MDR1 transfectant P-gp+ breast carcinoma lines had increased LAK susceptibility compared to their parent line. Some part of the increased LAK susceptibility of drug-resistant cell lines was at the binding/recognition level as shown by conjugate formation assays. This suggests that differences may exist between paired cell lines with respect to the expression of cell adhesion molecules (CAMs). Monoclonal antibodies (mAbs) to CAMs and flow cytometry were used to quantitate these antigens. The CAMs studied were those previously found to be upregulated by stimulating NK cells with (interleukin-2) IL-2; ICAM-1 (CD54), LFA-3 (CD58), N-CAM (CD56), and the β chain of LFA-1 (CD18). Although no differences in these CAMs were found between the breast carcinoma line and its MDR1-transfected variants, the target susceptibility results given above suggest that IL-2 treatment could be effective in combination with current protocols using chemotherapeutics, monoclonal antibodies (mAbs) and stem cell transplantation.Reports showing susceptibility of multidrug resistant (MDR) cancer cells to immune effectors, together with P-glycoprotein (P-gp) expression in immune effector subsets, including immature natural killer (NK) cells, and some activated T cells, suggest P-gp or some changes associated with it, have implications in immune-mediated mechanisms. A series of experiments were done to determine the nature of alterations associated with susceptibility to immune effector cells of MDR tumor cells. A cell line isolated from the malignant pleural effusion of a breast cancer patient was transfected with human and murine MDR1 genes, and four variants with different levels of MDR were obtained. Lymphokine-activated killer (LAK) activity was measured by a  Breast cancer is a serious health concern and a major public health challenge with an incidence of 217.500 and breast cancer deaths of 40.600 in USA being estimated in 2004 [One aspect of breast cancer biology that hinders cure is tumor cell heterogeneity and phenotypic variability. Some tumor cells may be killed by chemotherapy, but the ability of a tumor cell population to adapt to a new environment results in progression of the disease. This arises from either the tumor cells that are already resistant to the therapeutic drug or tumor cells that develop resistance during the course of the treatment.After the impressive progress in the understanding of the basic tumor biology of the breast cancer, demand for additional markers of biologic relevance for the prediction of breast cancer outcome and of response to various chemotherapy, radiotherapy and immunotherapy regimens has increased in the recent years.Over the ensuing decade, systemic administration of c-erbB-2 monoclonal antibody trastuzumab in patients with c-erbB-2 over-expressing tumors was found to produce objective regressions in 11 to 26 percent of patients with metastatic breast cancer . Recentl+ MDR tumor cell populations in ovarian, SCLC, malignant melanoma and renal cell carcinoma [+ MDR tumor cell populations, c-erbB2(+) positive breast cancer cells may be eradicated to a better extent with c-erbB2 mAb + LAK cells since LAK cells have strong antibody-dependent cell-mediated cytotoxic (ADCC) activity [LAK cell immunotherapy has been shown to have the potential to eradicate P-gparcinoma -16. In aThe objectives of the current study were:1. To quantitate the LAK cell-mediated lysis (CML) susceptibility of drug sensitive and drug resistant breast cancer cell lines.2. To study the binding/recognition step of LAK CML in these cell lines and its contribution to differences in LAK CML of the paired sensitive and MDR cell lines.3. To measure the level of expression of CAMs shown to be involved in the binding/recognition step of lysis and to compare these levels within the paired cell line groups.51Cr-release assay.Peripheral blood lymphocytes collected from normal volunteers were activated with human recombinant interleukin 2 and tested for LAK cell activity against the drug sensitive human breast carcinoma cell line MDA-MB-231, and its MDR-transfected variants MPAM-26, MPAHS-1-10, MPAHS-1-300, and MPAHS-DOX150. LAK cell cytotoxicity was also tested against the NK-sensitive, LAK-sensitive cell line K562, and the NK-resistant, LAK-sensitive RAJI cell line. LAK cell-mediated cytotoxicity against target cells was evaluated by an 18 hr 51Cr release), and in Fig. The percent chromium release, lytic unit values and cytotoxicity relative to the parental line were calculated in order to determine if there were differences in LAK cell susceptibility between paired cell lines. The susceptibility of MDA-MB-231 and its MDR variants to LAK cell lysis is shown in Fig. + transfected lines MPAHS-1-10, MPAHS-1-300 and MPAHS-DOX150 were found to have a four to five fold increased sensitivity to LAK CML in comparison to the drug sensitive parent line MDA-MB-231. Lysis of MPAM-26 was significantly higher than that of MDA-MB-231, as shown by both Student's paired "t" test  (log-transformed data) and the Wilcoxon signed-ranks matched-pairs non-parametric test . The correlation between the two sets of paired data was r = 0.95 for the log-transformed data. Lysis of MPAHS-1-10 was significantly higher than that of MDA-MB-231, as determined by both Student's paired "t" test  (log-transformed data) and the Wilcoxon signed-ranks matched-pairs non-parametric test . The correlation between the two sets of paired data was r = 0.96 for the log-transformed data. Lysis of MPAHS-DOX-150 was also significantly higher than that of MDA-MB-231, as determined by the Wilcoxon signed-ranks matched-pairs non-parametric test , and by the Student's paired "t" test  (log-transformed data). All of the above statistics were calculated with n = 15.In these experiments, the murine MDR1-transfected variant MPAM-26 and the human P-gp51Cr might be actively pumped out of MDR cells during the 51Cr-release assay by the P-gp. This might complicate the interpretation of changes in the susceptibility of P-gp+ cells to LAK CML. In order to examine whether 51Cr is actively pumped out of MDR cells by P-gp during the assay, the "spontaneous" release (SR) of 51Cr from MDR breast cell lines and their sensitive counterpart was determined  for the duration of the assay. MDR carcinoma cell lines exhibited unchanged SR with respect to their sensitive counterparts. It is concluded that, in general, P-gp expression was not associated with any significant change in spontaneous 51Cr release during the time of experiment. These results are in agreement with similar results for the effect of P-gp expression on 51Cr efflux in MDR SCLC and ovarian carcinoma cell lines [P-gp is postulated to function as an energy-dependent efflux pump in MDR resistant cells. Therefore, ll lines ,16.In order to examine whether the increased or decreased LAK susceptibility of MDR breast cell lines is at the binding/recognition or post-binding level, conjugate formation assays were performed. PBMCs were activated with human rIL-2 and tested for LAK cell binding activity against the breast carcinoma cell line MDA-MB-231 and its MDR variants MPAM 26, MPAHS-1-10, MPAHS-1-300, and MPAHS-DOX150. The data obtained from 4 separate experiments are shown in Fig. All of the MDR variants had increased LAK:Target binding that was statistically significant. The parent line had approximately 27% of LAK cells bound to it versus 41% to 61% for the MDR cell lines. MPAM 26, MPAHS-1-10 and MPAHS-DOX150 had 50% to 80% increased binding compared to the drug sensitive parent line and MPAHS-1-300 had approximately 120% increased binding. The results are in agreement with previous studies for the SCLC and ovarian carcinoma cell lines . IncreasIn order to determine whether or not differences at the binding/recognition step of LAK CML are associated with differences observed in the LAK susceptibility of drug sensitive and resistant paired breast cell lines, the level of surface expression of adhesion molecules ICAM-1, LFA-3, N-CAM, and CD18, as well as P-gp and HLA Class I were measured in conjunction with the LAK CML of the cell lines. The level of expression of the surface molecules was measured using commercially available mAbs and flow cytometric analysis. Data are expressed in terms of percent positive cells and monoclonal antibody binding sites values calculated as described in the Methods.There was no significant difference in the expression of CAMs or HLA Class I antigen between the drug sensitive parent line MDA-MB-231 and its MDR1-transfected counterparts. Differences were observed in the level of P-gp expression. P-gp determined by MRK16 mAb was not expressed on the parent line or on murine MDR1-transfected MPAM 26. However, when MDA-MB-231 and MPAM-26 cells were stained with mAb JSB-1 which, unlike MRK16, recognizes a conserved cytoplasmic epitope of P-gp , P-gp exMPAHS-1-300 and MPAHS-DOX150 had significantly increased levels of P-gp compared to the parent line , and MPAHS-1-10 was higher at p = 0.03 . Relative Fluorescence Intensity (RFI) values were calculated according to the equation : RFI = RFI (test sample) – RFI (negative control).Samples were prepared by incubating the cell lines with each mAb listed followed by FITC-labeled secondary Ab. Samples were read on a Becton Dickinson FACS IV. Anti-CD54 (ICAM-1), anti-CD58 (LFA-3), anti-CD56 (N-CAM) or Leu19, anti-CD18, which is the β chain of LFA-1, MAC-1 and P150/95 were the anti-CAM mAbs studied. Levels of anti-P-gp mAb MRK16, which labels the surface epitope of P-gp, and W6.32  were also measured.The percentage of cells expressing surface antigen was determined using flow cytometric analysis. A lower gate channel was set on the negative control sample so that 5% of the total cells lay above the gate. The same gate was set on each test sample, and the percentage of cells which lay above the gate was determined. This number, after 5% was subtracted from it, was reported as the percent positive cells in each test sample. The data are shown in Table In order to assess the absolute quantitative level of expression of the different surface antigens studied, the number of actual Ab binding sites on the cell surface was determined. Differences exist between the number of secondary antibodies that may bind to a primary Ab and thus, differences exist in the measure of fluorescence obtained. In order to make accurate comparisons between the expression of different surface antigens and to obtain values that are consistent and comparable between laboratories, it is necessary to correct for this difference. In order to accomplish this, a microbead quantitation system was used. After the standardization of the FACS with calibrated fluorescence microbeads, the effective fluorescence-to-protein ratios for each mAb are calculated. Values obtained from three separate experiments for the MDA-MB-231 breast carcinoma cell line series are presented in Table In this study, the following observations were made:1. Three human MDR1-transfected, and one mouse MDR1-transfected breast carcinoma cell lines had increased sensitivity to LAK cell-mediated lysis compared with the parental line. The increases of these targets were statistically significant.2. There were no significant differences in the expression of adhesion molecules ICAM-1, LFA-3, CD18, and CD56 or HLA Class I antigen between drug-sensitive and MDR breast carcinoma paired cell lines.3. The MDR breast tumor cell lines demonstrated increased binding to LAK cells in comparison to the parent line.Immunotherapy using LAK cells is a candidate treatment modality which may be effective in patients with tumors that are resistant to chemotherapy in breast, ovarian, and colon carcinoma, small cell lung cancer and melanoma -16,18-20+ MDR during incremental drug challenge of parental drug sensitive cell lines; human and murine MDR1 cDNA were transfected into a metastatic breast carcinoma cell line and P-gp was allowed to demonstrate itself under the selecting pressure of 3 different drugs at four different concentrations in a short time compared to the previous MDR lines developed without MDR1 transfection. Increased LAK susceptibility was observed in all of the four MDR1-transfected cell lines. It should be emphasized that increased LAK susceptibility was observed for both human and murine P-gp expressing targets. Previously, increased human LAK effector killing against P-gp+ Chinese hamster ovary target cell lines was also shown [In order to eliminate factors other than P-gp that may develop during the long time necessary for a cell line to develop P-gpso shown . In the + cells be due to a change in adhesion molecules? CAMs may bind either to self (homophilic adhesion) or to other ligands (heterophilic adhesion). After in vitro incubation with IL-2, human NK cells demonstrated four- to sixfold increases in surface levels of CD18, CD54, CD58, and CD56 [+ within 3 days of exposure to IL-2. In addition, IL-2 dramatically increased the levels of CD56 and CD58 on NK cells. In contrast, T cells did not demonstrate comparable up-regulation of these CAMs after incubation with IL-2. Increases in NK cell CAM expression were associated with enhanced formation of effector to target cell (E/T) conjugates, enhanced killing of NK-sensitive targets, and the induction of cytotoxicity for previously NK-resistant targets (LAK activity).Could this increased LAK susceptibility of P-gpand CD56 . FurtherIn 1991, a new association between P-gp expression in invasive colon carcinoma cells and increased metastases to lymph nodes was described . Althoug+ the ovarian carcinoma cell line 2780.AD645 and SCLC line H69/LX4 [MHC, ICAM-1, LFA-3, NCAM and CD18 were examined in MDR1-transfected breast carcinoma cell lines in this study, and no significant correlations were found for the breast carcinoma cell lines neither between LAK CML and these CAM expressions, nor between LAK CML and MHC Class I molecule expression. Similar results were also observed previously in MDR P-gp H69/LX4 ,16.++ and PKC involvement [Another possibility for the mechanism may involve gangliosides. In 1991, Mazzoni et al. found an involvement of cell membrane gangliosides in the development of the A2780-Doxorubicin MDR phenotype, and suggested a possible involvement of the ganglioside GD1a in the increased sensitivity of MDR cells to immuno-mediated cytolysis . Indirecolvement . Alteratolvement ,30.The differentiation stage of tumor cells could also influence susceptibility of target cells. A decrease of NK sensitivity is detectable after induction of a more differentiated phenotype in colon-carcinoma lines by treatment with differentiating agents  or by trFor MDR1-transfected breast carcinoma cell lines, the increased binding of MDR cell lines in comparison to the sensitive parent line for LAK:Target binding suggests that differences in the LAK cell-mediated lytic susceptibility of these cells are due in part to differences at the binding/recognition level.The possible increased LAK susceptibility and binding of P-gp(+) MDR metastatic breast cancer cells as suggested in this paper encourages studies toward mAbs and LAK cells for the treatment of breast cancer patients since LAK cells have strong ADCC. The development of "designer" mAbs, chimeric mAb to particular antigens and receptors, and their potential to target cancer cells and enhance their susceptibility to the effector arm of the immune system are currently providing important new opportunities. The potential for the use of monoclonal antibodies such as P-gp and C-erb-B2 for the better therapeutic efficacy of LAK immunotherapy in breast cancer is particularly encouraging.Our results demonstrated that four MDR1-gene transfected metastatic breast carcinoma cell lines have increased susceptibility and binding to the lymphokine-activated killer cells. Interleukin-2 administration seems to be a promising chemoimmunotherapy improving modality in MDR1 over-expressing tumors.Unless otherwise indicated, media used to culture cell lines consisted of RPMI 1640 containing 10% FCS supplemented with 10 mM L-glutamine . Media used for the generation of LAK cells consisted of culture media without FCS, supplemented with 10% autologous plasma. Recombinant IL-2 was used at a concentration of 1,000 U/ml , which had been pre-determined to yield maximum LAK cell generation. Vincristine and doxorubicin were purchased from Sigma, St. Louis, MO. Geneticin was purchased from Gibco/BRL.2 fragment of goat anti-mouse Ig  .Anti-CD54 (ICAM-1) and anti-CD58 (LFA-3) were purchased from AMAC Inc., Westbrook, ME, anti-CD56 (N-CAM) was purchased from Becton Dickinson, Mountain View, CA, and anti-CD18 (β chain of LFA-1) was purchased from Dakopatts, Glostrup, Denmark. Anti-P-gp mAb MRK16, which labels the first extracellular domain of P-gp which is heavily N-glycosylated and shows the least homology between human and murine counterparts  was a ge6/mL) were incubated in 25 cm3 cell culture flasks (Corning) for 5–8 days in a 5% CO2 environment at 37°C with RPMI medium containing 10 mM L-glutamine, 10% autologous serum and 1000 U/mL human recombinant IL-2, and were used as LAK effectors [Venous blood was collected from healthy donors in heparinized Vacutainer tubes. Mononuclear cells were prepared by centrifugation  over Ficoll-Isopaque and were depleted of monocytes by incubation on autologous plasma coated plastic dishes for one hour at 37°C. These cells were recognized as PBL and were used in NK studies. PBL , which was isolated from pleural metastases of a woman with breast cancer ,37 was tCells were split using EDTA (Gibco/BRL) at 80% confluence. Cells were fed 2 times a week. The continuous cell line drug supplements were as follows: MPAM-26 G418  400 μg/ml; MPAHS-1-10 Vincristine Sulfate (Sigma) 10 ng/ml; MPAHS-1-300 Vincristine Sulfate (Sigma) 300 ng/ml; MPAHS-DOX150 Doxorubicin Hydrochloride (Sigma) 150 ng/ml.K562  is a human erythroleukemia cell line isolated from a pleural effusion of a patient with chronic myelogenous leukemia in blast crisis . K562 wavs drug-resistant pairs, breast target cell lines were resuspended at a concentration of 0.3–0.6 × 106 cells/flask in new flasks 5 days prior to the experiments in drug-free medium. The culture medium was changed each of the 5 days, and the experiments were done while the target cells were in the exponential growth phase.In order to create similar conditions for the comparison of the cytotoxic cell susceptibility and adhesion molecule expression of drug-sensitive 51Cr release assay [-), MPAM 26 (murine MDR1-transfected), MPAHS-1-10, MPAHS-1-300, MPAHS-DOX150 (human MDR1-transfected). Single cell suspensions of target cells in a logarithmic growth phase were labelled with 51Cr. Effector/target cell concentrations were adjusted to 40:1, 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.62:1, and 0.31:1 in 96-well microtiter plates, then incubated for 18 hours at 37°C. The plates were then centrifuged , and supernatants from each well were counted in a Beckman 5500 gamma-counter.LAK activity was assessed in an 18 hr se assay  against Percent specific release of each target was calculated with following formula.cpm = radioactivity count per minute in each well.51Cr release data were expressed in lytic units (LU) calculated according to the equation: y = A(1-e-kx) where y = percent of chromium release, x = Effector/target cell ratio, A = Maximal lysis by effector cells, k = a constant proportional to effector cell number, and equal to the negative slope of the target survival curve obtained by plotting ln (A-y) vs x [-y) vs x . Statist6 cells were placed in 12 × 75 mm test tubes  and centrifuged at 300 g for 6 min. The supernatant was removed and the mAb was added, tubes were vortexed and then incubated on ice in the cold room for 30–45 min. The concentrations of mAb added were: anti-ICAM-1 at 20 μl/test (5 × 105 cells/test), anti-LFA-3 at 10 μl/test (5 × 105 cells/test), anti-N-CAM at 20 μl/106 cells, anti-CD18 at 20 μl of 206 μg/ml stock, MRK16 at 10 μg/ml (5 × 105 cells) and W6.32 (ascites) diluted 1:500 (5 × 105 cells). Following this they were washed twice by centrifugation at 300 g for 6 min. with ice cold wash medium containing RPMI, 5% FCS and 1% sodium azide (Sigma). 100 μl of GAM-FITC (goat-antimouse Ig conjugated to FITC) (12.5 μg/ml) was added to the cell button, vortexed and incubated on ice for 30–45 min. Tubes were then washed 3 times at 300 g for 6 min. with cold wash medium (containing 1% azide). Finally, cells were resuspended in 0.5 ml RPMI (no FCS) and 1% paraformaldehyde (Sigma). Samples were analyzed on a Becton Dickinson FACS IV instrument, using an argon-ion laser running at 350 mW with the wavelength of excitation set at 488 nm and a 530/30 bandpass emission filter. Histograms were displayed on a 256-channel log scale. Uniform cell-sized quantitative fluorescent microbead standards  were used to calibrate the instrument. The percentage of cells expressing surface antigen was determined using flow cytometric analysis. In the analysis of the flow cytometry data, a lower gate channel was set on the negative control sample so that 5% of the total cells lay above the gate. The same gate was set on each test sample, and the percentage of positive cells which lay above the gate was determined. This number, after 5 % of the negative population was subtracted from it, was reported as the percent positive cells in each test sample. The mean log fluorescence channel was determined for the cells which lay above the gate channel. Further details are described in the Results section. The percent positive cells was related to the relative fluorescence intensity of the positive cell population.0.25–0.5 × 10The level of target cell surface antigens was quantitated by determining the number of antibodies bound by measuring the effective fluorescence/protein (F/P) ratios. The F/P ratio (as determined by absorbance) is the number of fluorescein molecules conjugated to an antibody molecule. The effective F/P ratio is the average fluorescence intensity in Molecules of Equivalent Soluble Fluorochrome (MESF) per antibody molecule. The FACS was calibrated for each experiment in terms of MESF per channel with the FCSC Quantum Kit (Flow Cytometry Standards Corp.). These standards consist of fluorescein molecules covalently bound to alkyl spacer groups on the exterior of uniform 5.8 μm hydrophobic polymeric microbeads. Their size and range of fluorescence intensity was optimized for analysis of common surface markers on peripheral blood lymphocytes. For each mAb studied, the fluorescence intensity in MESF was determined using the calibration curve and the peak fluorescence channel number obtained above. This MESF value was then divided by the number of binding sites on the microbead to give the effective F/P ratio.Target cells were labelled and analysed on the FACS IV using the same indirect immunofluorescence technique as used for the microbeads. The mean channel number was used to obtain the MESF value again from the calibration curve, and this value was divided by the F/P ratio for the particular mAb to give the actual number of mAb binding sites on the target cell surface.6 in 1 ml) and target cells (1 × 106 in 1 ml) were mixed in ice-cold PBS and centrifuged at 200 g for 8 min in a 5 ml tube. After incubation for 25 min. on ice, the 1 ml cell pellet were removed, and gently resuspended. 50 μl conjugate suspension were mixed with 200 μl trypan blue, and an aliquot was transferred to a modified Cunningham chamber [Lymphocyte-target cell conjugates were analyzed visually using a modification of the method described by Pross . IL-2 ac chamber . The perThe author(s) declare that they have no competing interests.BS carried out the cell culture studies, and LAK cell generation-cytotoxicity-binding assays. PEK did the adhesion molecule profiles. HFP participated in the design of the study, statistical analysis, coordination and helped to draft the manuscript. All authors helped and approved the final manuscript.LAK cells. Demonstration of lymphokine-activated killer cells colonies which were formed after 5–8 days incubation of PBL with human recombinant IL-2.Click here for file


The in vitro effects of sodium n-butyrate (butyrate) on the growth, morphology and secretion of alpha-fetoprotein (AFP) and albumin by the human heptoma cell line PLC/PRF/5 were studied. Butyrate caused a marked reduction in the growth rate, colony forming efficiency in soft agar and de novo synthesis of DNA as well as remarkable morphological changes including cell enlargement, flattening and a decreased number of nucleoli. Secretion of AFP was reduced during culture with butyrate, while that of albumin was increased. The requirement of de novo protein synthesis for the increase in albumin and decrease of AFP by butyrate was demonstrated by inhibition studies with cycloheximide. These results suggest that butyrate caused the hepatoma cells to acquire in vitro properties that are considered to be more consistent with normal liver cells.


ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest's 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol®). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for  Artemisia annua L., which is used in traditional Chinese medicine for the treatment of fever and chills Plasmodium falciparum and Plasmodium vivaxPlasmodia parasites are located. The active moiety of artemisinin-like drugs is an endoperoxide bridge, whose reductive homolysis is promoted by iron(II)-heme leading to C4-centered alkylating radicals Artemisinin is a sesquiterpene isolated from The role of artemisinins and iron for malaria treatment has been intensively investigated during the past years The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are involved in iron homeostasis. They are located in the mitochondria and transport heme and protoporphyrins into these organelles In humans, 49 different ABC transporters have been identified, which are classified into seven sub-families (A–G) MDR1), ABCC1-C6 (MRP1-6) and ABCG2 (BCRP) confer resistance to cytostatic drugs of tumors and contribute to the failure of tumor chemotherapy ABC transporters have been intensively investigated in the past years. ABCB1  and iron(II) glycine sulfate (Ferrosanol®) from Sanol .2). Cells were passaged twice weekly.CCRF-CEM were cultured in RPMI medium and MCF-7 and MEL cells in DMEM medium each supplemented with 10% fetal calf serum under standard conditions  of the Developmental Therapeutics Program of the NCI has been previously described MEL cells were cultured with and without 1.0 µg/ml artesunate (2.6 µM) for 24 and 48 h. Cell numbers were determined by trypan blue exclusion.6 cells/ml) were cultures in the presence of 2% DMSO with or without 1.0 µg/ml artesunate (2.6 µM) for 28h. The content of heme was estimated by the conversion of heme to protoporphyrin with oxalic acid MEL cells (1×10Drug sensitivity of the NCI cell lines has been determined by the sulforhodamine B assay 50) values for artesunate have been reported http://dtp.nci.nih.gov).A modified propidium iodide assay Arrays of cell lines from the Oncotest Human Cell Line Collection were assembled using a tissue arrayer  as recently described Quantitative PCR was done as described 6 cells/ml) were cultured in the presence of 2% DMSO. After 24, 48, 72, or 96 h the cells were collected and lysed. The cellular proteins were analyzed by SDS-PAGE and transferred onto PVDF membranes. Immunoblotting was performed with antibodies for ABCB6, ABCB7, and actin as the primary antibodies MEL cells  as described Fisher's exact test was used to calculate significance values as a measure for the dependency of two variables. This test was implemented into the WinSTAT Program . The TfR expression in the Oncotest panel of tumor cell lines has been determined by a tissue micro-array technique adapted for the examination of cell line and immunohistochemistry TFR  determined by microarray analysis http://dtp.nci.nih.gov). The microarray-based expression levels of ABCB6 and ABCB7 have been validated by RT-PCR 50) for artesunate are shown in 50 values varied between 0.197 and 47.779 µg/ml (0.512 and 124.295 µM respectively). The mean IC50 value was 2.791 µg/ml (7.261 µM). Furthermore, we tested a combination of artesunate and iron(II) glycine sulfate   with a range from 0.013 and 22.96 µg/ml . The data in 50 values for artesunate. In the majority of cell lines, the combination of artesunate plus iron(II) glycine sulfate resulted increased inhibition rates. However, in 11 out of the 36 cell lines  the combination treatment was not superior to treatment with artesunate alone (degree of modulation <1.2). Furthermore, degrees of modulation (IC50 for artesunate divided by the IC50 for artesunate plus iron(II) glycine sulfate) did not correlate with IC50 values for artesunate, e.g. cell lines with high IC50 values for artesunate were not more modulated by the addition of iron(II) glycine sulfate than cell lines with low IC50 values for artesunate  Table 1.50 values for artesunate alone or artesunate plus iron(II) glycine sulfate.Then, we correlated the expression of TfR with the degrees of modulation. We found that cell lines with high TfR expression significantly correlated with high degrees of modulation Table 2 TFR for sensitivity or resistance to artesunate in 55 cell lines of the NCI drug screening panel. We found a significant relationship between cellular response to artesunate and TFR expression  and ABCB7 . As shown in ABCB6, but not for ABCB7.Since ABCB6 and ABCB7 are involved in iron homeostasis of cells, we investigated a role of these two proteins for cellular response to artesunate. Again, we used the ICABCB6 and ABCB7 determined by real time PCR. As can be seen in ABCB6 increased with increasing doses of artesunate, while the expression of ABCB7 decreased accordingly. The induction of gene expression by artesunate might indicate a role of these two genes for cellular response to artesunate treatment. This can, however, not be taken as evidence, that the two genes are causatively related to artesunate response, and an epiphenomenal association can not be excluded.Then, MCF-7 breast cancer cells and CCRF-CEM leukemia cells were incubated with artesunate in a range of 0.05 to 50 µg/ml (0.13 to 130 µM respectively) for 24 h. Afterwards, mRNA was isolated and the expression of Finally, we treated MEL cells with artesunate . Mouse erythroleukemia (MEL) cells were used as model to study the role of ABCB6 and ABCB7 during cellular differentiation. While cell growth was almost stopped, only 20% of cells died . We previously reported that an antisense oligonucleotide directed against ABCB6 depressed the erythroid differentiation of MEL cells to 75% The ICThe aim of the present study was to explore the role of proteins related to iron homeostasis for response of tumor cells to artesunate. For this purpose, we focused on TfR and the ABC transporters, ABCB6 and ABCB7.50 values and high degrees of modulation by iron(II) glycine sulfate. This indicates that iron(II) glycine sulfate is not a typical resistance modulator comparable to modulators of multidrug resistance, where modulating agents completely or partially overcome resistance of a drug-resistant sub-line to a cytostatic drug towards the level of a parental drug-sensitive cell line We observed a wide heterogeneity of tumor cell lines towards artesunate. Whereas iron(II) glycine sulfate was able to increase sensitivity of tumor cells to artesunate in most cell lines, there was no correlation between the ICIt is important to point out that several cell lines did not show increased inhibition by the combination of artesunate and iron(II) glycine sulfate compared to artesunate alone. Some of these cell lines did even reveal decreased efficacy to artesunate under co-treatment with iron(II) glycine sulfate. Iron is an important regulator of cell growth and tumor cells may take advantage of external iron supply to grow faster. Iron(II) glycine sulfate is an approved drug, Ferrosanol® applied for many years in the clinic to treat iron deficiency. Therefore, iron(II) glycine sulfate seems to be a safe and suitable adjunct for combination treatments with artesunate at first sight. Our results do, however, indicate that there may be patients, who would not profit from addition of iron(II) glycine sulfate during artesunate treatment. In this context, iron(II) glycine sulfate is a double edged sword improving treatment response in the majority of tumor cell lines but worsening it in others. Other iron preparations have also been reported to improve the response of tumor cells to artemisinins, e.g. ferrous sulfate or iron-loaded transferring (holotransferrin) The fact that in some cases iron(II) gylcine sulfate could also lead to reduced response of tumor cells towards artesunate opens an option for the testing of tumors before treatment in a clinical setting. If predictive tests, e.g., determination of TfR expression or sensitivity testing by MTT assay, indicate enhanced sensitivity, a combination therapy of artesunate and iron(II) glycine sulfate could be applied. If this is not the case, iron(II) glycine sulfate could be skipped, and artesunate could be applied alone. This situation adds to the attractiveness of individualized tumor therapy. The main idea of this concept is to adapt treatment protocols of each individual patient according to the results of pre-therapeutic testing of efficacy of a planned therapy 50<4.2 µM), and both more resistant melanomas (IC50>12 µM) were strongly sensitized (degree of modulation >15) by the addition of iron(II) glycine sulfate indicating that this entity is especially suited for this treatment regimen. Interestingly, these results are supported by recent clinical data. Artesunate has been applied to two patients with otherwise drug-resistant and refractory uveal mealoma On the other hand, three out of the five melanoma cell lines were clearly above-average sensitive to artesunate  are also relevant for response of tumor cells towards artesunate


The antibiotic drug novobiocin was evaluated for its anti-tumour properties in B16 melanoma cells. Novobiocin is shown to inhibit melanoma B16 cell proliferation. The anti-proliferative effect was gradually reversible upon removal of novobiocin from the culture medium. Growth inhibition by novobiocin was accompanied by phenotypic alterations, that included morphological changes, lipid accumulation and marked increases in the activities of NADPH cytochrome c reductase and gamma glutamyl transpeptidase. In vivo administration of repeated i.p. doses of novobiocin, to mice implanted with B16 melanoma cells resulted in growth retardation. The combined treatment of the B16 melanoma cells with novobiocin and other chemical inducers of differentiation was examined in a cell growth assay. Novobiocin and sodium butyrate inhibited cell growth in a near additive manner, while combination of novobiocin with the GTP-depleting agents, tiazofurin or mycophenolic acid resulted in a synergistic decrease in cell growth. Our results support the contention further that novobiocin and other differentiating agents might be of potential value in melanoma therapy.


An immunocytochemical method was used to test the reactivity of the anti-P-glycoprotein antibodies, C219, MRK 16, JSB-1 and 265/F4 against multidrug resistant (MDR) variants derived from the human small cell lung carcinoma line, NCI-H69, the mouse fibrosarcoma line, RIF-1 and the mouse mammary tumour cell line, EMT6. C219 produced positive staining in MDR variants of both human and mouse tumour cell lines. MRK 16 and JSB-1 however recognised P-glycoprotein only in the human MDR cell lines and not in the mouse MDR cells. 265/F4 appeared the most selective of the monoclonal antibodies used, producing positive staining of MDR variants derived from the RIF-1 line, but not of MDR variants derived from the EMT6 line. Total RNA was prepared from the mouse cell lines and, following reverse transcription, cDNA sequences were amplified by the polymerase chain reaction with primers specific for either the murine mdr1a or the mdr1b genes. From this it was possible to show that only the mdr1a gene is overexpressed in the resistant EMT6 lines that do not stain with 265/F4 whereas both mdr1a and mdr1b are overexpressed in the positively staining resistant fibrosarcoma line, RIF/1.0. Low level expression of mdr1b was detected in the sensitive parent RIF-1 cells and increasing levels of expression correlated with increasing resistance in the lines, RIF/0.1, 0.2, 0.4 and 1.0. Expression of mdr1a was found only in the more resistant fibrosarcoma cell lines. It seems that 265/F4 recognises only the mdr1b P-glycoprotein. Western blotting confirmed that this antibody detects a 170 kDa protein only in membranes derived from the resistant fibrosarcoma cells. 265/F4 may thus be used to distinguish between the murine P-glycoprotein isoforms so revealing differences between tumour cell lines in cellular localisation and in the time of appearance of mdr1a and mdr1b P-glycoprotein following drug exposure.


Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed.Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR.According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication.The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research. The search for differentially expressed genes in tumours has made extensive use of array technology. Most studies have involved tumour biopsy samples or established tumour-derived cell lines . Differein vitro can bias the expression profile when compared to native tissues. Another problem is that a number of widely-used cell lines have sub-lines that differ in their karyotypes and RNA expression levels [As noted by Dairkee et al. , one dran levels -5. BiopsIn this communication we report which genes, within a defined set of well-known cancer-related genes, are differentially expressed in freshly-derived breast cancer cell cultures compared with primary normal, established normal and breast cancer cultures. We compared expression patterns in cultures of finite life-span cells from pleural effusions or ascites of breast cancer patients (Mount Sinai School of Medicine: MSSM) with commercially-available finite life-span normal breast epithelial cells (HMECs). To ensure comparability, cultures were used at similar passage numbers and the karyotypes were analyzed. We also compared the expression profiles of some of the most widely-used established breast cancer cell lines and three putatively normal established breast cell lines.a): HMECs: Different batches of normal human mammary epithelial cells cultures were obtained . HMEC specimens were enumerated serially and maintained with MEGM  supplemented with 2.5% FCS .b): MSSMs: Breast cancer cells obtained from ascitic fluids or pleural effusions of patients with breast cancer were seeded and maintained in our laboratory. They were designated MSSM 3 through MSSM 14 and maintained in MEGM supplemented with 2.5% of the corresponding original fluid when available and 2.5% FCS . The full characterization of these cultures will be published elsewhere (manuscript in preparation).HMECs were passaged only twice or thrice in our laboratory. MSSMs were harvested at the 5th or 6th passage after initial plating.a): Normal-established (N-est.): the cell lines used were derived from normal tissues: MTSV1-7 (a gift from J. Taylor-Papadimitriou), MCF10A (from R Mira y Lopez) and MCF10F .b): Tumour-established (T-est.): cell lines established from breast cancer pleural effusions or tumour tissues: MDA-MB231, MDA-MB453, MDA-MB468, two strains of MCF 7 (designated MCF 7N and MCF 7P), T47D, BT20 and BT474 . They were maintained in medium and supplements as recommended by ATCC.All cell cultures were harvested 48–72 h after plating at about 80% confluence. Sources and markers are given in Table The Atlas Human Cancer 1.2 cDNA expression array  is a nylon membrane printed with 200–600 bp fragments of 1176 characterized genes involved in cancer, 9 housekeeping genes and 6 negative controls. RNAs were extracted and labelled with the Atlas pure total RNA labelling system and hybridized according to the manufacturer's instructions.All the cell lines used for the arrays  were probed twice in separate assays. The accuracy of the duplicates was assessed by Pearson's correlation coefficient based on the adjusted intensities of all the genes spotted on the membrane, which ranged from 0.93 to 0.99.Hybridizations with 30 μg of total RNA were performed according to the manufacturer's instructions. The hybridized membranes were exposed to a phosphorimager screen and were read at 100 μm resolution in a Storm Phosphorimaging system . The scanned images were transformed to TIFF files with a PC bit order and then aligned and analyzed using AtlasImage 2.01 software . To average or compare the samples, the adjusted intensity signal was normalized using the global normalization mode featured in the software. We report only (a) those genes with significant (p < 0.01) differential expression of 2-fold or more, and (b) genes that were undefined for all the cell lines belonging to one type of sample, but were detected in other types with a difference at least equivalent to one background (540 units in intensity). .The AtlasImage software compares only two samples at a time. When we used it to determine the differences between cell classes, we first averaged the cell lines in the four classes  and then performed the comparisons as instructed by the manufacturer.Statistical analyses  were performed using Microsoft Excel 2000. Further analyses were performed with Significance Analysis of Microarrays (SAM) , Predictm of the product as determined from its dissociation curve. Product size was confirmed by agarose gel electrophoresis. The efficiency of each pair of primers for amplification was determined and expression of each gene relative to G3PDH was assessed by the program Qgene [ms of the products are given in To validate the results of the cDNA array experiments, some of the genes found to be differentially expressed were further examined by real-time PCR in 10 HMECs, 9 MSSMs, 3 N-est and 8 T-est cell lines. Five μg of total RNA (corresponding to about 100 ng of mRNA) were reverse-transcribed with oligo(dT)  in a 20 μl reaction volume, and after 125-fold dilution, 1.25 μl were used for a 40-cycle PCR on an ABI Prism 7900 thermal cycler. The reaction was carried out in a 384-well plate with a QuantiTect SYBR Green PCR kit  at an annealing temperature of 63°C and detection at 2–5°C below the TSamples were tested twice in triplicate. Pearson correlation coefficients for the duplicate Q-PCR results ranged between 0.89 and 0.99.All the MSSM cultures displayed similar growth rates and had no or minimal chromosomal changes (data not shown). Table Each cell line was probed twice. Averages of the duplicates after normalization of the adjusted intensities  were used to obtain the expression values for further analysis. The overall gene expression profile, as determined by Pearson's correlation coefficient, discriminates between HMECs and cells derived from metastasis of breast cancer (MSSMs). As seen in Table Hybridization signals of 928 genes (78.9%) were represented in at least three cell lines.  We used this subset of 928 genes, plus the 9 control genes, to construct a classification model using two prediction programs: PAM and SVM. Since these algorithms do not work well when the numbers of members differ among classes, we trained the SVM program using comparable numbers of lines from three classes  and treated the three N-est cell lines and the remaining HMECs and MSSMs as unknowns to be classified. The model thus generated had a classification accuracy of 100% (24/24) by the leave-one-out cross-validation method. The prediction for the three N-est lines was T-est, suggesting that these cells have an expression profile resembling those of the other established cell lines rather than the cultures of finite life-span cells. The remaining HMEC and MSSM cell cultures were correctly assigned to their respective classes see .The same analysis with PAM, applying a threshold of 3.5, which gives the maximum number of significant genes yielding no misclassification error upon cross-validation, predicted MTSV1-7 and MCF 10F as members of the "T-est" class and MCF 10A as an HMEC-class member with a probability of 1. Again, HMEC 13 and MSSM 14 were properly assigned Fig . FurtherOf the 1176 genes in the array, 862 (73.3%) gave hybridization signals in at least three of the finite life-span cell lines used. Of these, 123 (14.3%) showed differential expression when HMEC and MSSM cells were analyzed using the AtlasImage software (ratio >2 and difference in adjusted intensity > 540), and 101 were deemed significant genes with SAM  .Of these 123 from Atlas and 101 from SAM, 75 genes were differentially expressed with at least a 2-fold change according to SAM and AtlasImage, a minimum difference in intensity of 540 units and a p-value < 0.01 (Excel). These two programs use different algorithms to calculate the ratio (AtlasImage) and fold (SAM) values, so the outputs are not exactly the same. Fig. Some genes showed a broad range of expression values among cell lines belonging to the same group: one or two individual cell lines over-expressed the gene (>5 fold the average of the cell class), while others in the same class gave no signal or were barely above background. Since we were looking for genes that could enable us to differentiate among types of cell lines, and hence be useful as markers for each class, we inspected the values of these genes manually. The aim was to exclude from the 75 genes in Fig Fifty genes showed significant >2-fold (according to SAM software) and >2.5-fold (according to Atlas) differences between the MSSMs and the established breast cancer cell lines. Twenty-five were up-regulated and 25 down-regulated in the established breast cancer cell lines compared to the finite life-span cultures. Most of the differentially expressed genes can be classified under the GO terms "metabolism" and "cell communication". In particular: most of the down-regulated genes seem to be related to remodelling of the extracellular matrix, cell adhesion and receptor-linked signal transduction, while the up-regulated genes are related to general signal transduction pathways and cell proliferation Tables .To validate the differences seen in the arrays, some genes were tested by real time PCR. We selected several genes of which the expression was significantly increased or decreased, according to SAM and Atlas, for both the MSSM/HMEC and T-est/MSSM comparisons: 14-3-3σ , SPINT2, FES, SPARC, BIGH3 (or TGFBI), TIMP1, TIMP2, MMP11 and DAB2. Also, we analyzed some genes that were deemed significant for only one of the comparisons: NOTCH1, PLAU, CDA and SERPINB2 (or PAI2), and a few genes that were non-significant but somehow related to some of the aforementioned genes: tPA, PAI1, uPAR, DCK.It has been reported that correlation between Q-PCR and array data is highly variable . It depeOne of the genes excluded from the HMEC/MSSM comparison was CYR61, which was tested by Q-PCR. Even though the overall up-regulation value for this gene was consistent , its expression levels crossed over to the values of the other cell class in 4/17 cell lines, thus invalidating CYR61 as a reliable marker by itself.Established breast cancer cell lines have been widely used to study signal transduction pathways, test new pharmaceuticals and determine expression profiles that might predict the metastatic capabilities of primary tumours. In many cases MCF10A has been chosen as the "normal" control, even though this cell line has been reported to possess markers for both myoepithelial and luminal phenotypes .One of the controversies about the use of HMECs as controls is the probable myoepithelial origin of these cells  based onBoth classes of non-immortalized cell lines, HMEC and MSSM, are more homogeneous than the established ones (N-est and T-est), as seen in Table As seen in Fig We tested only two sub-lines of MCF-7, well-known to be highly variable -5, and aWhen the MSSMs are compared with the HMECs, several genes display differential expression in a mode consistent with previous publications, where they have been shown to be significant for malignant progression or metastasis: down-regulation of the tumour suppressor and inhibitor of mitotic phase entry 14-3-3σ (SFN) ,17, the SPARC expression was detected in 17/17 human breast tumour biopsies and to a lesser extent in some established cell lines . It has in vitro.We also found genes of which the behaviour did not fully agree with previous descriptions, i.e. NOTCH1, CYR61 and DAB2. The many and varied functions of Notch signalling, achieved through activation or down-regulation, have been recently reviewed . In the Epithelial-mesenchymal transition (EMT) is considered a mechanism for carcinoma progression and metastasis, and the expression of vimentin (VIM) is its main marker. This view has now been extended to include whole pathways and a more complex relationship with the microenvironment of the cell ,39. AmonUsing arrays, we have studied 10 finite lifespan breast cancer cell lines freshly isolated from metastatic pleural or peritoneal fluids, 9 finite lifespan normal breast cell lines, 7 established breast cancer cell lines, and 3 established normal breast cell lines. We tested 1176 genes considered to be related to cancer. Within each cell class there was significant homogeneity of gene expression. Two clusters of genes distinguished the MSSMs from the HMECs. These 48 genes, which were differentially expressed by at least 2 fold, concerned cell-cell interactions and remodelling of the extracellular matrix. Fifty genes that were differentially expressed at least 2 fold between MSSMs and established breast cancer cell lines are generally considered to be involved in cell communication and metabolism. Established breast cancer cell lines have been used to model biochemical and pharmacological responses in human breast cancer; the differences from freshly isolated breast cancer lines imply they are not wholly satisfactory models.ER = oestrogen receptor, FCS = foetal calf serum, FDR = false discovery rate, KT = keratin, MGB = mammoglobin, SVM = support vector machines, VIM = vimentin.The author(s) declare that they have no competing interests.MFC designed and performed all the experimental work and drafted the paper. JFH provided fundamental advice on the clinical aspects of the project and participated in the final writing of the paper. BGTP has directed the entire project and participated in the final writing of the paper.The pre-publication history for this paper can be accessed here:Fig S1.doc: Pearson's correlation coefficient between cell line expression profilesClick here for fileFig S2.doc: Additional Plots and list of significant genes generated by PAM.Click here for fileFig S3: SVG . Go Miner DAG  view of the changed genes within the GO hierarchy.Click here for fileTable S1:.doc Primer sequences and PCR-product details.Click here for fileTable S2:.doc: Support Vector Machines: Validations and predictions.Click here for fileTable S3:.doc: SAM output.Click here for fileTable S3:.doc: False discovery rates and p-values of the Pomelo Tool applied to the comparison HMEC-MSSM.Click here for fileTable S5:.xls: p-values of Go Miner.Click here for file


The potential of mouse monoclonal antibodies for recognising different antigens associated with human small cell lung cancer (SCLC) to form active immunotoxins was assessed by an indirect in vitro screening assay. The screening agent used was a conjugate made by linking ricin A chain to a sheep anti-mouse IgG Fab' fragment via a disulphide bond. The monoclonal antibodies SWA11 and SWA20 both mediated the toxic effects of ricin A chain against the HC12 classic SCLC cell line in dose-dependent fashion. The SWA11 antibody was the more effective; in combination with the screening agent at a concentration of 1 x 10(-7) M, it inhibited the incorporation of [3H] leucine into HC12 cells by 94% compared with only 44% inhibition in the case of SWA20. An immunotoxin made by the direct chemical conjugation of ricin A chain to SWA11 exhibited selective toxic effects upon HC12 cells in tissue culture inhibiting the incorporation of [3H] leucine by 50% at a concentration (IC50) of 6.2 x 10(-10) M and by 98% at 1 x 10(-7) M. SWA11-ricin A chain had an IC50 of 4.4 x 10(-10) M against the NCI-H69 classic SCLC cell line but showed no cytotoxic activity against the human lung adenocarcinoma cell line NCI-H23 at a concentration of 1 x 10(-8) M.


We have studied the growth inhibition, DNA synthesis inhibition and cell incorporation of the new anthracycline 4'-iodo-4'-deoxydoxorubicin (4'-iododoxorubicin) and of its 13-dihydroderivative in a model of doxorubicin-sensitive and -resistant rat C6 glioblastoma cells; results were compared to those obtained with doxorubicin and doxorubicinol in the same model. 4'-Iododoxorubicin was 7.5 times more potent than doxorubicin on the wild cell line and 45 times on the doxorubicin-resistant line, indicating that cross-resistance was only partial between the two drugs. Whereas doxorubicinol presented only a very faint cytotoxic activity, 4'-iododoxorubicinol retained the same activity as the parent drug against sensitive cells and a lower activity against resistant cells. DNA synthesis inhibition occurred for much higher doses than growth inhibition in the sensitive cells, but for similar doses in resistant cells. In both cell lines, 4'-iododoxorubicin and its metabolite were incorporated to a higher extent than doxorubicin and doxorubicinol respectively. Incorporation of metabolites was always lower than that of their parent compound. We have studied the metabolism of doxorubicin and 4'-iododoxorubicin by sensitive and resistant cells; only traces (less than 5%) of metabolites were identified in the cells as well as in the culture medium. A new cell line was selected for resistance in the presence of low amounts of 4'-iododoxorubicin. It presented a 6-fold resistance to 4'-iododoxorubicin and an 85-fold resistance to doxorubicin. Doxorubicin incorporation was markedly reduced in this cell line while 4'-iododoxorubicin was incorporated to the same extent as in the sensitive line. Measurements of drug efflux were performed in the three cell lines. No significant difference was exhibited between the efflux of doxorubicin and that of 4'-iododoxorubicin in each cell line; these effluxes were very rapid in the doxorubicin-selected resistant line, slow in the wild line and intermediate in the 4'-iododoxorubicin-selected line.


We demonstrated that four human oesophageal squamous cell carcinoma cell lines  produced matrix metalloproteinase-1 (proMMP-1/tissue collagenase), 2 (ProMMP-2/'type IV collagenase'), 3 (proMMP-3/stromelysin), and 9 (proMMP-9/92-kDa gelatinase) as members of a matrix metalloproteinase (MMP) family, which degrades extracellular matrix macromolecules. Under normal culture conditions, in immunoblot analysis, proMMP-1 of M(r) = 53,00 was detected in one cell line (TE8), proMMP-2 of M(r) = 72,000 in three cell lines , and proMMP-3 of M(r) = 57,000 in all four cell lines. In addition to these enzymes, in enzymography, a gelatinolytic activity around M(r) = 92-kDa, likely to be proMMP-9, was detected in only one cell line (TE10) under normal culture conditions. When these cell lines were treated with epidermal growth factor (EGF), however, the agent stimulated three cell lines  to produce proMMP-9 in a dose-dose dependent manner. Oesophageal carcinoma-conditioned medium stimulated oesophageal fibroblasts to produce proMMP-1, -2, and -3, suggesting that the interaction between oesophageal carcinoma and stromal fibroblasts also plays a role in the production of MMPs by the latter. Our present study illustrates that oesophageal squamous cell carcinoma produces a variety of MMPs including proMMP-1, -2, -3, and -9 in vitro, suggesting that the ability of MMP production of the tumour may play an important role in its malignant behaviour and that the production of proMMP-9 may be regulated by EGF via overexpression of EGF receptors.


Stanley Riddell and Frederick Appelbaum review progress in preventing graft-versus-host disease following allogeneic hematopoietic cell transplantation for malignancies or other life-threatening blood diseases. This year approximately 20,000 individuals will receive an allogeneic hematopoietic cell transplant (HCT) as treatment for a malignant, or life-threatening non-malignant, hematopoietic disease. The process of HCT generally begins with administration of a preparative regimen to eradicate the underlying disease and immunosuppress the patient in order to prevent rejection of the subsequently transfused hematopoietic stem cells. Following HCT, donor T cells transplanted with or developing from the hematopoietic stem cells react with cells of the human leukocyte antigen (HLA)-matched but genetically non-identical host, providing a beneficial graft-versus-tumor (GVT) response but also resulting in possibly life-threatening graft-versus-host disease (GVHD). The manifestations of GVHD vary over its course. Acute GVHD usually appears within several weeks of HCT and is characterized by a diffuse maculopapular rash, mucosal inflammation causing crampy abdominal pain and diarrhea, and elevated liver function tests . GVHD thIf no immunosuppression is given after allogeneic HCT, life-threatening or fatal GVHD inevitably develops. The first successful application of allogeneic HCT to treat human leukemia in the early 1970s was made possible by the use of methotrexate, administered early after transplantation as prophylaxis against GVHD ,3, and sAn elusive goal of research has been to find ways to prevent GVHD without dramatically increasing other transplant complications. Most clinical studies to date have focused on the use of alternative immunosuppressants or removal of T cells from the donor stem cell source. More intensive post-transplant immunosuppressive regimens and T cell depletion are both capable of dramatically reducing the incidence and severity of GVHD, but do so at the cost of an increased incidence of fatal post-transplant infections and tumor recurrence. Increased graft rejection may also occur if donor T cells are removed from the donor stem cell graft, because the reaction of these cells against residual host immune cells contributes to engraftment. More intensive preparative regimens can overcome this problem but are associated with increased toxicity. More potent post-transplant immunosuppressive regimens and T cell depletion suppress or remove immunocompetent cells responsible for protection against many pathogens as well as cell populations that contribute to the beneficial GVT response. Accordingly, in retrospective analyses and prospective randomized trials, neither more intensive post-transplant immunosuppression nor T cell depletion has been demonstrated to improve survival following allogeneic HCT . While iOne area of progress, as noted above, has been in HLA-typing technology and donor selection. Historically, HLA typing was conducted using serologic methods, but with the advent of the polymerase chain reaction assay in the 1980s, it became possible to perform molecular typing of donor and recipient. When patients previously transplanted from serologically matched donors were retrospectively analyzed using molecular typing, approximately 30% were found to be mismatched with the donor for one or more alleles, and such mismatching was shown to lead to more GVHD and poorer survival . Thus, mIL-10 promoter region as well as in the donor IL-10 receptor beta gene have each been found to be associated with a lower risk of GVHD and non-relapse mortality [The pathogenesis of GVHD involves the expansion and differentiation of donor T cells reacting in peripheral lymphoid tissues against host antigen-presenting cells that display disparate minor histocompatibility antigens. These antigen-presenting cells are activated as a consequence of tissue injury and the resulting release of proinflammatory cytokines . Polymorortality ,9. PolymFerrara et al., 2006 [l., 2006  This revWagner et al., 2005 [l., 2005  A multicAnderson et al., 2003 [l., 2003  Stem celDickinson et al., 2002 [l., 2002  This papBonnet et al., 1999 [+ minor histocompatibility antigen–specific T cells recognize and eliminate human acute myeloid leukemia stem cells.l., 1999  The firsIn an effort to provide a more global assessment of whether a donor is likely to induce GVHD, Baron et al. have examined gene expression profiles of CD4 and CD8 T cells from donors and report that it is possible to segregate donors into those likely to cause GVHD and those who are relatively safe . If thisN), antigen-experienced memory T cells, (TM), and regulatory T cells (TREG). Recent studies have begun to dissect the contribution of these individual T cell subsets to GVHD and have identified opportunities for more refined manipulation of the T cell content of stem cell grafts that may reduce GVHD without the severe T cell deficiency associated with complete depletion (N from allogeneic stem cell grafts abrogated GVHD in both CD4- and CD8-dependent multiple minor histocompatibility antigen–mismatched mouse models, and the remaining TM provided reconstitution of immunity to pathogens [N and TM can also be distinguished based on phenotype— TN are CD45RA+ and CD62L+, while TM are CD45RO+ and either CD62L+ or CD62L-, and emerging data suggest that alloreactivity for minor histocompatibility antigens is predominantly contained in the TN repertoire subset [M comprises less than 1% of the overall T cell receptor diversity and consists of large numbers of T cells specific for cytomegalovirus, Epstein Barr virus, and other pathogens that cause opportunistic infections in HCT recipients [CD4+CD25+Foxp3+ TREG cells suppress T cell responses in vitro and in vivo suggests another attractive approach to donor graft manipulation for preventing GVHD. The importance of TREG in GVHD is supported by murine studies showing that their depletion from stem cell grafts exacerbates GVHD and that the infusion of additional TREG at the time of HCT reduces lethal GVHD, apparently by limiting the initial activation of alloreactive T cells in lymph nodes [REG confer a lower risk of GVHD [REG that might be used to supplement stem cell grafts and abrogate GVHD [Stem cell grafts contain distinct functional and phenotypic subsets of T cells, including antigen-inexperienced naïve T cells (Tepletion . For exaathogens ,13. Humae subset . The humcipients . Thus, uph nodes ,17. Clin of GVHD , and effate GVHD .Although these new approaches to allogeneic HCT are likely to reduce the severity of GVHD, an important concern for patients undergoing HCT for a malignant disease is whether reducing GVHD might increase the risk of tumor recurrence. Like GVHD, GVT is the result of donor T cells reacting with disparate minor histocompatibility antigens, and elimination of GVHD would seem almost certain to diminish the GVT effect. Elucidation of the molecular structure, HLA restriction, and tissue expression of human minor histocompatibility antigens, and the identification of non-polymorphic leukemia-associated antigens that can be recognized by T lymphocytes offers the exciting prospect that targeted T cell therapy after HCT might selectively augment GVT activity . An incrN subsets, retaining TM subsets, and possibly supplementing TREG subsets are just being initiated. In the long term, perhaps the most exciting potential lies in the segregation of GVHD from the GVT reaction based on increased recognition of minor histocompatibility antigens exclusively expressed on normal versus malignant hematopoietic tissues. Whether these new advances will lead to the hoped-for clinical victories will become apparent in the next few years.The human graft-versus-host reaction continues to both fascinate and frustrate clinical investigation with its lack of predictability, possibly lethal toxicity, but potentially lifesaving anti-tumor effects. The ability to select optimal donors based on improved HLA-typing technologies and better understanding of non-HLA contributions to immune reactivity seems at hand, and should substantially reduce the risk of developing severe GVHD. Clinical studies of donor graft manipulation removing T


Integrin-linked kinase (ILK) is a ubiquitously expressed protein kinase that has emerged as one of the points of convergence between integrin- and growth factor-signalling pathways.In this study we identify the ILK isoform expressed in five human oesophageal squamous cell carcinoma cell lines of South African origin as ILK1, and demonstrate its cellular distribution. ILK expression, although similar in the majority of the cell lines, did show variation. Furthermore, the ILK expressed was shown to be catalytically functional. The effect of growth factors on ILK expression was examined. An increase in ILK expression, following EGF and TGFβ1 exposure, was a trend across all the five oesophageal carcinoma cell lines tested.These results suggest that growth factor modulation of ILK expression relies on the internalisation/recycling of growth factor receptors and stimulation of the PI3K pathway, which may have implications with regards to cell adhesion and tumourigenesis. While responsiveness to growth factors is central to a host of normal cellular events, aberrant activity plays a key role in tumour development -3. AmongIn normal epithelial cells, appropriate cellular attachment is necessary for signalling pathways to be elicited ,10. MoreThe ILK protein is a ubiquitously expressed serine/threonine protein kinase that is activated in response to PI3K stimulation ,11-17. OSince our focus concerns oesophageal carcinoma, ILK expression is particularly intriguing since this tumour has a propensity to invade and metastasise. Carcinoma of the oesophagus has an extremely high incidence with high mortality and a poor prognosis -27. It hBamH1 to two fragments of approximately 823 and 536 bp respectively . However, differences in expression were observed for cell lines WHCO1 (1.3 fold higher) and WHCO6 (1.5 fold lower) than the average Figure .1 integrin subunit, it was noted that both ILK and the β integrin subunit followed a similar focal adhesion distribution pattern , not unlike the effect of EGF. However, variations were noted in the WHCO1 and SNO cell lines where fluctuations in ILK expression were observed Fig. . After 0The WHCO3, WHCO5 and WHCO6 cell lines demonstrated a similar trend in increasing ILK expression in response to TGFβ1 treatment. The WHCO3 cell line produced a substantial increase in ILK expression reaching and maintaining a 7 fold increase over 3 hours exposure. Surprisingly, exposure to TGFβ1 for 6 hours did not produce a higher increase in ILK expression, although ILK expression was still 5.6 fold higher than the untreated control Figure . ILK exp1 integrin peptide, was used to demonstrate the basal kinase activity of ILK. The five cell lines displayed a range of activities with the WHCO5 cell line exhibiting highest ILK activity (higher by 2 fold than average ILK activity) and the WHCO3 demonstrating much lower activity levels (2.5 fold lower than average) , which contains a β Figures  and 8B. Interestingly, it was observed that treatment of cell cultures with 10 ng/ml EGF and TGFβ1 for a period of 24 hours resulted in a decline in ILK kinase activity of 1 to 3 fold in cell lines WHCO1, WHCO6 and SNO , share similar structural and functional properties but, whereas ILK1 is expressed in all normal tissues, ILK2 has only been identified in two highly metastatic fibrosarcoma and melanoma cell lines  and ILK2,23. ThesWith the exception of the WHCO1 and WHCO6 cell lines, the expression levels of ILK were relatively similar among the WHCO3, WHCO5 and SNO cell lines  atmosphere.Five South African moderately differentiated human oesophageal squamous carcinoma cell lines WHCO1, WHCO3, WHCO5, WHCO6  and SNO ® solution (GibcoBRL). For reverse transcription, 2μg of total RNA was converted into cDNA by MMLV reverse transcriptase at 37°C for 1.5 hours in a 25μl RT reaction. Briefly, PCR amplification mixtures contained cDNA, ILK antisense primer (5'-CTCGAGCTACTTGTCCTGCATCTTCTCAAG-3'), sense primer (5'-GAATTCGTATGGACGACATTTTCACTCAGTGC-3') and Expand High FidelityPLUS Taq . Thermal cycle conditions included denaturation at 95°C for 10 minutes, and 30 cycles between 95°C for 15 seconds, 50°C for 1 minute and a 2 minute extension step at 72°C, followed by a final elongation at 72°C for 10 minutes. RT-PCR products were visualised on 2% agarose gels following ethidium bromide (EtBr) staining.Cells were grown to 80% confluence in sterile 10 cm Nunc™ tissue culture dishes and total RNA isolated using TRIzol® QIAquick Purification Kit. Thereafter, restriction digests were performed by the addition of BamH1 and HincII restriction enzymes to purified ILK products, and incubated for 1.5 hours at 37°C. Subsequent digestions were cleared of protein by the addition of phenol/chloroform and centrifuged at 6000 rpm for 2 minutes and aqueous phase transferred to a sterile tube. Both 0.7 M EtOH and 3 M NaAc were added and allowed to precipitate DNA for 12 hours at -20°C. Samples were centrifuged at 6000 g for 20 minutes, the supernatant decanted, and the DNA pellet resuspended in dH2O. Products were visualised on 2% agarose gels following EtBr staining.ILK RT-PCR products were purified using a QIAGEN®, USA) and polyclonal horseradish peroxidase (HRP)-bound anti-rabbit secondary antibody  antibodies were used for immunoprecipitation and immunoblotting procedures. Polyclonal rabbit anti-ILK antibody and a fluoroscine isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody  were used for indirect immunofluorescence.Polyclonal rabbit anti-ILK antibody  followed by incubation with an anti-rabbit Fluoroscine Isothiocyanate (FITC)-conjugated anti-rabbit antibody (1:1000) for ILK. Slides were viewed under a Zeiss LSM 410 confocal microscope .Nuclear extractions were performed utilising the CelLytic™ Nuclear™ Extraction Kit , which is based on a hypotonic buffer treatment followed by a high salt buffer release of nuclei.To examine growth factor effects on the WHCO1, WHCO3, WHCO5, WHCO6 and SNO HOSCC cell lines, cell cultures were first serum starved for a period of 16 hours. Cells were then treated with either 10 ng/ml EGF or TGFβ1 for a period of 0.5, 1, 3, 6 and 24 hours respectively. Experiments were repeated on three separate occasions.® Bayer, SA) stock solution. The cell suspension was centrifuged at 1000 g for 2 minutes. The supernatant was decanted, and the cell pellet resuspended in an extraction buffer  and incubated for 2 hours on ice. Thereafter, samples were centrifuged at 6000 g for 10 minutes. The resulting cell lysates were stored at -70°C. Total protein content was determined using the Bradford assay  ATP  and 10 μg myelin basic protein (MBP)  for 2 hours at 30°C with gentle agitation. The reaction was terminated by the addition of an equal volume of 2 × laemmli buffer and incubation at 95°C for 5 minutes. Phosphorylated MBP products were recovered by centrifugation at 6000 g for 10 minutes and resolved on 10% SDS-PAGE, following Coomassie Blue staining, destaining, and rinsing in dH2O and 10% ethanol respectively. Gels were subsequently dried on a slab gel drier SE1160 . Phosphorylated MBP was detected via autoradiography.Immunoprecipitated ILK was incubated in a kinase reaction buffer , to be an appreciable increase in ILK expression levels. All subsequent fold increases are compared to an untreated control, unless otherwise stipulated.GAD and RBV contributed equally to the conception and design of the study. GAD performed all experimental work. RBV participated in the coordination of the study. Both authors read and approved of the final manuscript.


The DNA repair enzyme, O6-alkylguanine-DNA-alkyltransferase (ATase), is thought to be the principal mechanism controlling resistance to nitrosoureas and related alkylating agents. We compared the sensitivities of five human testis and five bladder tumour cell lines to two nitrosoureas (N-nitroso-N-methylurea (MNU) and mitozolomide) with cellular levels of ATase. Enzyme levels ranged from 3 to 206 fmol mg-1 protein (0.1 x 10(4) to 5.1 x 10(4) molecules/cell) in the testis lines and from 11 to 603 fmol mg-1 (0.4 x 10(4) to 9.1 x 10(4) molecules/cell) in the bladder lines. Based on IC50s in an MTT assay, the testis tumour cell lines were, on average, four times more sensitive to MNU and six times more sensitive to mitozolomide than the bladder cell lines. The cytotoxicities of MNU and mitozolomide were closely related (R = 0.9). In the testis cell lines ATase activity (molecules/cell) was related to IC50s for mitozolomide (R = 0.97) but not MNU (R = 0.78). In the bladder cell lines and overall, ATase activity correlated with cellular sensitivity to neither agent. Relatively high levels of resistance occurred in cells expressing low levels of ATase, and amongst cell lines expressing high levels of ATase, large differences in IC50s were observed. These results support the suggestion that resistance to nitrosoureas can be mediated by mechanisms other than ATase and that at relatively high levels of expression, ATase does not confer resistance in proportion to its activity.


Previous data from our laboratory has indicated that there is a functional link between the β-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer.GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay.2-adrenergic antagonist ICI 118,551 (100 μM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the β-adrenergic agonist isoproterenol (10 μM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 μM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK  mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK  mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells.GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with βWe feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and β-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results. Xenopus laevis oocytes coexpressing β2-adrenergic receptors and GIRK1/GIRK4 subunits [1 or β2 adrenergic receptors, the β-adrenergic agonist isoproterenol (Iso) stimulated GIRK currents, whereas this stimulation was not seen in non-transfected cells [Recent studies in human cancer cell lines or in animal models have shown that the growth of adenocarcinomas of the lungs, pancreas and colon are under β-adrenergic control -5. The tsubunits . In addied cells . Previoued cells ,10. AddiEstablished risk factors for breast cancer include age, increased hormone exposure, alcohol consumption and family history as well as many other factors . Smoking+, Na+ and Ca2+ channels have been shown in the small cell lung cancer (SCLC) cell lines NCI-H128, NCI-H69 and NCI-H146 by measurement of currents [+ channels in the SCLC cell lines have a role in modulating cell proliferation [In order to investigate possible similarities between breast cancer and lung cancer, we wanted to determine if GIRK channels are expressed in human lung cancers and if β-adrenergic signaling is also involved in lung cancer. Voltage gated Kcurrents . These vferation . A classferation . Inwardlferation . GIRK1 eferation . Prior t2. A549 cells were grown in Hams F12 media with supplements as above. Human small airway epithelia cells (SAEC) were purchased from Clonetics/BioWhittaker . These primary cells were maintained in SAEC basal medium with supplements (Clonetics) at 37°C in an atmosphere of 5% CO2. Fresh surgical tissue samples were collected from patients at the University of Tennessee (UT) Graduate School of Medicine's Cancer Center and processed for reverse transcription polymerase chain reaction (RT-PCR). The collection of tissue was approved by the UT Institutional Review Board, and the authors have been certified by the NIH Office of Human Subjects Research. Cultures of fetal hamster pulmonary neuroendocrine cells (PNEC) were established from fetal lung periphery harvested on day 15 of gestation as previously described [The human SCLC cell lines NCI-H69 (H69), NCI-H146 (H146), NCI-H187 (H187), NCI-H209 (H209), and NCI-H526 (H526), the human adenocarcinoma cell lines NCI-H322 (H322), NCI-H441 (H441), and A549, the carcinoid cell line NCI-H727 (H727), and the squamous cell lines NCI-H226 (H226), NCI-H2170 (H2170), and NCI-H520 (H520) were purchased from the American Type Culture Collection . The human SCLC cell line WBA  was a giescribed  . PCR conditions are 94°C, 30 sec; 55°C, 30 sec; 72°C, 45 sec for 40 cycles. Cyclophilin primers were used an internal control .RNA from cell cultures, adult hamster brain, PNEC and from fresh surgical tissue samples was isolated by guanidine isothiocyanate/cesium chloride ultracentrifugation  or by anNM002239) and the internal TaqMan probe is 6-FAM-tcaagtggcgctggaacctc-TAMRA (bases 429–449), annealing 62°. GIRK2 primers-forward 5'-gacctgccaagacacatcag-3' and reverse 5'-cggtcaggtagcgataggtc-3'  and the internal TaqMan probe is 6-FAM-gtgcaatgttcatcacggcaac-TAMRA (bases 837–859), annealing 56°. GIRK4 primers-forward 5'-agcgctacatggagaagagc-3' and reverse 5'-aagttgaagcgccacttgag-3'  and the internal TaqMan probe is 6-FAM-accggtacctgagtgacctcttca-TAMRA (bases 301–324), annealing 62°. Reactions were run on a Cepheid SmartCycler . Reaction conditions are 200 μM dNTPs, 0.3 μM gene specific primers, 0.2 μM TaqMan probe, 4 mM (GIRK1) or 6 mM (GIRK2or4) magnesium acetate, 2 μl cDNA and 1.5 U MasterTaq  and MasterTaq buffer in a final volume of 25 μl. In some experiments, 18S primers were used an internal control .Real-time PCR was done as previously described . GIRK1 pCells were counted and plated in RPMI without serum or phenol red at a density of 50,000 cells/well with appropriate treatments in a volume of 500 μl. Cells were allowed to grow for 48 hours, and proliferation was measured by a cell proliferation assay for 5-bromo-2'-deoxyuridine (BrdU) according to the manufacturer's instructions , N = 5 for each treatment group.Cell pellets were collected and membrane protein was isolated with the ReadyPrep protein extraction kit  . Protein levels were determined using the RCDC kit (Biorad). Aliquots of 20–30 μg protein were boiled in 3× loading buffer  for two minutes, then loaded onto 12% Tris-glycine-polyacrylamide gels , and transferred electrophoretically to nitrocellulose membranes. Membranes were incubated with the primary antibody . In all western blots, membranes were additionally probed with an antibody for actin (Sigma) to ensure equal loading of protein between samples. The membranes were then incubated with appropriate secondary antibodies . The antibody-protein complexes were detected by the LiCor Odyssey infrared imaging system .T values for the samples. We wished to compare GIRK1 expression in SCLC to normal primary cells. GIRK1 was not expressed in the normal human SAE cells  cancers. GIRK1 was not expressed in two adenocarcinoma cell lines of Clara cell phenotype, H322 and H441  had no effect on gene expression when H69 cells were treated for seven days . As indicated above, we wanted to determine if β-adrenergic ligands altered proliferation in H69 cells. We determined the effects of the β-adrenergic agonist Iso and the GIRK channel inhibitor U5 [Since β-adrenergic ligands affected both gene expression of GIRK1 and cell proliferation for breast cancer cell lines in our laboratory , we wantys Table . HoweverCR Table . Calcula Formula , ICI trebitor U5  on proli+, Na+ and Ca2+ channels have been shown in SCLC cell lines [+ channels have a role in modulating cell proliferation [This is the first report that describes the expression of mRNA for G-Protein Inwardly Rectifying Potassium Channels (GIRKs) in lung cancer cell lines. Previous reports have indicated that voltage gated Kll lines , and theferation . Classicferation ,23. Sincferation . All sixferation , and thiferation .Differences in GIRK channel mRNA expression was seen in different types of lung cancer. The three adenocarcinomas from the lung cancer patient samples did not express GIRK1, and a subset of lung adenocarcinoma cell lines  also did not express GIRK1. However, GIRK1 expression was seen in an adenocarcinoma cell line with alveolar type II cell phenotype (A549). These variations may be due to differences between the Clara cell and the alveolar type II lineage, or it may be due to the small sample size. The cell linage of the lung cancer patient samples is unknown. Data from our laboratory has also indicated differences in responses to anticancer agents between alveolar type II and Clara cells in the lungs . FurtherThe two samples from squamous cancer patients also did not express GIRK1 mRNA. However, squamous carcinoma cells lines and a carcinoid cell lines do express GIRK1. In all NSCLC cell lines that express GIRK1, either GIRK2 or GIRK4 mRNA was expressed, indicating that functional GIRK potassium channels are possible in these non-SCLC cancer cell lines. Our data showing lack of GIRK1 expression in tumors from human adenocarcinomas and squamous cell carcinomas in is in contrast to Takanami et al. . They foThe mRNA for GIRK1 & GIRK2 channels have not been detected in the normal cell lines tested, although the mRNA for GIRK4 channels was found in SAE normal cells. Our data indicates more differential expression of GIRK1 and GIRK2 than of GIRK4 in both SCLC and NSCLC cell lines. It is our hypothesis that GIRK4 is less important in lung cancer due to the fact it was expressed in all normal and cancerous cell lines. Further study is needed to determine the importance of GIRK1 and GIRK2 in lung cancer. In addition GIRK3 was found in all cell lines tested, including SAEC and normal hamster PNEC. Since GIRK3 expression was seen in all cells tested, we also feel it is unlikely to be a factor in tumor growth or progression. This hypothesis is supported by data indicating that one of the functions of GIRK3 is to inhibit plasma membrane expression of other GIRK subunits . FurtherSince protein expression would also be necessary for functional GIRK channels, we determined GIRK1 protein expression in cell lines that expressed GIRK1 mRNA. Expression of GIRK1 protein was seen in the three SCLC cell lines that express GIRK1 mRNA. This is the first report of GIRK1 protein expression in SCLC cell lines. We also determined GIRK1 protein expression in NSCLC cell lines. None of the NSCLC cell lines that expressed GIRK1 showed protein expression for GIRK1. We feel that this indicates that GIRK1 expression may be a larger factor in SCLC than in NSCLC. Further study is needed to elucidate these differences.2 adrenergic antagonist ICI daily for seven days. In contrast to this data, exposure of the breast cancer cell line MDA-MB453 for six days to the β-adrenergic antagonist propranolol (1 μM) increased the GIRK1 mRNA levels [1 antagonist, atenolol, for the same time period had no effect, indicating that the β2-adrenergic receptor is more important for changes in GIRK mRNA. GIRK currents have been shown to be increased in cells stimulated with the β-adrenergic agonist Iso in rat atrial myocytes transfected with β1 or β2 receptors [In the present study, we demonstrated a small decrease in GIRK1 mRNA expression (1.5×) in the H69 SCLC cell line after treatment for one week with the βA levels . Treatmeeceptors . Heteroleceptors . The difThe tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for β-adrenergic receptors, stimulated DNA synthesis in two breast cancer cell lines, an effect inhibited by ICI . In the Further studies are needed with RNA interference (RNAi) or small interfering RNA (siRNA) and additional β-adrenergic agonists and antagonists to further determine the effects of GIRK1 and GIRK2 on β-adrenergic signaling in human lung cancer, especially studies on differences in GIRK protein expression. However, it appears that β-adrenergic signaling and GIRK channels are important in both SCLC and breast cancer. These findings may be important in devising therapies based on β-adrenergic signaling and GIRK channel expression in lung cancers that express GIRK channels or β-adrenergic receptors.We feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and β-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results.The author(s) declare that they have no competing interests.HP carried out the majority of experiments, designed the study, and helped draft the manuscript. MD carried out the GIRK western blots. MD and MC were involved in proliferation studies. MC was also involved in RT-PCR. HS helped draft the manuscript.The pre-publication history for this paper can be accessed here:


The reduced cellular uptake, and subsequent reduced nuclear availability, of cytotoxic agents is a factor in the resistance of mammalian cells to anti-cancer drugs that act by interaction with DNA. The whole cell uptake, nuclear binding and cytotoxicity of a DNA-specific ligand, Hoechst dye number 33342 (Ho342), has been studied in cytotoxic drug resistant variants of a murine tumour cell line. Cell lines showing various degrees of cross-resistance to adriamycin as a part of the phenotype of classical multi-drug resistance (MDR) demonstrated a reduction in intranuclear Ho342 content, up to a maximum of 35% of the level found in the parent as assessed by flow cytometry, despite similar levels of whole cell uptake determined using radiolabelled ligand. Ability to limit nuclear accessibility of Ho342 correlated closely with cellular resistance to Ho342 and to adriamycin. All drug resistant cell lines showed a significant increase in nuclear accessibility to Ho342 after verapamil treatment, including a methotrexate resistant cell line. The methotrexate resistant variant, not demonstrating MDR, showed reduced nuclear binding of Ho342 but increased cell kill associated with a propensity to develop a population of cells showing extra DNA replication in response to Ho342 exposure. Differences between cell lines in the relationship between Ho342-induced cell cycle perturbation and cell kill supported the conclusion that modulation of several pathways of response to cytotoxic agents had occurred in the development of drug resistance.


The ability of the anti-oestrogens tamoxifen, toremifene and their 4-hydroxy and N-desmethyl metabolites to modify doxorubicin (dox) toxicity to intrinsically resistant and multidrug resistant cell lines was compared, using human breast and lung cancer, and Chinese hamster ovary cell lines. The anti-oestrogens significantly enhanced dox toxicity to multidrug resistant, P-glycoprotein-positive cell lines, but did not affect toxicity to intrinsically resistant, P-glycoprotein-negative cells. Modification was observed at clinically achievable anti-oestrogen concentrations. Toremifene and tamoxifen would therefore appear to be good candidates for in vivo studies as MDR modulating agents in selected patients with P-glycoprotein-positive tumours.


DNA repair processes play an important role in the determination of radiation response in both normal and tumour cells. We have investigated one aspect of DNA repair in a number of human cell lines of varying radiosensitivity using the adenovirus 5 host cell reactivation assay (HCR). In this technique, gamma-irradiated virions are used to infect cells and the ability of the cellular repair systems to process this damage is assayed by a convenient immunoperoxidase method recognising viral structural antigen expression on the cell membrane 48 h after infection. Reduced HCR was exhibited by radioresistant HeLa cells and by a radiosensitive neuroblastoma cell line, HX142. In contrast, an ataxia telangiectasia cell line, AT5 BIVA, did not show reduced HCR. On the basis of these results we can make no general conclusions about the relevance of HCR to cellular radiosensitivity. We have extended these studies to determine whether our cell lines exhibited enhanced viral reactivation (ER) following a small priming dose of gamma-radiation given to the cells before viral infection. No evidence for this phenomenon was found either in normal or tumour cell lines.


Two human colon cancer xenografts (EC and AC) were established in tissue culture only after long-term passage in nude mice. Earlier attempts to establish cell lines were unsuccessful. The epithelioid cells retain their tumourigenicity after in vitro growth, giving rise to tumours with a take rate of 60-80%. After reimplantation, the xenografts retain a similar morphology to that of the original human tumours. Both cell lines show human karyology. Comparative mapping of Concanavalin-A acceptor glycoproteins provides a fingerprint characteristic of each cell line. These glycoprotein patterns are similar to those shown by HT-29, an established colon cancer cell line.


The sensitivity of cultured and mouse-passaged cloned lines of chemically-induced murine fibrosarcomas to killing by NK and NC cells, and to cell-mediated immunity, has been studied in in vitro assays, using target cells labelled with 51Cr or 125IUDR. None of the lines tested proved sensitive to NK cells. Three cultured lines were, at most, only slightly sensitive to NC cells; a fourth cultured line was moderately sensitive and became less so, but not completely insensitive, after passage in susceptible hosts. The primary object of these experiments was to test the hypothesis that cultured cell lines which ordinarily fail to grow in normal mice are able to grow after being passaged in a susceptible immunodeficient host because, during this passage, they become resistant to NK or NC cells. This has been shown to occur with one clone, but will not serve as a general explanation because, with other clones, both cultured and mouse-passaged lines were NC-insensitive. The cell-mediated immunity assays confirm our previous conclusion that cultured and mouse-passaged lines of the same clone differ little, if it all, in immunogenicity.


The interaction of membrane-active amphiphiles with a series of MDR Chinese hamster ovary (CHO) cell lines was investigated. Cross-resistance to cationic amphiphiles was observed, which was effectively sensitised by verapamil. MDR cells showed collateral sensitivity to polyoxyethylene amphiphiles (Triton X-100/Nonidet P-40), which reached a maximum at 9-10 ethylene oxide units. Resistant lines were also highly collaterally sensitive (17-fold) to dibutylphthalate. mdrl transfectants showed cross-resistance to cationic amphiphiles, but no collateral sensitivity to nonionic species. Triton X-100/Nonidet P-40 inhibited 3H-azidopine photoaffinity labelling at low concentrations, perhaps reflecting a specific interaction with P-glycoprotein. Further investigation of the molecular basis of collateral sensitivity revealed that association of 3H-Triton X-100 with MDR cells reached steady state levels rapidly, and occurred by a non-mediated mechanism. The equilibrium level of X-100 uptake was inversely related to drug resistance. Collateral sensitivity is thus not a result of decreased Triton X-100 association with the cell. The fluorescent probe merocyanine 540 was used to examine the MDR plasma membrane microenvironment for physicochemical changes. Increasing levels of drug resistance correlated with a progressive shift in the mean cell fluorescence to lower levels, which suggests that the packing density in the outer leaflet of MDR cells is increased relative to that of the drug-sensitive parent.


Aromatase is a key enzyme in the conversion of androstenedione and testosterone to oestrone and oestradiol. Intratumoral aromatase activity is expressed by around 70% of breast carcinomas, but it is not clear what effect this has on the tumour phenotype. To address this question we expressed human aromatase in hormone-dependent MCF-7 breast cancer cells. Clone Arom. 1 expressed aromatase at 1,000 times the endogenous level in wild-type (WT) cells. Clone Arom. 2 incorporated the expression construct but did not express aromatase at levels above WT. There was no morphological difference between the two clones and WT, all three cell lines expressed oestrogen receptor at equivalent levels, and all manifested a mitogenic response to oestradiol. In steroid-depleted medium Arom. 1 cells showed significant growth enhancement over WT and Arom. 2, and this growth advantage was increased by exogenous androstenedione or testosterone. Both the enzyme activity and androgen-stimulated growth of Arom. 1 cells were completely reversible by aromatase inhibitor CGS 16949A. The Arom. 1 cell line may contribute to the development of an in vivo model of intratumoral aromatase, to study the biological significance of this phenomenon.


A novel arotinoid with a morpholine structure in the polar end group Ro 40-8757 (4-[2-[p-[(E)-2propenyl]phenoxy]ethyl]-morpholine) was tested for its anti-proliferative activity against nine human cancer cell lines in vitro. The lines included two estrogen receptor positive breast cancer lines (MCF-7 and ZR-75-1), two estrogen receptor negative breast cancer lines (MDA-MB-231 and BT-20), one cervix carcinoma line (KB-3-1), two lung adenocarcinoma lines (A549 and HLC-1), one large cell lung cancer line (LXFL 529) and two colorectal lines (CXF 243 and CXF 280). Proliferation of all the lines, except the two lung adenocarcinoma lines, was inhibited by lower concentrations of Ro 40-8757 than those of all-trans retinoic acid (RA) or 13-cis RA giving the same level of inhibition. The degree of inhibition of RO 40-8757 was concentration and time dependent. The arotinoid was not cytotoxic and morphological signs by differentiation were not evident in cultures treated with Ro 40-8757 for up to 2 weeks. Because this compound is active on cells such as KB-3-1 that are not inhibited by all-trans RA and because it does not bind to nuclear retinoic acid receptors, it may represent a novel class of anti-proliferative agents.