraffin-embedded samples of mesothelioma from patients who were subsequently treated with pemetrexed.Cell lines were treated with pemetrexed to determine the concentration that reduced growth to 50%  and RS5 cells having a GI50 value greater than 10 μM. No FRα protein was detected in any cell line, and there was no relationship between sensitivity and expression of folate transporters. FRα was detected in 39% of tumour samples, generally in a small percentage of cells. There was no correlation between the presence of FRα and the outcome of pemetrexed treatment, and no significant difference between histological subtypes.A wide range of GIα.Response to treatment with pemetrexed does not depend on the presence of FR Malignant pleural mesothelioma (MPM) is an aggressive tumour of the pleura and usually has a poor prognosis. It is increasing in many countries worldwide as a result of widespread exposure to asbestos in the past and is expected to peak around 2015 in the United Kingdom . Inhibition of TS leads to depleted levels of thymidine, which are crucial to DNA synthesis. Pemetrexed also inhibits, to a lesser degree, glycinamide ribonucleotide formyltransferase (GARFT) (α) is an energy-dependent high-affinity, low-capacity folate-binding protein that delivers folates and antifolates into cells. The low capacity is due to endocytosis of the receptor that has to return to the cell surface in order to function. Expression is limited to specific tissues and is expressed in a characteristic distribution, for example, on syncytiotrophoblastic cells of the placenta and the proximal convoluted tubule of the kidney  and was 14-fold more sensitive when maintained in sub-physiological levels (1 nM leucovorin) (Folic acid and antifolates enter cells by three mechanisms. The reduced folate carrier (RFC) is a bidirectional anion exchanger that has a high affinity for reduced folate co-factors and antifolates, but a low affinity for folic acid. It has a neutral pH optimum and is ubiquitously expressed ( cancers . Howevercovorin) . Membranα to pemetrexed transport is likely to be significant in cells in which FRα is highly expressed. In these situations, transport of pemetrexed via FRα may enhance delivery of the drug to the tumour and potentially enhance response. Recently, a new monoclonal FRα antibody has been developed that can be used for immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) tissues  (Sigma-Aldrich) or 10% dialysed FBS (DFBS) . Dialysis of serum removes low molecular weight compounds such as thymidine and homocysteine, which may affect the response of cells to pemetrexed. The concentration of folic acid in the medium used is 2 μM. All cell lines were subcultured regularly and were negative for mycoplasma.Eight human mesothelioma cell lines were used in this study. Five cell lines were of epithelioid type: NCI-H28 (H28), NCI-H2052 (2052), NCI-H2452 (2452) , NCI-H226 (H226)  and JL1 ; MSTO-211H (MSTO) (ATCC) was of biphasic origin; two cell lines, DM3 and RS5 (DSMZ) were of sarcomatoid type. Cells were grown in RPMI 1640  containing 2 m3 cells per well and treated with 10 nM to 10 μM pemetrexed for approximately three cell doubling times, apart from the slower growing RS5 and DM3 cells that were treated for 6 days. The concentration of pemetrexed resulting in 50% growth inhibition (GI50) for each cell line was calculated using GraphPad Prism software.Growth-inhibition studies using pemetrexed were carried out using the SulfurRhodamine B (SRB) .Cell lysates required for western blotting were prepared after washing cells with ice-cold PBS and addition of lysis buffer  from each sample were loaded on a 4–20% gradient polyacrylamide gel (Invitrogen). After SDS–PAGE and electroblotting , as well as universal mastermix. The expected amplicon sizes were 89, 106, 72 and 187, respectively. PCR was performed as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min using the ABI Prism 7900HT sequence detection system (Applied Biosystems). Results were quantified relative to 18S, and calibrated to the IGROV1 FRα estimation.RNA was extracted from cells using the Qiagen RNAeasy minikit  and reverse transcribed using TaqMan reverse transcriptase reagents . A volume of 2.5 −2, i.v. administered over 3 h on day 1) and pemetrexed administered at a dose of 500 mg m−2 i.v. for 10 min on day 1. Cycles were repeated every 21 days to a maximum of six cycles. Patients also received folic acid supplementation at a dose of 400 μg daily, beginning at least 7 days before the first dose of pemetrexed and one injection of 1000 μg vitamin B12 intramuscularly 7–14 days before beginning the treatment. Folic acid was continued, and vitamin B12 was repeated every 12 weeks, throughout the treatment until 21 days after the last dose of pemetrexed. Dexamethasone, 4 mg twice daily, was given orally on the day before, the day of and the day after therapy. Our antiemetic regimen included a serotonin antagonist. Chemotherapy was discontinued in the event of disease progression, unacceptable toxicity or after completion of six cycles. Cycles were repeated at 100% of the intended doses if neutrophil and platelet counts were ⩾1.5 × 109 l−1 and ⩾100 × 109 l−1, respectively. If any dose reduction occurred, patients continued to receive the reduced dose throughout their treatment. All patients underwent planned radiographic evaluation.Eligible patients were identified from chemotherapy prescription records. All patients underwent histological diagnosis of malignant pleural mesothelioma and were treated according to our institutional protocol, in which carboplatin was administered to produce a value of the area under the curve (AUC) of 5, i.v. over 30 min on day 1  and allowed to dry at 56°C. The slides were dewaxed in xylene, rehydrated in graded ethanol and incubated for 10 min in 0.5% hydrogen peroxide solution to block endogenous peroxidase activity. The slides were then treated with an endogenous biotin blocking kit (Dako) before high-temperature antigen retrieval (microwaved thrice for 5 min in citrate buffer pH 6.0). Blocking with a 1 in 5 dilution of normal goat serum for 10 min preceded overnight incubation in a 1 in 20 dilution of FRα antibody (Sections of 4 Staining was graded independently by two assessors (JN and AR) using a composite index. The intensity of staining was scored 0–3, with 0 being no staining, 1 weak staining, 2 moderate staining and 3 intense staining. This was multiplied by the percentage of tumour area stained, using 10 for upto 10%, 25 for 11–25%, 50 for 26–50%, 75 for 51–75% and 100 for 76–100% of the tumour area stained, giving a composite score range of 0–300. The staining obtained in the renal tissue was used as a reference for intense staining, with a score of 300.α immunohistochemistry of tumour samples was correlated to clinical parameters such as response (objective decrease in tumour size on CT imaging), disease control rate , time-to-treatment failure  and overall survival .The composite score for the FRThis study was approved by the local research ethics committee and the Newcastle Hospitals Caldicott Guardian.50±s.d. value from three replicate experiments is shown in 50 values ranged from 14 nM in the H2452 epithelioid cell line to greater than 10 μM in the sarcomatoid cell line RS5. In most cases, apart from sarcomatoid RS5 cells, those grown in medium containing DFBS were more sensitive to pemetrexed than cells grown in medium containing normal FBS, with the largest difference observed in JL1 and DM3 cell lines. This is probably because of the removal of molecules such as folates, thymidine and homocysteine from the serum on dialysis, which are able to rescue cells from the effects of pemetrexed.Pemetrexed inhibited the growth of mesothelioma cell lines to variable extents. Representative graphs showing patterns of growth inhibition for each cell line in medium containing FBS or DFBS are shown in α protein in any of the mesothelioma cell lines, but was shown to be present at high levels in the IGROV1 ovarian cell line positive control. Similarly, the results from real-time PCR showed FRα mRNA to be undetectable in three cell lines , whereas extremely low levels at the limit of detection were present in the other mesothelioma cell lines. In these cell lines, the level of expression for FRα was approximately 100 times less than the highly expressing IGROV1 ovarian cancer cell line.The results for western blotting and quantitative RT–PCR are shown in 50 of pemetrexed in the cell lines.The results from real-time RT–PCR for other folate transporters, RFC and PCFT, in the cell lines are shown in In all, 62 patients were eligible for this study. A summary of demographics and treatment details is shown in α expression was considered to be positive when the composite score was >0. FRα expression was positive in 24 of the 62 samples examined . Both membranous and cytoplasmic staining was seen. When present, however, FRα expression was usually weak as 12 (50%) stained samples had a score of 30 or less. Only two samples had a composite score of more than 100. FRα expression was seen to be higher in the epithelioid disease subtype but was not significant , biphasic, n=6 (35%) and sarcomatoid, n=2 (22%), × 2 1.614, P=0.45).A sample of the positive control and various staining intensities are depicted in α. Responses were seen in 8 (33%) and 9 (24%) patients with positive and negative FRα-stained tumours , whereas 75 and 61%, respectively, had disease control . The TTF and OS rates were not statistically different between patients with positive FRα tumours and those that were negative , as shown in α.There were no significant differences in the proportion of patients with a response or disease control when subdivided into those with a positive or negative expression of FRα in the uptake of both folates  and antifolates has been the subject of numerous publications with contrasting results  were less sensitive to pemetrexed than the other cell lines. Clinically, sarcomatoid tumours tend to be more aggressive than epithelioid tumours . It has been reported that the effect of pemetrexed is enhanced in cell lines when there is a low or physiological concentration of folate in the medium  in mesothelioma of differing histological type. Following the development of an FRα antibody for use on paraffin-embedded tissues , involved in α expression in mesothelioma, or to the expression of RFC and PCFT in the cell lines. Other reasons for differences in sensitivity to pemetrexed remain to be established, but this study shows that there is no simple relationship between the sensitivity of cell lines to pemetrexed and the expression of any of the folate transporters; other potential molecular markers for pemetrexed response in mesothelioma are currently under investigation.In conclusion, the use of a varied panel of eight mesothelioma cell lines and the investigation of a large number of tumour samples from patients with mesothelioma treated with pemetrexed in this study demonstrate that there is no relationship between response to pemetrexed and FR


Cooperative synergistic effects by these two ligands have been reported, and it is, therefore, possible that greater therapeutic effects could be achieved if these compounds were administered together. The role of retinoid-dependent anti-activator protein 1 (anti-AP-1) effects in controlling cancer cell proliferation appears significant. We have utilized an anti- AP-1 retinoid , which does not transactivate through a retinoic acid response element (RARE), and a potent vitamin D3analogue  together at low, physiologically safer doses against a panel of prostate cancer cell lines that represent progressively more transformed phenotypes. The LNCaP (least transformed) and PC-3 (intermediately transformed) cell lines were synergistically inhibited in their clonal growth by the combination of LH and SR11238, whereas SR11238 alone was essentially inactive. DU-145 cells (most transformed) were completely insensitive to these analogues. LNCaP cells, but neither PC-3 nor DU-145, underwent apoptosis in the presence of LH and SR11238. Transactivation of the human osteocalcin vitamin D response element (VDRE) by LH was not enhanced in the presence of SR11238, although the expression of E-cadherin in these cells was additively up-regulated in the presence of both compounds. These data suggest the anti-AP-1 retinoid and the vitamin D3 analogue may naturally act synergistically to control cell proliferation, a process that is interrupted during transformation, and that this combination may form the basis for treatment of some androgen-independent prostate cancer. © 1999 Cancer Research CampaignThe secosteroid hormones, all-


Decreased accumulation of the fluorescent dye BCECF  characterized murine and human multidrug-resistant cell lines overexpressing the multidrug resistance protein (MRP). Indomethacin (10 microM), a known cyclo-oxygenase and glutathione-S-transferase inhibitor as well as a modulator of anion transport, increased accumulation and blocked efflux of BCECF in MRP-expressing murine and human cells. The drug did not affect P-glycoprotein (P-gp)-mediated export of rhodamine 123. The indomethacin effect on BCECF efflux was not reversed by the addition of exogenous prostaglandins, suggesting that the drug acts by a mechanism other than decreasing prostaglandin synthesis. Indomethacin also increased multidrug susceptibility of both murine and human cell lines overexpressing MRP, but not those displaying P-gp-associated resistance. In addition, indomethacin modulated the decreased vincristine accumulation in cells expressing MRP, but not in those expressing P-gp. These data suggest that indomethacin is a specific inhibitor of MRP, possibly functioning by inhibition of glutathione-S-transferase or, alternatively, by direct competition with the drug at the transport site.


Attempts have been made by a number of methods to eliminate minimal residual disease from bone marrow to be reinfused in autologous transplantation. In this paper we describe a conjugate containing a monoclonal antibody, named 8A, recognising a plasma cell-associated antigen, and momordin, a ribosome-inactivating protein similar to the ricin A-chain. This immunotoxin is active on target cell lines and on neoplastic plasma cells, while myeloid progenitors are fairly resistant. The conjugate is shown to be acceptable for ex vivo purging in autologous bone marrow transplantation in multiple myeloma patients.


Thromboplastic and fibrinolytic activities of 14 lines of cultured human cancer cells were estimated by modified Astrup's methods. High tissue thromboplastic activity was found in one line of urinary-bladder cancer, 2 lines of gastric cancer and one line of lung cancer, but no activity was found in 6 lines of lung cancer. High fibrinolytic activity was noted in one line of gastric cancer and 2 lines of lung cancer, but no activity was seen in 6 lines of lung cancer and one line of gastric cancer. No plasmin activity was found. The tumour cell lines could be classified into 3 groups on the basis of the 2 activities. Cancer cell lines could also be classified into 2 groups: with high or low release of thromboplastin into culture media. Fibrinolytic activity was found in the culture media of all cell lines with high fibrinolytic activity. Fibrinolytic activity, but not thromboplastic activity, seemed to be influenced by the constituents of culture media. No definite correlation was found between the 2 activities and the histological types of the parent tumours of the cultured cells.