In the present review, we concisely explore the conceptual framework of stem cell biology and recent advances pertinent to the lungs. We illustrate lung diseases in which manipulation of stem cells may be physiologically significant and highlight the challenges facing stem cell-related therapy in the lung.Fueled by the promise of regenerative medicine, currently there is unprecedented interest in stem cells. Furthermore, there have been revolutionary, but somewhat controversial, advances in our understanding of stem cell biology. Stem cells likely play key roles in the repair of diverse lung injuries. However, due to very low rates of cellular proliferation  According to Greek mythology, the immortal Prometheus stole fire from the Gods as a gift for humankind. As punishment, he was shackled to a rock, whereupon each day for 30,000 years an eagle consumed as much of his liver as would regenerate. There is some debate whether the eagle ate his liver or heart, but what if the bird had a taste for lung? And what if Prometheus was a mere mortal?Analogous to Prometheus and the eagle, the ambient air-exposed lung is subject to an array of potentially damaging agents, including chemical oxidants and proteolytic enzymes. Presumably, daily oxidant and protease wear and tear on structural components such as elastin and collagen contributes to inevitable age-related declines in pulmonary function in normal individuals ,2. Acute. During embryonic development, the inner cell mass of the blastocyst forms three primary germ layers, which generate all fetal tissue lineages , or preparations enriched for hematopoietic stem cells (HSC). Whole body irradiation, which may injure lung tissue, is typically used to deplete the host bone marrow. Importantly, lung injury apparently enhances engraftment into lung -29. Wholculation .Sex-mismatched lung and bone marrow transplantation in humans provides a natural model for analysis of donor and recipient cell behavior. Bronchial epithelial and gland cells and type II pneumocytes of host origin were reported in one study of lung allografts , but notin vitro and in vivo, which likely explains why some of the cells contain both donor and lung cell markers [see [Many questions remain unanswered. The mechanism whereby cells assume lung cell phenotypes remains uncertain. Several studies have demonstrated that cell fusion occurs both ers [see  for a sters [see ,41 for rers [see . New, moers [see  and the ers [see , while aers [see . MitigatMajor lung diseases likely involving stem cells and the cellular targets for stem cell therapy are summarized in Table in vivo [What are the realistic prospects for beneficial stem cell therapy of the lung? First, we must conclusively identify lung diseases/cases/timing in which cell and tissue damage occurs in excess of the capacity for timely endogenous repair. Second, we must establish standardized sources of relevant stem/progenitor cells and methods for their delivery to the appropriate lung sub-compartment. Once delivered, therapeutic cells must home to microscopic sites of need and integrate to serve a beneficial function. There is clearly potential for adverse effects, as exemplified by the propensity of embryonic stem cells to form teratomas when implanted in vivo . Major lProvocative, but controversial, recent evidence suggests that circulating stem cells may home to the lung. There is great excitement and hope that exogenous and/or mobilized endogenous stem cells may be harnessed to prevent or treat acute and chronic lung diseases and even regenerate abnormally developed or lost tissue. Our understanding of lung stem cells and the regulation of lung morphogenesis is still rudimentary, and the complex, integrated function of multiple cell types underlying normal lung structure and function poses unique challenges. Thus, the therapeutic prospects for stem cell therapy in lungs appear more distant than in some other organs. This realization should stimulate meaningful new studies from the lung research community. Unlike the mythical hero Prometheus, patients with lung disease cannot wait 30,000 years!None declared.


The urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC), the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype.We screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS), fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers.These data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC. Small cell lung cancer (SCLC) is the most aggressive type of lung cancer and has a uniformly poor prognosis. Metastases develop quickly, primarily to bone marrow and brain, and are usually present at the time of diagnosis. In untreated patients, median survival is two months from the onset of symptoms In several types of tumors increased levels of urokinase plasminogen activator (uPA) and its receptor uPAR (CD87) strongly correlate with poor prognosis and unfavorable clinical outcome et al underlines the importance of uPAR signaling in prevention of apoptosis by resistance of cancer cells to anoikis (apoptosis induced by loss of anchorage). uPAR expression promotes cell survival by activating anti-apoptosis factor Bcl-xL transcription through the MEK/ERK- and PI3K/Akt-dependent pathways A recent study by Alfano We report here the presence of a rare population of uPAR-positive cells in human SCLC cell lines that demonstrate significant drug resistance to traditional chemotherapeutic agents such as 5-fluorouracil (5-FU), cisplatin and etoposide. The uPAR-positive cells expressed stem- and cancer cell markers, including CD44 and MDR1. Identification and targeting of uPAR-positive cells in SCLC may provide valuable insight into biology of human lung cancer and may establish novel critical targets for more effective anticancer therapies.6 cells/ml. Fifty µl (2.5×104 cells) was added to each well of a 96-well v-shaped plate. Antibodies (FITC- or PE-conjugated) were added in concentrations recommended by the manufacturer (20 µl/106 cells). Antibodies to CD133, CD34, CD44, CD87 and MDR1 have been individually titrated. The 96-well plates were placed on ice and cells were stained with antibodies for 30 min in dark. After staining, 150 µl of wash buffer /well was added and the plates were centrifuged at 500× g for 5 min at 4°C. The cell pellets were re-suspended in SB, supplemented with propidium iodide (PI) (1 µg/ml) to exclude nonviable cells, followed by flow cytometric analysis.Primary (lung) small cell lung carcinoma (SCLC) cell lines , bone marrow (BM) metastatic SCLC  and brain metastatic SCLC (H250) cell lines were obtained from human primary lung and metastatic tissues ( ATCC), grown in RPMI 1640 modified medium  supplemented with 10% Fetal Bovine Serum (FBS). The BM metastatic cell line (H1882) was cultured in complete HITES medium . Cells were grown for two weeks and were analyzed by flow cytometry using the following antibodies: CD59 (CBL467P), CD109 (CBL585P), CD62E (CBL180F) from Chemicon, CD87 (3936CJ) from American Diagnostica, CXCR4 (FAB170F) from R&D Systems, CD24 (555427), CD90 (555596), CD38 (347680), CD44 (555478), CD45 (555482), CD13 (555394), CD49b (555498), CD29 (555443), CD3 (30104X) from BD Pharmingen, ABCG2/BCRP1 (10400) from Stem Cell Technologies, CD133/2 (clone 293C3) and CD133/1 (clone AC133) from Miltenyi Biotec, CD34 (347660) from Becton Dickinson, CD105 (326–050) from Alexis, MNF116 (F0859), Cyt18 (F7212) from DACO, and CD166 (3FT) from RDI. For FACS analysis each cell line was detached by trypsinization and re-suspended in staining buffer (SB)  supplemented with 2% FBS and 10 mM HEPES at a density of 5×106 cells were collected and then re-suspended in 800 µl of SB, followed by staining with uPAR (CD87)-FITC-conjugated antibody as described above. uPAR-positive and uPAR-negative cells were sorted by FACS, followed by culture in “base” methylcellulose media  for 16 days at 37°C, 5% CO2.SCLC cell lines  were grown in RPMI 1640 medium and 4×103, 1×103, or 1×102 cells/ml. Cells were cultured at 37°C, 5% CO2 in a humidified incubator for 16 days. Colonies were counted using an inverted brightfield microscope at 4× magnification.Complete RPMI 1640 was added to MethoCult H4100 medium (40 ml) to achieve a final volume of 100 ml. uPAR-positive and uPAR-negative cells derived from SCLC cell lines  were plated in MethoCult medium in triplicates containing 3×103 cells/well, in triplicates) with final concentrations of drugs 0, 3, 10, 100, 200 µg/ml. After incubation for 72 hr, both viable and dead cells were counted by using Guava ViaCount assay, and only the viable cells were included in data analysis. The Guava ViaCount assay distinguishes between viable and non-viable cell based on the differential permeability of DNA-binding dyes in the ViaCount reagent, and thus fluorescence of the dyes allows quantitative assessment of both viable and non-viable cells in suspension. Alternatively, non-sorted cells were applied to 48-well plates (1×104 cells/well) and treated with cisplatin, etoposide and their combination in concentrations of 0, 3, 10, 100 µg/ml. The seeding densities per unit area (mm2) were the same for both the 48- and 96-well plates (density = 125 cells/mm2). After 72 hrs of incubation, viable cells were stained with uPAR-FITC antibodies and evaluated by flow cytometry.Cells derived from three SCLC lines were immunostained and sorted by flow-cytometry analysis (as described above). Cell lines  were counted and placed into 96-well plates (4×105 cells/1 µg of antibody). Iso-type matched PE- or FITC-conjugated antibodies were used as controls. Stained cells were analyzed by flow cytometry.Cell lines were stained with CD44-PE and MDR1-PE, were washed and then stained with uPAR-FITC  with a panel of antibodies for surface determinants of tumor cells, including uPAR, CD13, CD29, CD44, CXCR4, CD105, CD109, CD166, and for stem cell markers CD34, CD90, CD133, ABCG2/BCRP1. SCLC cell lines displayed heterogeneous phenotypes with regard to tumor surface determinants. All six SCLC cell lines were positive for CD29 (20–99%), CD44 (8–98%), CD105 (3–34%), CD166 (85–98%), and negative for CD90, and CXCR4. uPAR (CD87) was the only cell surface antigen expressed on a small sub-population of cells (1–4%) in each of the six SCLC cell lines, when analyzed by FACS using anti-uPAR-FITC antibody  (We hypothesized that the uPAR-expressing cell population may be resistant to chemotherapeutic agents. We performed cytotoxicity assays on three selected cell lines using non-sorted (bulk) as well as sorted uPAR-positive and uPAR-negative cell populations. Increasing concentrations of 5-fluorouracil (5-FU)  were added to these cell cultures and incubated for 72 hrs, followed by counting of both viable and killed cells. Data was normalized to 100%, which signified the number of uPAR-positive and uPAR-negative cells without drug added. A cell-killing effect was detected in all three non-sorted cell lines , where 40–80% of cells were killed by 5-FU . Importactively) .in vitro studies   were used in cytotoxic assays with cisplatin and etoposide in 0 µg/ml) . 60–80% 0 µg/ml) . Again, In order to confirm that uPAR expression confers tumor resistance to cisplatin and etoposide, we performed cytotoxicity assays on the same non-sorted SCLC cell lines and looked for enrichment of uPAR-positive cells in the surviving cell population. Indeed, we detected a significant enrichment of uPAR-positive cells (40–70%) among surviving cells, compared to only 1–5% of uPAR-positive cells in control cultures grown in the absence of these drugs . These dTo show that uPAR-positive cells possess high clonogenic and self-renewal potential, we sorted primary lung , metastatic bone marrow (H211) cell lines by FACS using anti-uPAR-FITC-conjugated monoclonal antibodies. After sorting (97% purity), uPAR-positive and uPAR-negative cells were plated in methylcellulose media at densities of 3000, 1000, 100 cells/well (6-well plate). uPAR-positive cells from these primary and metastatic SCLC lines formed multiple distinct colonies . We estiWe further hypothesized that if uPAR-positive cells represent drug-resistant and clonogenic population in SCLC, they may express CD44 and MDR1 (ABCB1) genes, which are involved in development of cancer stem cell phenotype and multi-drug resistance. To further characterize the uPAR-positive population, we performed double-labeling experiments with uPAR-FITC, and CD44-PE as well as MDR1-PE on H211, H69AR, H1417 cell lines. uPAR-positive cells derived from SCLC cell lines  expressed CD44 on 50–80%, whereas MDR1 on 10–40% of cells . ExpressThe main finding of this study is identification of a rare subpopulation of uPAR-positive cells in SCLC cell lines derived from lung and metastasis to bone marrow and brain. It is well documented that uPAR over-expression in various malignant tumors is strongly correlated with most invasive cancer phenotypes and poor prognosis Several studies suggest the presence of a rare population of cells in solid tumors and leukemia that possess the capability to regenerate and propagate the tumor We found that uPAR-positive cells were more resistant to treatment with the cytotoxic agent 5-FU, while uPAR-negative cells were killed much more efficiently. The cytotoxic effect of 5-FU has been ascribed to misincorporation of fluoronucleotides into RNA and DNA and to inhibition of the nucleotide synthetic enzyme thymidylate synthase, which mainly targets the fast dividing cells uPAR-positive cells isolated from three SCLC lines were able to proliferate and form multiple colonies in methylcellulose media, while uPAR-negative cells displayed little or no clonogenic potential. We also showed co-expression of uPAR with CD44 and MDR1, which may explain the association between advanced malignancy and drug resistance. Several studies have investigated individually the role of uPAR, CD44 and MDR1 in various malignancies ATP-binding cassette (ABC) drug transporters have been shown to protect cancer stem cells from chemotherapeutic agents Future studies in animal models will address whether the uPAR-positive cells are responsible for primary tumor growth and formation of distant metastases in SCLC. In summary, we have identified the uPAR-positive subpopulation of SCLC cells that possess high multi-drug resistance and clonogenic activity, while the uPAR-negative cells are sensitive to chemotherapeutic drugs and display little or no clonogenic activity. We also provide evidence of association of uPAR, CD44 and MDR1 expression on SCLC cells. Further investigation is warranted to determine if targeting of this cell population will be critical for therapeutic success.


A subline (COR-L23/R) of the human large cell lung line [corrected] COR-L23, derived by in vivo exposure to doxorubicin, exhibits an unusual multidrug resistant (MDR) phenotype. This subline shows cross-resistance to daunorubicin, vincristine, colchicine and etoposide but does not express P-glycoprotein. Interestingly, COR-L23/R [corrected] shows little or no resistance to a range of structurally-modified analogues of doxorubicin comprising 9-alkyl and/or sugar modified anthracyclines. We have previously identified these same compounds as effective agents against P-glycoprotein-positive MDR cell lines. In contrast to typical MDR cell lines, COR-L23/R [corrected] shows only minimal chemosensitisation by verapamil and no collateral sensitivity to verapamil. Compared to the parental cell line, COR-L23/R [corrected] displays reduced accumulation of doxorubicin and daunorubicin. Accumulation defects were apparent only after 0.5-1 h of incubation of cells with these agents. The rate of daunorubicin efflux was shown to be enhanced by COR-L23/R [corrected] and this efflux was demonstrated to be energy-dependent. The use of anthracyclines which retain activity in MDR cells thus appears to be a valid approach for the circumvention of MDR, not only in cells which express P-glycoprotein, but also where defective drug accumulation is due to other mechanisms possibly involving an alternative multidrug transporter.


In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines  by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines  or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects.


A new cell line (CAL51) was isolated from a malignant pleural effusion of a woman with metastatic breast cancer. These cells grow in continuous culture and exhibit the morphological, ultrastructural and immunohistochemical features of epithelial cells of mammary origin. They are tumorigenic in nude mice and clone in soft agar. Oestrogen receptors are not detected. CAL51 consists of a homogeneous population of cells with normal chromosomes even after the use of high resolution banding. Cytogenetic analysis of the cells from the tumour induced by CAL51 in the nude mouse confirmed the normality and the stability of the karyotype. All breast cancer cell lines established to date present abnormal karyotypes; CAL51 cell line may be more informative than cell lines with aberrant karyotypes for investigating essential genetic differences between normal and malignant mammary gland cells.


Viscum album) are the form of cancer treatment that is most frequently used in the complementary medicine. Previous work has shown that these preparations are able to exert cytotoxic effects on carcinoma cells, the extent of which might be influenced by the host tree species and by the content of mistletoe lectin.Preparations of mistletoe  obtained from mistletoe growing on oak , apple tree , pine  or white fir , on the in vitro growth of breast and bladder carcinoma cell lines. While MFM-223, KPL-1, MCF-7 and HCC-1937 were the breast carcinoma cell lines chosen, the panel of tested bladder carcinoma cells comprised the T-24, TCC-SUP, UM-UC-3 and J-82 cell lines.Using colorimetric assays, we have now compared the cytotoxic effects of 50) varied between 0.6 and 0.03 mg/ml. Higher concentrations of VAP-P were required to obtain a comparable effect. Purified mistletoe lectin I (MLI) led to an inhibition of breast carcinoma cell growth at concentrations lower than those of VAPs, but the sensitivity towards purified MLI did not parallel that towards VAPs. Bladder carcinoma cells were in most cases more sensitive to VAPs treatment than breast carcinoma cells. The total mistletoe lectin content was very high in VAP-Qu (54 ng/mg extract), intermediate in VAP-M (25 ng/mg extract), and very low in VAP-P (1.3 ng/mg extract) and in VAP-A (1 ng/mg extract). As to be expected from the low content of mistletoe lectin, VAP-P led to relatively weak cytotoxic effects. Most remarkably, however, the lectin-poor VAP-A revealed a cytotoxic effect comparable to, or even stronger than, that of the lectin-rich VAP-Qu, on all tested bladder and breast carcinoma cell lines.Each of the VAPs inhibited cell growth, but the extent of this inhibition differed with the preparation and with the cell line. The concentrations of VAP-Qu, VAP-M and VAP-A which led to a 50 % reduction of cell growth (ICThe results suggest the existence of cytotoxic components other than mistletoe lectin in VAP-A and reveal an unexpected potential of this preparation for the treatment of breast and bladder cancer. Breast cancer ranks as the most frequently diagnosed form of malignant disease and the second most relevant cause of cancer-related death in women living in Europe and North America . The proIn the United States of America, bladder cancer is the sixth most common malignant disease and the ninth leading cause of cancer-related deaths . The majViscum album preparations (VAPs) are the most commonly used form of complementary/alternative cancer therapy. These preparations are often used in the adjuvant setting, together with standard chemo- or radiotherapy [Given that the conventional therapies often lead to only partial success, a relatively high proportion of cancer patients have been trying treatments originating from complementary and alternative medicine. In the case of breast cancer, a recent clinical study involving patients from 11 European countries revealed that some form of complementary/alternative treatments had been used in 45 % of the cases . Viscum otherapy . Post-opotherapy ,7.In vitro experiments with cell lines and with primary cultures have shown that the various VAPs can be cytotoxic to a variety of carcinoma cells, either through the activation of the apoptotic cascade, or by leading to necrosis [in vitro toxicity of VAPs does not always correlate with their lectin content, suggesting that other components [necrosis -11. Furtnecrosis -16. Prepnecrosis . Furthernecrosis . The cytnecrosis ,19. Nevemponents  might almponents ,17.in vitro growth of several breast and bladder carcinoma cell lines were studied.In the present study, the effects of VAP-A, VAP-M, VAP-P and VAP-Qu obtained from mistletoe growing on Abies (white fir), Malus (apple tree), Pinus (pine), and Quercus (oak), respectively, on the 2SO4-precipitates of extracts from V. album ssp. album grown on apple trees (elution by 0.1 M lactose); MLI was not contaminated by MLII and MLIII as controlled by SDS-gel-electrophoresis; the concentration of MLI was determined using an ELLA-test. 3--2, 5-diphenyltetrazolium bromide) (MTT) and fetal calf serum (FCS) were purchased from Sigma Chemical Co. Aldrich (Switzerland), dimethyl sulfoxide (DMSO) and trypan blue from Fluka (Switzerland), Dulbecco's Modified Eagle's Medium high glucose (DMEM), RPMI 1640, Dulbecco's phosphate buffered saline (PBS) and trypsin-EDTA solution (0.25% trypsin/0.05% EDTA) from AMIMED BioConcept (Switzerland). The cell lines MFM-223 (ACC 422), KPL-1 (ACC 317), MCF-7 (ACC 115), HCC-1937 (ACC 513), T-24 (ACC 376), TCC-SUP (ACC 377) were supplied by DSMZ , whereas the cell lines UM-UC-3 (CRL-1749) and J-82 (HTB-1) were purchased at ATCC .VAP-Qu (Charge Nr. 41011), VAP-M (Charge Nr. 30611), VAP-P (Charge Nr. 40511) and VAP-A (Charge Nr. 41111), as well as the isolated MLI were provided by Hiscia AG ; total mistletoe lectin concentrations of the preparations were determined by the manufacturer. MLI was isolated by affinity chromatography on lactose-Sepharose using (NH4)2. The cells, all of which exhibited an adherent morphology, were sub-cultured twice a week by washing with PBS and digesting the monolayer with trypsin/EDTA at 37°C for 5 min.All cell lines were cultured as recommended by the suppliers and kept at 37°C in a humidified atmosphere with 5% CO5 cells/ml in 100 μl/well , in a 96-well polycarbonate plate and incubated for 24 hours at 37°C and 5 % CO2. Thereafter, the cells were exposed to different concentrations of appropriate dilutions of VAPs, purified MLI or blank medium (control) for 48 h, in a total volume of 200 μl/well. The previously described MTT-colorimetric assay was then used to estimate cell growth [Cells were seeded at the density of 1050) were collected in colorimetric MTT-assays performed as described above. The assays included the following concentrations of mistletoe preparations: 4, 2, 1, 0.5, 0.1, 0.01 and 0.001 mg/ml. In the case of the assays with MLI, 1000, 100, 10 and 1 ng/ml were tested. Computer-aided curve fitting and statistical analyses were performed using the commercially available software package IGOR Pro . The curves were modeled using the following sigmoid fit for a logarithmic scale: y = A2 + (A1-A2)/(1 + (x/x0)^p). The confidence level was set at 68 %.The data used for determining the concentrations of VAPs and of MLI which resulted in a 50% inhibition of cell growth , HCC-1937 cells to VAP-M (IC50 of 0.10 mg/ml) and VAP-Qu (IC50 of 0.11 mg/ml), and KPL-1 cells to VAP-Qu .Fig. rvations , VAP-P e50 values of MLI calculated for the various breast carcinoma cell lines are depicted in Table Since MLI has often been considered to be responsible for the cytotoxic effect of mistletoe extracts, the inhibitory effect of isolated MLI on breast carcinoma cell growth was studied. Purification and characterization of ML I was performed as described under Methods. The IC50 values obtained with the various bladder carcinoma cells were determined after 48 hours of treatment and are depicted in Table 50 values of the VAPs ranged between 0.03 and 0.60 mg/ml  the sensitivity profile towards isolated MLI did not parallel that towards VAPs, and b) the mistletoe lectin content of the various extracts was not always proportional to the corresponding cytotoxic effect.The ICThe hypothesis that the mistletoe lectin content of a preparation determines the magnitude of its inhibitory effect on carcinoma cell growth seems to be confirmed by the data obtained with VAP-P. This preparation, which had a very low level of mistletoe lectin (1.3 ng/mg extract, Table Should the effect of VAP-A be mainly mistletoe lectin-independent and those of the other preparations mainly mistletoe lectin-dependent, one would expect that VAP-A would induce a different set of intracellular processes than VAP-Qu or VAP-M. In line with these expectations, very recent data on the gene expression profile in breast carcinoma cells indicate that VAP-A induces molecular changes on the cell-cell adhesion and cytoskeleton pathways, while VAP-Qu and VAP-M mainly affect the immune defense and stress response genes .Viscum album in breast and in bladder cancer therapy. To our knowledge, and in contrast to the situation in breast cancer, the data concerning the clinical use of mistletoe preparations on bladder cancer, even though very promising, are scarce [Our work suggests that the differences among the various VAPs might have been so far underestimated and that the assumption that their cytotoxic effect relies exclusively on the mistletoe lectin content is likely to be a simplification. It is conceivable that VAP-A opens new possibilities to explore the full potential of e scarce . The potWithin the last five years AV, LR and APSW have received occasional research funding from the pharmaceutical company Weleda AG, who produces VAP. MR has received salaries from this company. The other authors declare that they have no competing interests.JE coordinated the practical part of the project and carried out the experiments with the breast carcinoma cell lines, LB performed the data analysis, BS and DP carried out the cytotoxicity assays with the bladder carcinoma cell lines. MR provided scientific support. KU isolated and purified ML I. AV conceived the study and helped to draft the manuscript. APSW drafted and wrote the manuscript with LR. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The rapidly growing field of targeted tumor therapy often utilizes an antibody, sometimes tagged with a tumor-ablating material such as radioisotope, directed against a specific molecule.32P to colon cancer cells. The decapeptides vary from one another by one to three amino acids and demonstrate vastly different binding abilities. The most avidly binding decapeptide can permanently deliver very high levels of radioisotope to the adenocarcinoma cancer cell lines at an efficiency 35 to 150 times greater than to a variety of other cell types, including cell lines derived from other types of cancer or from normal tissue.This report describes the discovery of nine novel decapeptides which can be radioactively labeled, bind to, and deliver This experimental approach represents a new example of a strategy, termed peptide binding therapy, for the potential treatment of colorectal and other adenocarcinomas. Both utilize a monoclonal antibody directed against the B-cell marker CD20 and can deliver either 32P) to cell lines derived from several colorectal carcinomas. Under identical experimental conditions, very little  of the most efficient 32P-labeled decapeptides bind to cell lines established from a variety of other cancers or to normal colon, kidney, or esophageal cells.The decapeptides described herein bind to and transfer isotope . 32P-labeled peptides and the experimental design of assays to measure binding of peptides to cell lines.We have identified nine decapeptides, differing from one another by only a few amino acids, that when labeled with 32P counts per minute (cpm) remaining bound to eighteen different cell lines and blank wells after a two hour incubation with MA5, the most efficient binding decapeptide (see below). The Caco-2 colon adenocarcinoma cell line retained the greatest number of radioactive counts after a two-hour incubation and subsequent washes with complete medium, the average value of triplicate wells equaling 298,639 cpm per 10,000 cells. HCT116 colon adenocarcinoma cells retained an average value of 131,998 cpm per 10,000 cells. Blank wells and nonbinding cell lines had mean values of less than 550 cpm; bars representing these values are not visible at the scale used in Modified Adjuvant radioactive peptide) with five of these being colon adenocarcinoma cell lines , one being an esophageal adenocarcinoma cell line (SEG1), and one being a Barrett's esophagus cell line (QHTRT). In contrast, the eleven nonbinding cell lines were mostly squamous cell lines derived from carcinomas of the cervix (HeLa S3), colon (RKO), lung , esophagus (KYSE-70), a fibrosacroma (HT1080), or cells cultured from normal kidney (293H), colon (1459), or esophagus (HEEpiC). Nonbinding cell lines included T84, derived from a colon adenocarcinoma metastatic to lung, and SK-BR-3, isolated from a breast adenocarcinoma. The ratio of cpm retained by Caco-2  to the average of the eleven nonbinding cell lines (365) was 818∶1. Caco-2 cells retained approximately 18% of the total radioactive counts present in the incubation well after two-hour incubation.Seven of the eighteen cell lines demonstrated very strong retention of radioactivity when incubated with MA5 , still revealed similar levels of 32P-decapeptide release and retention for all nine MAs, as described for MA5 in To investigate how quickly overnight at 37°C. i.e., when extrapolated over a potential 2-week exposure period, equivalent to over 87,000 counts per cell).The peptide binding, washing and assay experiment described for 32P-labeled MA5 released into the medium is shown in 32P) and can bind avidly to several different adenocarcinoma cell lines, efficiently delivering this potential tumor-ablating material to the cells. The decapeptides, termed MA for Modified Adjuvant, are protein kinase substrates. Previously, it had never been shown or suspected that this substrate, when labeled with a tumor-ablating material such as 32P, could bind to and transfer the radioisotope to a cell line after one to two hours of incubation. Moreover, we have shown for the first time that transfer of isotope from these decapeptides is restricted to cell types derived from primary colon and esophageal adenocarcinomas. For example, exposure of certain colon cancer cell lines (e.g. Caco-2) to the most avidly binding labeled peptide, MA5, for a two-hour period resulted in the transfer of a radioactive dose of over 29 counts per minute per cell after a two hour incubation, wash, and immediate determination of the retained radioactivity.This report describes the discovery of decapeptides that can be labeled with a high energy (1.7 Mev) beta emitter  which yield various decapeptides after thrombin cleavage designated MA1 through MA9 (Modified Adjuvant). The protein sequences are: MA1, GSRRASVGSA; MA2, GSRGASVGGA; MA3, GSRRGSVGSA; MA4, GSRRGSVASA; MA5, GSRRASVASA; MA6, GSRRASVGSG; MA7, GSRGGSVGSA; MA8, GSRGGSVASA; MA9, GSRGGSVGSG. DH5-α bacteria containing these clones were grown overnight in LB (containing 100 µg/ml ampicillin), diluted 1/10 in LB-Amp and grown at 37°C for two hours. IPTG was added to 1 mM and the culture grown at 37°C for five hours. Ten ml of each culture were centrifuged and the cell pellet resuspended in 1 X TBS containing 100 µg/ml lysozyme. After two cycles of freeze-thaw, the lysate was centrifuged and the supernatant was mixed with 100 µl of Sepharose-Glutathione for two hours at RT. Each pellet was washed three times with 1 X TBS, and the bound recombinant fusion proteins were labeled with 32P using protein kinase A and 32P-γ-ATP according to the manufacturer's instructions . The pellet was washed four times with 1 X PBS and the labeled decapeptide was cleaved and released into the supernatant with thrombin .Production of the 32P-labeled decapeptides to cell lines: Cell lines were grown in complete medium containing 10% bovine fetal serum (heat inactivated). In each well of a 96-well plate, 10,000 cells from various cell lines were grown overnight in complete medium. Ten µl of the labeled-peptide in 1 X PBS and 90 µl of complete medium were added to each well and incubated at 37°C at various times of up to two hours. The peptide-medium was removed and one µl added to 100 ul gel loading buffer and counted by scintillation counting for the probe control or run on a polyacrylamide-SDS gel (Biorad).The adherent cells were briefly and gently washed with complete medium three times and some wells were assayed immediately by adding 100 µl of gel loading buffer to each well and run on a gel or counted in a scintillation counter. Other wells had 100 µl complete medium added and incubated for a further time period. Samples were either counted in a liquid scintillation counter or run on polyacrylamide-SDS gels, exposed to x-ray film, and the film developed.Assay of the binding of


In this study, we investigate the anti-tumor effect of YM529 on non-small cell lung cancer (NSCLC).YM529 is a newly developed nitrogen-containing bisphosphonate (BP) classified as a third-generation BP that shows a 100-fold greater potency against bone resorption than pamidronate, a second-generation BP. This agent is, therefore expected to be extremely useful clinically for the treatment of osteoporosis and hypercalcemia. Recently, YM529 as well as other third-generation BPs have also been shown to exert anti-tumor effects against various types of cancer cells both 50) values. YM529 induced apoptosis of NCI-H1819 was examined by DNA fragmentation of 2 % agarose gel electrophoresis and flowcytometric analysis (sub-G1 method). We examined where YM529 given effect to apoptosis of NSCLC cells in signaling pathway of the mevalonate pathway by western blotting analysis.Direct anti-tumor effect of YM529 against 8 NSCLC cell lines  were measured by MTS assay and calculated inhibition concentration 50 % .We found that there was direct anti-tumor effect of YM529 on 8 NSCLC cell lines in a dose-dependent manner and their ICOur study demonstrate that YM529 showed direct anti-tumor effect on NSCLC cell lines in vitro, which supports the possibility that third-generation BPs including YM529 can be one of therapeutic options for NSCLC. Lung cancer is the leading cause of death from cancer worldwide. Approximately 80% of lung cancers can be histologically classified as non-small cell lung cancers (NSCLCs). The majority of patients presents with locally advanced (37%) or metastatic (38%) disease at the time of diagnosis . Despitein vitro in a concentration- and time-dependent manner [Bisphosphonates (BPs) are structural analogues of pyrophosphoric acid, which is a biomedical component. Several studies have shown that BPs exert direct anti-tumor effects on a variety of human tumor cell lines  t manner -13. BPs t manner . The so-t manner -17. YM52in vitro or/and in vivo [Recently, YM529 as well as other third-generation BPs have also been shown to exert anti-tumor effects against various types of cancer cells both  in vivo ,13,18-22 in vivo .In this study, we examined the effects of YM529 on 8 NSCLC cell lines to investigate the potential usefulness of YM529 as a therapeutic agent against NSCLC.in vitro experiment.YM529 was kindly provided by Astellas Pharmaceuticals Co., Ltd. . YM529 was diluted in 10 mM of concentration and stored at -20°C for 2 in air as suspension cultures in RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 μg/ml streptomycin .Eight human NSCLC cell lines  were kindly provided by Dr. Adi F. Gazdar . All 8 cell lines were maintained at 37°C in a fully humidified atmosphere of 5% CO® AQueous One Solution Reagent . NSCLC cells were seeded on 96-well flat-bottomed tissue culture plates at a concentration of 5 × 103 cells/well with complete culture medium and allowed to adhere to the plate overnight. Then the cells were incubated in the presence of each concentration of 0(control), 0.01, 0.1, 1, 5, 10, 20, 50, 100, 200 μM of YM529 for another 72 hours at 37°C in a humidified atmosphere of 5% CO2 in air. After treatment, 20 μl of CellTiter 96® AQueous One Solution Reagent were dropped into each well of plates. After 90 minutes incubation, the optical densities (OD) of these samples were directly measured using an Immuno Mini NJ-2300  and reference wavelength of 490 nm. The OD of control samples was regarded as 100. Each condition was performed with 8 wells and each experiment was repeated twice. The anti-proliferative activities of YM529 are shown in terms of IC50s.Cell proliferation was determined by a modified MTS assay with CellTiter 962) irradiation. Genomic DNA was isolated by digestion with proteinase K followed by phenol:chloroform (1:1) extraction and ethanol precipitation from cell lines and primary tumor [NCI-H1819 cells were treated with concentrations 0 (negative control) and 10, 100 μM of YM529 for 48 hours. As positive control, NCI-H1819 was incubated for 24 hours after UV light  were separated on 2 % agarose gels.2HPO4, 4 mM citric acid) and incubated for 30 min at room temperature and resuspended in 1 ml of PBS containing 10 μg/ml of propidium iodide (PI) and 10 μg/ml of RNase A. The PI-stained cell samples were analyzed using FACSCalibur  and data analysis for the population of cells in sub-G1, G1, S and G2/M phase of the cell cycle was performed with CELLQuest . Cells undergoing apoptosis were determined as a percentage of cells with sub-G1 population.Subconfluent NCI-H1819 cells were cultured in 60 mm dishes with concentrations 0(control), 10 and 100 μM of YM529 for 24 hours. Cells were trypsinized, fixed in 70 % iced-cold ethanol, and stored at -20°C for 72 hours. After fixation, cells were suspended in 100 μl phosphate-citrate buffer (0.19 M NaSubconfluent NCI-H1819 cells were cultured in 60 mm dishes with concentrations 0(control), 10 and 100 μM of YM529 for 24 hours. Equal amount of protein (10 μg) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were probed with monoclonal anti-Ras , goat polyclonal anti-unprenylated Rap1A antibody , anti-ERK1/2 , anti-phospho-ERK1/2, anti-Akt, anti-phospho-Akt, β-actin  antibodies, and then with goat anti-rabbit, goat anti-mouse and rabbit anti-goat IgG-HRP coupled to horseradish peroxidase conjugated secondary antibodies , after which the membranes were developed by ECL Plus Western Blotting Detection Reagents .50 values. YM529 inhibited cell proliferation in a concentration-dependent manner in all the 8 cell lines , a significant increase in the population of cells in the sub- G1 phase was observed in the cells treated with 100 μM of YM529 (21.0%)  cell lines; it was shown that the agent exerted anti-proliferative effect in of the 8 cell lines with IC50 values in the range of 13–30 μM, whereas the remaining cell lines were resistant to zoledronic acid [Our studies showed that YM529 directly inhibited cell proliferation in NSCLC cell lines. The ICst NSCLC ,22,23. Y22–73 μM , which a1 arrest indicating apoptosis [1 population with YM529 treatment. Taking together these findings, it is speculated that YM529 exerts its anti-proliferative effect against NSCLC by induction of cellular apoptosis through the small GTP-binding proteins associated signal transduction pathway.In regard to the mechanism of action of nitrogen-containing BPs, including YM529, it is thought that they inhibit the activity of farnesyl diphosphate (FPP) synthase and geranylgeranyl diphosphate (GGPP) synthase which cause activation of FPP and GGPP, respectively. Since FPP and GGPP activation cause prenylation of small GTP-binding proteins including Ras resulting in anti-apoptosis, inhibition of their activation by YM529 induces cellular apoptosis -29. Indepoptosis ,31. Our Several studies have indicated the usefulness of the combined use of BPs with other cytotoxic agents ,22,32. M1 arrest cell cycle through down regulation of phospholyration of ERK1/2. These findings support the possibility that third-generation BPs including YM529 can be one of therapeutic options for NSCLC.Our study demonstrated that YM529 showed direct anti-tumor effect on NSCLC cell lines in vitro and induced apoptosis and GThe author(s) declare that they have no competing interests.RK drafted the manuscript and carried out the experiment. MA conceived the study. ST managed the experiment and edited the manuscript. FH carried out western blotting. MO and MT carried out extracting DNA and electorophoresis for DNA. YS gave valuable advice for flow cytometric assay. HD and NS conceived the study and participated in its coordination. All authors read and approved this manuscript.The pre-publication history for this paper can be accessed here:


We have detected somatostatin receptors (SSR) by autoradiography in 3/4 established small cell lung cancer (SCLC) cell lines but not in two non-SCLC cell lines. The growth of 1/3 SSR positive SCLC cell lines was significantly inhibited by the long-acting somatostatin analogue octreotide  10(-9) M. We treated 20 SCLC patients with octreotide 250 micrograms three times daily for 1 week prechemotherapy (six patients) or at relapse after chemotherapy (14). Octreotide was well tolerated, and serum insulin-like growth factor-I levels were suppressed to 62 +/- 7% of pre-treatment levels. However there was no evidence of anti-tumour activity measured by tumour bulk or serum levels of neuron-specific enolase. In one patient metastatic skin nodules were shown to be SSR positive before and at the end of 2 weeks octreotide. Despite this the patient had progressive disease, and tumour cells obtained by fine needle aspirate before and after treatment showed no growth inhibition when cultured with octreotide immediately or following establishment as a cell line. In summary we saw little correlation between SSR expression and growth inhibition by octreotide, either in vitro or clinically.


Analogues of the neurotransmitter substance P (SP) can interact with neuropeptide receptors, and are reported to inhibit growth of small cell lung cancer cell lines (SCLC CLs). We found  substance P (D-Phe5SP) significantly inhibited DNA synthesis by 10/10 human tumour CLs; six SCLC, one N-SCLC (squamous), two ovarian and one squamous cervical carcinoma, with inhibition to 50% control levels (IC50) of 20-50 microM. There was dose dependent inhibition of colony forming efficiency (CFE) in 3/3 SCLC and 1/1 N-SCLC CL, IC50s of 0.5-6.5 microM in 5% serum. Exposure of SCLC CL HC12 to 100 microM D-Phe5SP for 1-4 h caused a progressive fall in viable cell number; surviving cells, grown in the absence of peptide, showed a decreased growth rate. During 1 week's exposure of two SCLC CLs to 20 microM D-Ph5SP, growth was slower than control cultures, while 50-100 microM completely inhibited growth. These inhibitory effects were partially reversed by increasing serum concentration from 5 to 20%, but not by SP, vasopressin, bombesin or insulin-like growth factor 1. There was some inhibition of CFE by 3/3 normal human bone marrows, IC50s of 30-80 microM, compared with 8 microM for HC12 in 20% FCS. Therefore D-Phe5SP appears to have more potent antiproliferative effects in tumour cells than normal cells, suggesting a role for this analogue in tumour treatment.


The aim of this study was to characterize the presence and roles of CXCL12, CXCL8, CXCL1, and HGF in five human uveal melanoma cell lines, using different methods, in order to ascertain their significance in this disease.Five human uveal melanoma cell lines  of known proliferative, invasive, and metastatic potential were used in this experiment. A migration assay was used in order to assess the responsiveness of each cell line towards the four chosen chemotactic factors. Immunohistochemistry was then performed for all five cell lines (cytospins) using antibodies directed toward CXCL1, CXCL8 and their receptors CXCR2 and CXCR1 respectively. Quantitative real-time PCR was then performed on all five cell lines in order to establish the presence of these four chemotactic factors.All five human uveal melanoma cell lines migrated towards the four chosen chemotactic factors at a level greater than that of the negative control. Chemokines CXCL1 and CXCL8 resulted in the greatest number of migrating cells in all five of our cell lines. Immunohistochemistry confirmed the expression of CXCL1, CXCL8, and their receptors CXCR2 and CXCR1 in all five of the cell lines. Quantitative real-time PCR results established expression of CXCL8, CXCL1, and HGF in all 5 cell lines tested. CXCL1 and CXCL8 are highly expressed in SP6.5 and UW-1. None of the five cell lines expressed any detectable levels of CXCL12.in vivo.The migratory ability of the 5 human uveal melanoma cell lines was positively influenced by the four chemotactic factors tested, namely CXCL12, CXCL8, CXCL1, and HGF. Self-expression of chemotactic factors CXCL8, CXCL1, and HGF may indicate an autocrine system, which perhaps contributes to the cells' metastatic ability  Uveal melanoma is the most common primary intra-ocular 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 It is now well described and understood that chemo-attractive cytokines referred to as chemokines play a number of functional roles in tumor biology . Chemokiin vitro studies investigating these interactions are important to further gain an understanding of the steps involved in the metastatic cascade.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 angiogenic processes . InteracMembers of the CXC chemokine family have been shown to be involved in malignant melanoma cell growth and invasion in an autocrine dependent manner . ChronicDue to cytokine involvement in malignant progression of the primary tumor and the metastatic process, chemokines might represent a potential therapeutic target for cancer patients. Promising animal model data has shown that inhibiting interactions between CXCL12 and CXCR4 in a model of murine B16 melanoma cells will impair the metastatic process .in vitro  [Growth factor cytokines were also shown to be implicated into malignant growth of melanoma cells. Hepatocyte growth factor (HGF) has been previously shown to promote the migration of uveal melanoma cell lines  SOM269) . ExpressThe present study was designed in order to analyze the expression of CXCL12, CXCL8, CXCL1, and HGF in five human uveal melanoma cell lines  and to investigate the effects of these factors on the migratory ability of the 5 cell lines. Demonstration of a functional role for CXCL12, CXCL8, CXCL1, and HGF in uveal melanoma may yield novel therapeutic targets. Chemokine receptor inhibitors should be further investigated in the future, in order to possibly delay or inhibit tumor growth and 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.Five human uveal melanoma cell lines  were incubated at 37°C in a humidified 5% COThe uveal melanoma 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 ,12.6 cells/ml in serum free RPMI-1640 medium and placed in the upper well insert of a QCM™ 24-Well Colorimetric Cell Migration Assay (Chemicon). Each individual chemokine was reconstituted and diluted to predetermined, optimum concentrations in serum free RPMI-1640 medium  and placed in the lower chamber of the cell migration assay. All five cell lines were done in triplicate for each of the selected chemo-attractants. The 24-Well Plates were then incubated for 24 hours in a humidified 5% enriched CO2 atmosphere. Cells that migrated through the 8 μm pore membranes, located at the bottom of every well insert, were stained and eluted. The optical density of the stained cells was then read by a colorimetric plate reader at 560 nm.A QCM™ 24-Well Colorimetric Cell Migration Assay (Chemicon) was used for this experiment. Uveal melanoma cell lines were trypsinized and seeded at a concentration of 1 × 10Cytopsins of the 5 human Uveal Melanoma 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 antibodies against CXCL1 (clone #:20326), CXCL8 (clone #: 6217), and their receptor CXCR1 (clone #: 42705), and CXCR2 (clone #: 48311) using a standard Avidin-Biotin Complex method for each antibody (R&D Systems).® Primer Assays) for use with QuantiTect SYBR® green kits (Qiagen), for this experiment. Total mRNA was then extracted from all 5 human uveal melanoma cell lines from culture  using an RNeasy RNA extraction kit (Qiagen). A Chromo4 thermocycler (MJ Research) was used for all experiments and all results were analyzed using the GeneEx software. Beta actin was used as a housekeeper gene for purposes of normalization.Appropriate primers for CXCL12, CXCL8, CXCL1, and HGF were chosen,  migrated towards the selected chemo-attractants at a level greater than the selected negative control (serum-free RPMI media) (p < 0.05) except for cell line OCM-1 towards chemokine CXCL1 . Chemokines CXCL1 and CXCL8 resulted in the greatest average migration for all five cell lines followed by CXCL12 and HGF respectively.  initiates invasive oncogenic cellgrowth . HGF iniIn conclusion, characterizing the expression of CXCL12, CXCL8, CXCL1, and HGF in uveal melanoma cell lines leads us to a better understanding of how these cytokines are integral in the metastatic process. Further characterization and understanding of their roles may lead to novel therapies that target these factors, which may delay or inhibit the metastatic process.The author(s) declare that they have no competing interests.


Small cell lung cancer (SCLC) is treated primarily with combination chemotherapy. Despite high initial response rates, most patients eventually die with drug resistant disease. In some tumours, resistance to multiple chemotherapeutic agents is attributed to overexpression of P-glycoprotein (P-gp). However, this does not appear to be a frequent occurrence in drug resistant SCLC. Increased levels of glutathione (GSH) and related enzymes may play a role in resistance to alkylating agents as well as natural product drugs. We measured levels of GSH, glutathione S-transferase (GST), glutathione reductase (GSH Red), glutathione peroxidase (GSH Px), and gamma-glutamyl transpeptidase (gamma-GT) in a panel of 20 SCLC cell lines. Most of these lines were established from patients treated at this centre. Each cell line had a characteristic and reproducible profile of GSH and related enzyme levels. Immunoblot analysis indicated that the predominant GST in the cell lines was the anionic pi isoenzyme. The relative sensitivity of each of these cell lines to 16 different chemotherapeutic agents was measured using a modified MTT assay. Spearman rank correlation analysis was used to determine the relationships between the relative chemosensitivity of these cell lines and the levels of GSH and related enzymes. The number of positive correlations was no greater than expected by chance alone. Furthermore, there was no correlation with the treatment history of the patients from whom the cell lines were derived. These data suggest that alterations in glutathione metabolism do not play a major role in resistance to chemotherapeutic agents in these human SCLC cell lines.


The metastatic progression of cancer is a direct result of the disregulation of numerous cellular signaling pathways, including those associated with adhesion, migration, and invasion. Members of the Rac family of small GTPases are known to act as regulators of actin cytoskeletal structures and strongly influence the cellular processes of integrin-mediated adhesion and migration. Even though hyperactivated Rac proteins have been shown to influence metastatic processes, these proteins have never been directly linked to metastatic progression.in vitro adhesion, migration and invasion assays.To investigate a role for Rac and Cdc42 in metastatic breast cancer cell invasion and migration, relative endogenous Rac or Cdc42 activity was determined in a panel of metastatic variants of the MDA-MB-435 metastatic human breast cancer cell line using a p21-binding domain-PAK pull down assay. To investigate the migratory and invasive potential of the Rac isoforms in human breast cancer, namely Rac1 and the subsequently cloned Rac3, we stably expressed either dominant active Rac1 or dominant active Rac3 into the least metastatic cell variant. Dominant negative Rac1 or dominant negative Rac3 were stably expressed in the most metastatic cell variant. Cell lines expressing mutant Rac1 or Rac3 were analyzed using We show that increased activation of Rac proteins directly correlates with increasing metastatic potential in a panel of cell variants derived from a single metastatic breast cancer cell line (MDA-MB-435). The same correlation could not be found with activated Cdc42. Expression of a dominant active Rac1 or a dominant active Rac3 resulted in a more invasive and motile phenotype. Moreover, expression of either dominant negative Rac1 or dominant negative Rac3 into the most metastatic cell variant resulted in decreased invasive and motile properties.This study correlates endogenous Rac activity with high metastatic potential and implicates Rac in the regulation of cell migration and invasion in metastatic breast cancer cells. Taken together, these results suggest a role for both the Rac1 and Rac3 GTPases in human breast cancer progression. Cancer metastasis is a multi-faceted process requiring the disregulation of numerous signaling pathways, including those associated with cell adhesion and motility. The initial steps of metastasis require the acquisition of a motile phenotype in order to traverse tissue boundaries, while the later stages require dynamic adhesive interactions with the extracellular matrix to facilitate the extravasation of malignant cells . ActivatActivation of Rac and Cdc42 is critical for initiating cell motility and adhesion via the dynamic turnover of cell-substratum contacts  and the nucleation of actin monomers necessary for the assembly of actin filaments required for cell movement . AberranThe Rac subfamily includes Rac1, the myeloid-lineage specific Rac2, and the subsequently cloned Rac3 protein . ExhibitTo further understand the molecular mechanisms of the small GTPases Rac and Cdc42 in human breast cancer, we used a panel of cell variants, isolated from the MDA-MB-435 human metastatic breast cancer cell line, that varied in their ability to form secondary pulmonary and cerebral lesions in the nude mouse model of experimental metastasis . Within in vivo [Previously, Rac1 was shown to play a critical role in rat mammary tumor cell growth and metastasis in vivo . To esta2 atmosphere at 37°C.The human breast cancer cell line variants MDA-MB-435α6HG6, MDA-MB-435, MDA-MB-435α6LF9, and MDA-MB-435Br1 were cultured in Dulbecco's modified Eagle's medium  with 10% v/v FBS  and cultured in a humidified 5% CORac1 mutant cDNA (Myc-Rac1(G12V) and Myc-Rac1(T17N)) were generous gifts from Dr Gary Bokoch of the Scripps Institute . Rac3 mutant cDNA (Myc-Rac3(G12V) and Myc-Rac3(T17N)) were generous gifts from Dr Ulla Knaus of the Scripps Institute . Mutant Rac cDNAs were digested out of the pRK5myc vector and inserted into the multiple cloning site of the pIRESneo2 vector . pIRESneo2 vector alone, or vectors encoding myc-tagged Rac1(G12V), Rac1(T17N), Rac3(G12V), or Rac3(T17N) were transfected into cell variants using Lipofectamine Plus Reagent (Gibco™). Maximal expression was achieved 24 to 48 h post transfection with a transfection efficiency of approximately 70% at 48 h, as monitored by staining for myc expression. All experiments were conducted at 36 h following transfection and confirmed using stable cell lines.Cells cultured on glass coverslips were fixed in 3.7% formaldehyde , permeablized with 0.5% Triton X-100 , and blocked with 5% goat serum (Gibco™) and 5% BSA . Cells were then stained with rhodamine phalloidin  to visualize F-actin, and a mouse monoclonal anti-phosphorylated tyrosine antibody, clone 4G10 , followed by fluorescein isothyocianate (FITC)-conjugated goat anti-mouse IgG  as in . Cells wet al. [5) were added to the wells and allowed to adhere for 15 minutes. Non-adherent cells were removed, and the adherent cells were fixed in 3.7% formaldehyde . The number of cells per microscopic field for 30 fields per coverslip was counted with an Olympus upright microscope with a 40× phase contrast objective. Non-specific cell adhesion as measured on BSA-coated coverslips has been subtracted. Effects of the ectopic expression of Rac mutants were assessed 36 to 48 h post transfection.Cell adhesion assays were performed according to Klemke et al. . Brieflyet al. [®, Costar Corp., Cambridge, MA, USA) containing polycarbonate membranes were coated with matrigel (Fisher Scientific) or laminin (Gibco BRL) on the underside of the membrane (migration), or the upperside of the membrane (invasion). For invasion assays, cells chemotracted to media supplemented with 10% v/v FBS . Serum starved cells (106 cells) were added to the upper surface of each migration chamber and allowed to migrate to the underside of the membrane for 4 h (migration) or 24 h (invasion). The non-migratory cells on the upper membrane surface were removed, and the migratory cells attached to the bottom surface of the membrane were stained with propidium iodide (PI) . For PI staining, cells were fixed and permeablized in 70% ethanol and then incubated with 40 μg/ml PI in 1× PBS. The number of migratory cells per 30 microscopic fields per membrane was counted with an Olympus upright fluorescence microscope with a 40× objective for migration assays and a 10× objective for invasion assays. Non-specific migration as measured on chambers with no chemoattractant has been subtracted. Effects of the ectopic expression of Rac mutants were assessed 36 to 48 h post transfection.Cell migration and invasion assays were performed as described in Klemke et al. . Brieflyet al. [2.For guanine nucleotide binding, cell lysates were incubated for 15 minutes at 30°C in the presence of 10 mM EDTA and 100 μM GTPγS  or 1 mM GDP (Sigma) to facilitate nucleotide exchange as described in Knaus Rac and Cdc42 activity assays were performed as described in  with minClostridium difficile toxin B was purchased from Calbiochem and used as described in [ribed in ,18. CellP-values were calculated from unpaired or paired t-tests using Microsoft Excel  and considered significant at p-values less than 0.05.Data are expressed as means ± standard error of the mean (SEM). To understand the role of the Rho GTPases in metastatic breast cancer, we selected variants of the MDA-MB-435 metastatic breast cancer cell line and cycled them through the nude mouse model of experimental metastasis. The variants studied were chosen based on their ability to metastasize from tumors in the mammary fatpad to distant organs, predominantly lungs and lymph nodes. The MDA-MB-435α6HG6 variant produced the highest number of distant metastases from primary mammary tumors, followed by the parental MDA-MB-435, then MDA-MB-435α6LF9, and finally MDA-MB-435Br1. The MDA-MB-435Br1 cell line readily forms experimental brain metastases following injection into the intra-carotid artery, yet it shows significantly lower ability to metastasize in the more stringent assay of spontaneous metastasis .An invasive cellular phenotype can be indicative of metastatic behavior . Rac-indSubsequent to the epithelial to mesenchymal transition, cells must first migrate away from the primary tumor through the basal lamina to begin the process of establishing sites of secondary tumor formation. Therefore, increased cell migration in malignant cells is thought to be closely linked to invasion and metastasis ,2. Upon Increases in activity levels of the Rho proteins Rac and Cdc42 have been shown to be accountable for the promotion of tumor cell invasiveness ,7. ThereClostridium difficile toxin B to inhibit the Rho family in these cell variants. Toxin B was used as described in [Because the Rho family of small GTPases, namely Rho, Rac, and Cdc42, are essential to cell motility, we used ribed in ,18. InhiHyperactive Cdc42 has been implicated in tumor cell invasion due to its effects on the actin cytoskeleton ,20. To dInvasive malignant cell morphology includes an increased number of focal adhesions, as well as an increase in actin structures such as cross-linked actin fibers and membrane ruffles ,2. The min vitro. For each of these assays, cells expressed equal amounts of activated Rac1 or Rac3 mutant protein (data not shown).Because metastatic progression results from increased migration of malignant cells out of the basal lamina, subsequent adhesion to the extracellular matrix, and final invasion into distant tissues to establish secondary sites of metastasis, we measured the effect of Rac mutants on these processes in vitro. Expression of dominant active Rac1(G12V) or Rac3(G12V) causes a significant increase in adhesion to basal lamina when expressed in the low metastatic MDA-MB-435Br1 variant compared to the vector alone control, whereas dominant negative Rac1(T17N) or Rac3(T17N) cause a significant decrease in adhesion when expressed in the high metastatic MDA-MB-435α6HG6 variant  or Rac3(G12V) caused a significant increase in migration and invasion when expressed in the low metastatic variant , Arp2/3 (actin-related protein), and subsequent actin polymerization and filopodia formation . Furtherin vitro. Moreover, we found that augmenting endogenous Rac activity by expressing dominant active Rac1 or Rac3 led to a significant increase in adhesion, migration, and invasion. Taken together, these data substantiate not only a vital role for Rac1 in cell functions relevant for breast cancer metastasis, but also a vital role for Rac3. In fact, expression of a dominant active Rac3 in the MDA-MB-435Br1 low metastatic cell variant increased invasion through basal lamina 1.5 times compared to expression of dominant active Rac1. This difference suggests an enhanced ability of the cells expressing Rac3(G12V) to invade, perhaps by a more efficient degradation of the extracellular matrix compared to the cells expressing Rac1(G12V). Because we found that expression of dominant active Rac1 or Rac3 results in a similar motile phenotype, it is possible that Rac3 is more efficient at activating proteins that degrade extracellular matrix proteins, or matrix metalloproteinases, than is Rac1.In this study, we also demonstrate the efficacy of both the Rac1 and Rac3 isoforms in the malignant progression of human breast cancer. Because Rac1 and Rac3 both have been implicated in breast cancer ,11,30, wRac1 and Rac3 differ in their carboxyl terminus region, which is essential for subcellular localization . Even thin vitro, whereas RhoC overexpression in mammary epithelial cells resulted in a significant increase in cell migration [Studies have also demonstrated that, in addition to the Rac proteins, Rho proteins, especially RhoC, may contribute to breast cancer cell invasion. RhoC was demonstrated to be overexpressed in the human inflammatory breast cancer cell line SUM 149 and transient inhibition of RhoC in inflammatory breast cancer cells by treatment with farnesyl transferase inhibitors reduced invasion and motility igration . Interesin vivo model. By using variants of the same cell line, we have minimized genetic variation. It remains to be investigated, however, whether or not similar results will be observed in other cell lines with different genetic backgrounds.We found a direct correlation between increased metastatic potential and increased endogenous Rac activity in a panel of metastatic human breast cancer cells that vary in their ability to form secondary metastases in an in vitro metastatic properties in human breast cancer cells. Currently, we are testing the hypothesis that Rac3 activation alone can increase breast cancer metastasis in vivo by using the nude mouse model of experimental metastasis.The research presented here establishes a direct role for Rac3 in cell functions relevant for breast cancer progression. We found that Rac3 activation alone can significantly increase Arp2/3 = actin related protein 2/3; BSA = bovine serum albumin; FBS = fetal bovine serum; FITC = fluorescein isothyocianate; PBS = phosphate-buffered saline; PI = propidium iodide; WASP = Wiskott Aldrich Syndrome protein.The authors declare that they have no competing interests.PB characterized the morphology and motile phenotypes of MDA-MB-435 variants and the dominant active (RacGV) cell lines, carried out activity assays, generated the dominant active stable cell lines, participated in the design of the study, and drafted the manuscript. LK generated the dominant negative stable cell lines and characterized their motility phenotypes. JP provided the cell variants and participated in the coordination of the study. SD conceived of the study, participated in its design, supervised experiments, and helped to draft the manuscript.


The physiology of the multidrug transporter P-glycoprotein (Pgp) is still poorly understood. We now show evidence that cell lines with a high expression of Pgp display a reduced accumulation of cortisol and an ATP-dependent outward transport of the hormone. Cortisol efflux from Pgp negative cells does not have such an active component. Further we show that the steroid hormones cortisol, testosterone, and progesterone cause an immediate, dose-dependent increase of daunorubicin accumulation in Pgp overexpressing cells. These effects are particularly apparent for the more lipophilic steroids. These results demonstrate that Pgp may function as a transporter for cortisol and suggest a physiological role of the protein in steroid handling by organs such as the adrenal.


The growth inhibitory effect of tumour promoters on human leukaemia and lung cancer cell lines was examined using the [3--2, 5-diphenyl-tetrazolium bromide (MTT) assay. The four cell lines used were the K562 human leukaemia cell line, its adriamycin (ADM)-resistant subline (K562/ADM), which shows the mdr phenotype, PC-9 (a human lung adenocarcinoma cell line) and its cisplatin (CDDP)-resistant subline (PC-9/CDDP), which does not show the mdr phenotype. Phorbol 12-tetradecanoate-13-acetate (TPA) and the TPA-type tumour promoters, aplysiatoxin and debromoaplysiatoxin, inhibited the growth of the two parental cell lines, K562 and PC-9. The non-TPA-type tumour promoter, okadaic acid, also inhibited the growth of the two parental cell lines in a dose-dependent manner. TPA-type and okadaic acid inhibited the growth of K562/ADM more weakly than that of K562, and showed no growth inhibition in PC-9/CDDP. Anhydrodebromoaplysiatoxin, an inactive derivative of the TPA-type tumour promoter, could suppress the growth of K562 and K562/ADM only at high concentration (more than 50 pM) and it showed similar growth inhibitory effects on the two cell lines. Okadaic acid tetramethyl ether, the inactive form of the non-TPA-type tumour promoter did not inhibit the growth of any of the cell lines. The growth inhibitory effect of these compounds was well correlated with their tumour-promoting activity. A study of the accumulation of okadaic acid revealed that the amount of 3H-okadaic acid in K562/ADM and PC-9/CDDP was similar to that in their parental cells indicating that cross-resistance to this tumour promoter in the drug-resistant cell lines is not due to a difference in the amount of drug accumulated in sensitive and resistant cells. These results suggest the presence of another common mechanism for resistance to ADM and CDDP as well as to TPA- or non-TPA-type tumour promoters.


Results from this study indicates that altered expression of the genes Atp1b1, CARD 10, KLF4, Spint2, and Acly may play a role in the aggressive phenotype seen in breast cancer in African American women.African American (AA) women have a lower overall incidence of breast cancer than do Caucasian (CAU) women, but a higher overall mortality. Little is known as to why the incidence of breast cancer is lower yet mortality is higher in AA women. Many studies speculate that this is only a socio-economical problem. This investigation suggests the possibility that molecular mechanisms contribute to the increased mortality of AA women with breast cancer. This study investigates the expression of 14 genes which have been shown to play a role in cancer metastasis. Cell lines derived from AA and CAU patients were analyzed to demonstrate alterations in the transcription of genes known to be involved in cancer and the metastatic process. Total RNA was isolated from cell lines and analyzed by RT-PCR analysis. Differential expression of the 14 targeted genes between a spectrum model (6 breast cancer cell lines and 2 non-cancer breast cell lines) and a metastasis model (12 metastatic breast cancer cell lines) were demonstrated. Additionally, an  Cancer is characterized by excessive growth and spread of abnormal cells. It affects all populations in the United States and ranks second only to heart disease as the leading cause of death . More thMetastasis is the main cause of morbidity and mortality in cancer patients. The selective distribution of metastases is dictated by numerous factors, including complementary adhesive contacts, the pattern of vascular flow from the primary site, and molecular interactions between the tumor cell and the stroma at the secondary site . Our labCell lines were purchased from American Type Culture Collection  and Coriell Cell Repositories . Cells were propagated in the recommended media and given new media every 2 to 3 days until 90% confluent. The spectrum model Table  containsRNA was extracted from the cell lines using RNAqueous . Cells were collected by low speed centrifugation and lysed by adding 200 μl of Lysis/Binding Solution. An equal volume of 64% ethanol was added to the lysate. The lysate/ethanol mixture was transferred to the RNAqueous Filter Cartridge and centrifuged for 1 minute at 13,400 rpm. The flow through was discarded and 700 μl of Wash Solution 1 was added to the RNAqueous Filter Cartridge and centrifuged for 1 minute. The column was washed twice with 500 μl of Wash Solution 2/3 and eluted with 110 μl Elution Solution. Isolated RNA was quantitated using the NanoDrop Spectrophotometer . All RNA samples were stored at -70°C in RNA elution solution .The RT-PCR reactions were performed in a P/E GeneAmp 9700 thermocycler , using the Access RT-PCR system . The reaction mixes were prepared by combining 27.5 μl of nuclease free water, 10 μl of AMV, 1 μl Tfl 5X reaction buffer, 2 μl dNTP mix, 50 pM of upstream primer, 50 pM of downstream primer in 1.5 μl volume each, 3 μl 25 mM MgSO4, 1.0 μl AMV reverse transcriptase, Tfl DNA polymerase and 1 μg of total RNA in a 0.5 ml thin walled Eppendorf tube on ice. The reaction mixes were then vortexed for 5 seconds and centrifuged. The PCR cycling profile was as follows: 48°C for 1 minute for reverse transcription of the RNA into cDNA, 94°C for 4 minutes to deactivate the reverse transcriptase, and 30 cycling sequences of denaturing at 94°C for 45 seconds, annealing at 55°C-58°C  and β  subunits are known [1 and β1 isoforms are produced in most tissues, whereas α2, α3 and β2 are produced predominantly in the brain [The data show that the mRNA transcript encoding Na K-ATPase (Atp1b1) was elevated in the cancer cell lines compared to the normal cell lines and exhibited altered expression profiles between AA and CAU cell lines. The active form of Atp1b1 is composed of a 100–112 kDa catalytic ct-subunit and a 45–60 kDa glycoprotein 13-subunit . Three dre known -18. At tre known  while 50he brain ,19. The he brain .We observed decreased levels of both Caspase recruitment domain family (CARD) and Kruppel-like-factor 4 (KLF4) expression in both primary and metastatic cell lines compared with the normal cell lines, with the lowest expression levels occurring in the metastatic cell lines. CARDs are protein modules found in many proteins that regulate apoptosis -23. A toDrosophila melanogaster, is thought to control embryogenesis [Krüppel, a group of zinc-finger-containing transcription factors found in ogenesis . Among togenesis . KFL4 waogenesis . KLF4 haogenesis . The sigogenesis , gastricogenesis , bladderogenesis , and lunogenesis . The expogenesis , suggestin vivo functions of SPINT2/HAI-2, but disruption of the HAI-2 gene has resulted in embryonic lethality in mice indicating a potentially important role of this inhibitor in cellular development [Elevated expression of Serine protease inhibitor, Kunitz type 2 (SPINT2)/Hepatocyte growth factor activator inhibitor type 2 (HAI-2) (SPINT2/HAI-2) in both primary and metastatic cell lines compared with the normal cell lines with the highest expression levels occurring in the metastatic cell lines. We additionally observed lowered expression in AA cell lines compared to their CAU counterparts. SPINT2/HAI-2 has a broad inhibitory spectrum and was independently reported to be a placental bikunin . SPINT2/elopment . SPINT2/elopment  and ovarelopment  and has elopment  and breaelopment .The data revealed elevated levels of ATP citrate lyase (Acly) expression in both primary and metastatic cell lines compared to the normal cell lines with the highest expression levels occurring in the metastatic cell lines. We also observed elevated expression found in AA cell lines compared to their CAU counterparts. Acly is critical for the conversion of glucose to cytosolic acetyl CoA and therefore for glucose-dependent lipogenesis. Acly deficiency in mice has resulted in embryonic lethality, with no viable embryos detectable even at early stages of development . PreviouIn conclusion, we have amassed two cell line models that could be used to identify biomarkers and treatments unique to AA women with breast cancer. The significance of the study is that Atp1b1, CARD 10, KLF4, Spint2, and Acly are not only useful in serving as possible biomarkers of metastasis, but also provide support for the concept that they could serve specifically as biomarkers for AA women. To date, this is the only study that has used an AA model system specifically for gene mining to investigate cancer and metastatic gene expression in cell lines derived from African American patients.AA-African American women, CAU-Caucasian, RT-PCR-Reverse transcriptase Polymerase Chain Reaction, ECM-Extracellular matrix, MMP-Matrix MetalloproteinaseThe author(s) declare that they have no competing interests.HFY conceived the study and participated in its design, was responsible for primer design, performed data analysis, densitometric readings of RT-PCR, and prepared the first draft of the manuscript. JAM participated in the study design, was responsible for primer design, performed the RT-PCR experiments, was involved in tissue culture, and prepared the first draft of the manuscript. SP performed RT-PCR experiments, data analysis, densitometric readings of the RT-PCR, and assisted in the editing of this manuscript. CETIII performed RT-PCR experiments, data analysis and densitometric readings of the RT-PCR. GKL participated in the funding, as well as data analysis and editing of the manuscript. MJ participated in study concept, design and data analysis. AAD participated in the study concept, study design, secured funding, interpretation of the data, as well as preparation of the manuscript. All authors read and approved the final manuscript.


Gap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition.We investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC) in 5 primary lung fibroblast lines derived from normal subjects (NF) and from 3 histologically proven IPF patients (FF).Here we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF.These results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice. Idiopathic pulmonary fibrosis (IPF) is the most common among interstitial pneumonias of unknown origin and one of the most aggressive interstitial lung diseases. Although the pathogenesis is incompletely understood, the activation and proliferation of lung fibroblasts which lead to excessive extracellular matrix components (ECM) accumulation and altered mesenchymal cell interactions, are believed to be critical events driving the chronic and progressive course of IPF .The presence of aggregates of actively proliferating fibroblasts termed "fibroblast foci" is a hallmark of usual interstitial pneumonia (UIP) in IPF ,3. It haGap junctions are specialized membrane regions composed of aggregates of transmembrane channels that directly connect the cytoplasm of adjacent cells -7. The pUncontrolled fibroblast proliferation and altered fibroblast phenotypes are considered crucial events in the onset and evolution of IPF. Nevertheless, to date, the role of GJIC in the pathogenesis of IPF has not been investigated.In the present study the expression of Cx43 in normal fibroblasts and in fibrotic fibroblasts from patients with IPF/UIP was studied. Moreover, to better understand the role played by gap junctions in the pathogenesis of IPF, we functionally evaluated the gap junctional intercellular communication in normal and fibrotic fibroblasts by measuring gap-junctional coupling using flow cytometry.Primary lines of normal human lung fibroblasts were established by using an outgrowth from explant following the method described by Jordana et al. . Five noTotal RNA extraction and cDNA synthesis were performed as previously described . The folNM_004004): a 290-bp fragment, encompassing nucleotides 146–435, was amplified using the following primers: forward primer: 5'-TTCCTCCCGACGCAGAGCAA-3'; reverse primer: 5'-ACACGAAGATCAGCTGCAGG-3'. Human liver RNA  was used as positive control sample.1) Cx26 mRNA (accession number NM_000166): a 221-bp fragment, encompassing nucleotides 26–246, was amplified using the following primers: forward primer: 5'-AGGTGTGGCAGTGACAGGGA-3'; reverse primer: 5'-TGTTGCAGCCAGGCTGGAGT-3'. Human liver RNA (Ambion Inc.) was used as positive control sample.2) Cx32 mRNA (Accession number NM_001101): a 336-bp fragment, encompassing nucleotides 158–493, was amplified using the following primers: forward primer: 5'-ACTTGGCGTGACTTCACTAC-3'; reverse primer: 5'-CATGAGCCAGGTACAAGAGT-3'. Human heart RNA (Ambion Inc.) was used as positive control sample.3) Cx43 mRNA . The following oligonucleotides were used: – forward primer: 5'-TTCATTTTACTTCATCCTCCAAGGA-3', – reverse primer: 5'-CAGTTGAGTAGGCTTGAACCTTGTC-3', – fluorogenic probe: 5' FAM-ACTTGGCGTGACTTCA-TAMRA 3'. Commercially available normal human lung, kidney, heart RNA (Ambion Inc.) and cerebral cortex RNA, extracted from an autoptic sample as previously described , were aldecribed .Cells grown in 100 mm culture dishes of five normal and three fibrotic lung fibroblast lines were washed with PBS buffer and were homogenized in ice-cold 40 mM Tris-HCl buffer, pH 7.4, containing 2.5% SDS detergent, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a cocktail of protease inhibitors diluted at 1:200 (Sigma-Aldrich P8340). The homogenization medium was further supplemented with the phosphatase inhibitors sodium orthovanadate and sodium fluoride at 1 and 10 mM concentration, respectively. After homogenization, samples were sonicated for 30 sec. Protein concentration was determined with the bicinchoninic acid method, using BSA as the standard. Samples were separated on 10% polyacrylamide gels. Before being loaded onto gels, samples were boiled in sample buffer  for 4 min. Resolved proteins were transferred to nitrocellulose membrane (0.45 μm)  in transfer buffer  containing 0.05% SDS. Membrane was blocked for 2 hr at 22°C in 20 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 (TBS-T) containing 3% BSA and incubated with primary antibody overnight at 4°C in TBS-T containing 1% BSA. Blot was tested with a monoclonal mouse Cx43 antibody diluted at 1:250 . After incubation with primary antibody, membrane was washed in TBS-T and then incubated for 1 hour at room temperature in TBS-T containing 1% BSA and incubated with anti-mouse horseradish peroxidase-conjugated secondary antibody at dilution of 1:10000. Blot was washed in TBS-T and then incubated for 3 min using the SuperSignal chemiluminescence detection Kit system .Separate blots, loaded with the same samples, were also incubated with the mouse anti-β-actin monoclonal antibody  as a control for the quality of the protein preparations. Specific band was visualized by using the SuperSignal chemiluminescent detection system .Functional assay of the gap junctional activity was assessed with a dye-loading technique by means of flow cytometry . Donor cCocultured experiments were performed either using the same cell line as donor and recipient cells (homologous coupling) either using different cell lines as donor and recipient cells (heterologous coupling). Carbenoxolone (CBX) , a specific gap junction blocker, was used to ascertain whether the intercellular transfer assay described above was dependent on intercellular gap junction communication.Results are shown as means ± standard deviation (SD). GJIC assay results represent the number of calcein-positive recipient cells (CAL+DiI-) expressed as percentage of total recipient cells (DiI-). Comparisons between groups were made by means of two way ANOVA or Student's unpaired t-test where appropriate; P values of 0.05 or less were considered to be statistically significant.In order to establish whether, and identify which connexins were expressed in cultured lung fibroblasts, specific RT-PCR amplifications were performed and the products were separated by agarose-gel electrophoresis.We established and used 5 fibroblast cell lines derived from histologically normal areas of surgical specimens from 5 patients undergoing to resective lung surgery for cancer and 3 fibrotic fibroblast cell lines derived from surgical biopsies of 3 different patients with histologically proven UIP/IPF.Transcripts for Cx32 and Cx26 were absent in both normal and fibrotic fibroblasts, while Cx43 mRNA was intensely expressed in all the samples analyzed Fig. . AlthougConnexin 43 mRNA expression in normal fibroblasts was in perfect agreement with that of a commercially available human lung mRNA sample. However, Cx43 mRNA expression in fibrotic cell lines of different patients was significantly reduced when compared to normal fibroblast cell lines . This excludes the possibility that gross abnormalities in cell adhesion are responsible for the observed differences.In the experiments described thus far donor and recipient cells belonged to the same cell line (homologous GJIC). In order to exclude the possibility that differences in loading and retention of intracellular marker dye calcein in donor cells might account for the observed differences between fibrotic and normal fibroblasts, we performed experiments using a single normal fibroblast cell line as donor cells for both fibrotic and normal recipient cell lines (heterologous GJIC).In confirmation of our homologous GJIC experiments, heterologous GJIC was significantly reduced at 120 minutes in fibrotic fibroblasts when compared to normal recipient fibroblast lines  Fig. .The pathogenesis of IPF is still unclear. Nevertheless, the presence and the extent of fibroblastic foci in the lung of affected patients is one of the most prominent features associated with IPF progression and survival -35. The In this context, it is believed that abnormal cell-to-cell communication between parenchymal cells may lead to the emergence of altered fibroblast phenotypes and uncontrolled fibroblast proliferation. Among the structures that maintain cell contact, gap junctions are believed to be the main players in cell-to-cell communication, by allowing the sharing of small cytoplasmic molecules and ions between adjacent cells. This particular type of communication has been implicated in several important biological processes such as cell growth and proliferation, embryogenesis as well as senescence, neurotransmission, hormonal secretion, tissue repair, leukocyte adhesion and inflammation, and oncogenesis -39.in vitro model. Indeed, we have previously shown that fibrotic fibroblasts possess distinct features that characterize their unique phenotype. Not only they are able to differentially drive inflammatory cell responses . Ultimately, the time course depends on the number of active gap junctions and on their permeability for the selected dye. In some experiments, carbenoxolone, a specific gap junction blocker, was added to ascertain whether the intercellular transfer assay was effectively dependent on GJIC. Our results show that carbenoxolone abrogated calcein spreading into recipient cells, therefore confirming the reliability of our data on GJIC obtained by the dye-loading technique.in vivo the altered cell-to-cell communication in fibrotic fibroblasts releases these cells from the control of the sourrounding normal areas. Under certain conditions, this is believed to be of paramount importance in the cascade of events that drive an altered cell type to escape from the restraints of contact-inhibition, thus facilitating uncontrolled cell proliferation.Our main experimental setup employed donor cells homologous to recipient cells. This condition simulates the cell-to-cell communication between phenotypically similar cells belonging to the same tissue or microscopic area, but adds nothing to the cell-to-cell communication taking place in the boundary between healthy and diseased section of the same organ. Similarly to our homologous GJIC experiments, heterologous GJIC was reduced in fibrotic fibroblasts even if the donor cells used were characterized by a normal gap junctional activity. In this regard Zhang et al. [53] who studied defective heterologous dye spreading between normal and cancer cell lines clearly demonstrated that reduced GJIC might explain the tumorigenic potential of a cell line only when it reaches a minimal threshold of Cx43 coupling. The alteration of the gap junctional activity between normal donor and diseased recipient cells might thus suggest that In an experimental model of cisplatin induced damage, Cx43 expression and GJIC were found to be reduced in primary human lung fibroblasts [54]. Interestingly, p53, a well known lung cancer marker, was found to be elevated in conjunction with the aforementioned cell-to-cell alterations. Moreover, Cx43 deletion in mice results in a higher susceptibility to lung cancer . Recently, several studies have tried to correlate IPF with lung cancer with conflicting results . It would be tempting to hypothesize that the reduction of Cx43 and defective cell-to-cell communication we found in IPF fibroblasts is similar to what is described in cancer. This lends support to the idea that the uncontrolled proliferation of fibroblasts that characterize IPF may have some similarities to lung cancer.in vitro model representing cell-to-cell communication cannot fully reflect the in vivo communication between cells in either normal or diseased lung. Further research is needed to unravel the steps influenced by gap junction dysfunction in IPF. However, this study proposes a novel pathogenetic alteration in primary lung fibroblasts from IPF/UIP patients: impairment of cell-to-cell communication. These phenomena may well explain some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical treatment.Of course an


A class of new 5-fluorouracil (FU) analogues, the 5-fluoro-5,6-dihydro-6- alkoxy-uracils was synthesised with a modification at the 6-position of the pyrimidine ring. At this position the analogues have a hydroxy or alkoxy group of different chain lengths either in the cis- or trans-configuration. The antiproliferative effect of these compounds was tested on five cell lines of different origin. Generally, the analogues with a cis-configuration had a higher activity than those with a trans-configuration. The growth inhibitory effect of the compounds decreased with increasing alkoxy chain length, but the compound with a hydroxy group had the lowest growth inhibitory effect. One analogue, cis-5-F-5,6-dihydro-6-methoxy-uracil had a higher antiproliferative effect than FU in one of the cell lines. Effects on thymidylate synthase (TS), the possible target of these analogues, were evaluated by thymidine rescue of growth inhibition and incorporation of tritiated deoxyuridine (3H-UdR) into DNA. In solid tumour cell lines addition of TdR reversed the antiproliferative effect. Inhibition of TS in intact cells was determined by measuring 3H-UdR incorporation in two cell lines. The effect of cis-5-F-5,6-dihydro-6-methoxy-uracil on incorporation of 3H-UdR was 2- to 5-fold stronger than that of FU in both cell lines. All other compounds produced a higher 3H-UdR incorporation than FU both at equimolar and equi-toxic concentration. Concluding from these results we regard cis-5-F-5,6-dihydro-6-methoxy-uracil as the most promising FU analogue of this series, because of its higher antiproliferative activity than FU and marked inhibition of TS in intact cells.


Thirty permanent and more than 60 primary tumour cell lines were established from pleural and pericardial exudates or wedge biopsies from human bronchial carcinoma. The permanent cell lines have their origin in 6 small cell, 5 large cell, 9 squamous and 5 adeno carcinomas of the lung. Tumour cells were purified from non tumour cells by direct cloning in fluid cultures or by soft agar cloning. In vitro secretion of ACTH, bombesin, calcitonin, and neurotensin was demonstrated for lung tumour cells belonging to the four major histological types. Cell suspensions of peptide hormone secreting permanent cell cultures were grown to solid tumours after xenotransplantation into nude mice. Comparative ultrastructural examination of the primary tumour and of cells grown in tissue culture and in xenografts demonstrated the preservation of most tumour type specific structural criteria in the ex vivo/in vitro systems. The present data show that not only tumour cells from small cell carcinoma but also from other histological types are capable of synthesizing a broad spectrum of immunoreactive peptide hormones. This result might be interpreted as indicating a common expression of hormone biosynthesis and secretion by all lung tumours.


The relationships between resistance to adriamycin, vincristine, colchicine and etopside, expression of P-glycoprotein and CP22 (sorcin), and resistance modification by verapamil and cyclosporin A have been studied in a panel of multidrug-resistant (MDR) mouse tumour cell lines. Whereas there was a generally good correlation between the degree of resistance and the amount of P-glycoprotein, no relationship between resistance and CP22 expression was seen. At 3.3 microM verapamil, the sensitisation of the MDR cell lines was no greater than that of the parent line. At 6.6 microM verapamil, however, sensitisation of the MDR lines generally exceeded that of the parent line, although the line CR 2.0, expressing very high levels of P-glycoprotein was an exception. Little sensitisation to etoposide was seen in any of the lines. When cyclosporin A was used as the sensitiser at either 2.1 or 4.2 microM, there was a greater effect in lines expressing moderate to high levels of P-glycoprotein than in the parent line, although this tendency was less for adriamycin than for the other cytotoxics. Sensitisation to etoposide was much greater with cyclosporin A than with verapamil. At low levels (less than 1 microM) of CsA, however, sensitisation to colchicine was greater in the parent line than in cell line CR 2.0. These studies indicate that chemosensitisation by verapamil and cyclosporin A is extremely complex, depending upon sensitiser dose, the particular cytotoxic and the cell line. At low doses of the sensitisers, the sensitisation may be greater in lines expressing low levels of P-glycoprotein than in lines showing high levels.


In this study we report detection of mdr1 gene expression in the liver metastases of 7/11 patients with colon carcinoma and characterise the MDR phenotype associated with a panel of 19 human colon carcinoma cell lines. Within this panel, mdr1 mRNA biosynthesis and surface localisation of Pgp were assessed with respect to MDR functionality where the cell lines are representative of different clinical stages of tumour progression, metastatic potential and differentiation. The data indicates that constitutive levels of mdr1 mRNA/Pgp expression may not necessarily result in the functional expression of the MDR phenotype. While low levels of mdr1 mRNA/Pgp were detected in 5/8 well differentiated colon cell lines, only 2/8 were functionally MDR. In contrast, 10/11 moderate and poorly differentiated lines expressed mdr1 mRNA/Pgp and of these, 9/11 were functionally MDR. The phosphorylation status of the mature 170 kD P-glycoprotein and the surface localisation of this glycoprotein showed the strongest correlation with functionality. Analysis of cell lines for cross-resistance and chemosensitivity profiles against a battery of chemotherapeutic drugs suggests multiple mechanisms, in addition to Pgp, contribute to the overall resistance of colorectal cancer.


The cytotoxic activity profile of an immunotoxin, SWA11-ricin A chain, recognising a cell-surface antigen associated with human small cell lung cancer (SCLC), was examined in detail using a panel of SCLC, non-SCLC and non lung tumour cell lines in tissue culture. SWA11-ricin A chain was potently and selectively active against three SCLC cell lines of both classic and variant morphologies, inhibiting the incorporation of 3H-leucine with an IC50 of 5 x 10(-11) M. At a concentration of 1 x 10(-8) M, the SWA11 immunotoxin could selectively eliminate in excess of 99.9% of clonogenic tumour cells. Intoxication proceeded rapidly following a 4 h lag phase; the initial rate of protein synthesis inhibition occurred with a t50 of 2 h and a t10 of 7 h. The cytotoxic activity of SWA11-ricin A chain was potentiated by 100-fold in the presence of the carboxylic ionophore monensin at 1 x 10(-7) M. Kinetic studies revealed that monensin enhanced the rate of protein synthesis inhibition by two-fold and eliminated the lag phase suggesting a rapid effect on either the rate or route of internalisation. Studies with SWA11 could detect no influence of monensin on the rate of antibody internalisation and a transient delay in the delivery of internalised antibody to lysosomes was observed by immunoelectron microscopy.