CD40L was found to reduce doxorubicin-induced apoptosis in non Hodgkin's lymphoma cell lines through caspase-3 dependent mechanism. Whether this represents a general mechanism for other tumor types is unknown.The resistance induced by CD40L against apoptosis induced by a panel of cytotoxic chemotherapeutic drugs in non Hodgkin's lymphoma and breast carcinoma cell lines was investigated.Doxorubicin, cisplatyl, etoposide, vinblastin and paclitaxel increased apoptosis in a dose-dependent manner in breast carcinoma as well as in non Hodgkin's lymphoma cell lines. Co-culture with irradiated L cells expressing CD40L significantly reduced the percentage of apoptotic cells in breast carcinoma and non Hodgkin's lymphoma cell lines treated with these drugs. In breast carcinoma cell lines, these 5 drugs induced an inconsistent increase of caspase-3/7 activity, while in non Hodgkin's lymphoma cell lines all 5 drugs increased caspase-3/7 activity up to 28-fold above baseline. Co-culture with CD40L L cells reduced (-39% to -89%) the activation of caspase-3/7 induced by these agents in all 5 non Hodgkin's lymphoma cell lines, but in none of the 2 breast carcinoma cell lines. Co culture with CD40L L cells also blocked the apoptosis induced by exogenous ceramides in breast carcinoma and non Hodgkin's lymphoma cell lines through a caspase-3-like, 8-like and 9-like dependent pathways.These results indicate that CD40L expressed on adjacent non tumoral cells induces multidrug resistance to cytotoxic agents and ceramides in both breast carcinoma and non Hodgkin's lymphoma cell lines, albeit through a caspase independent and dependent pathway respectively. Primary or secondary chemoresistance to cytotoxic chemotherapy is a frequent phenomenon in patients with solid or hematological malignant tumors and is a major cause of death of these patients. The most common cytotoxic agents used for the treatment of advanced cancers act by inducing the apoptosis of tumor cells through activation of the caspase cascade -4, althoThe acquisition by tumor cells of resistance to the apoptosis induced by cytotoxic drugs involves various biological mechanisms shared by tumors of different histological types -11,3. ThAmong these, several observations indicate that integrins may protect tumor cells against the cytotoxic effects of anticancer agents after interaction with glycoproteins expressed in the extra cellular matrix or at the surface of adjacent non tumoral cells ,14. In aCD40 is expressed not only on cells of the hematopoietic system but also on melanoma and on epithelial cells, in particular breast, lung, or ovarian carcinoma cell lines -37. As fIn the present report, we investigated the capacity of CD40L to modulate apoptosis induced by a variety of cytotoxic agents with different modes of action in breast and NHL cell lines. The results indicate that CD40L induces a multidrug resistance to apoptosis in both breast cancer cell lines and NHL cell lines, through caspase independent and dependent pathways.5 to 106 cells/ml in RPMI 1640  for NHL cell lines and carcinoma, containing 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamin . Breast carcinoma cell lines  were grown at 105–-2 × 105 cells/ml in DMEM . The MCF-7 cell line did not express detectable levels of CD40 and was therefore used as negative control for CD40/CD40L effect. CDw32/FcγRII and CD40 ligand (CD40L) transfected Ltk (-) cell lines (CDw32 L cells and CD40L L cells) were kindly provided by Schering-Plough . The former being the negative control to the latter, FcγRII had no counterpart on NHL and BCC cell lines. 95% of the transfected L cells expressed CD40L as previously reported TdR  for 18 hours. [3H]TdR incorporation was measured by tritium detector using standard liquid scintillation counting techniques on a β counter . Of note, after irradiation, CD40L L cells did not interfere with the proliferation assay with no significant [3H]TdR incorporation.Thymidine incorporation in the different cell lines was tested in the presence of lower, cytostatic, concentrations of DOX (0.5 μg/mL), ETO (5 μg/ml), CDDP (5 μg/ml), VIN (30 nM), TAX (30 nM), C2 and C6 ceramides (30–50 μM). NHL or BCC cell lines  and then resuspended in the lysis buffer  at 4°C for 30 min and finally centrifuged at 4°C for 15 min at 13 000 g. Caspase-3/7 activity was measured using caspase-3/7 cellular activity assay kit plus . OD's measurements were performed at 0, 30, 60, 90, 120, 150, 180 min at 405 nm. Caspase-3/7 activity was calculated with the following formula:Drugs were used at the same concentrations than in apoptosis experiments. After 72 h of culture, 10pmol/min = Slope (OD/min) × conversion factor (μM/OD) × assay volume (μl) where conversion factor is: 50 μM/Average A405 nm .Total protein extraction was obtained by resuspended treated cells (in the same conditions of apoptosis induction), in lysis buffer CSH  and protease inhibitors cocktail from SIGMA  .Protein extracts (30–50 μg) were heated 5 min at 100°C in loading buffer . Denatured samples were run on a 8% acrylamide gel. After transfer onto PVDF membranes , the membranes were blocked overnight with blocking buffer: I-Block 0.2% , PBS 1× and Tween-20 0.1%. Then, the membranes were incubated with the first antibody anti-Poly (ADP-Ribose)-Polymerase  at 1/1000, washed and incubated with alkaline phosphatase-conjugated secondary antibody. The reaction was revealed with chemoluminescence detection method with CSPD substrate .Statistical analyses were performed using paired Student t test.CD40 was found detectable in 2 of the 3 breast carcinoma cell lines (BCC), in all 6 renal cell carcinoma lines (RCC), in the HTB81 prostatic carcinoma and the HCT116 colon carcinoma cell lines as well as in 5 of 5 NHL cell lines tested (not shown). We investigated whether the apoptosis induced by a panel of cytotoxic agents with various modes of action (doxorubicin (Dox), paclitaxel (TAX), vinblastin (VIN), etoposide (ETO), cisplatin (CDDP) was affected by co-culture with CD40L expressing L cells (CD40L L cells). All 5 drugs tested induced the apoptosis of the 2 breast carcinoma cell lines  induced a modest increase 2 to 4-fold) of caspase-3/7 activity in the 2 breast carcinoma cell lines, while these 5 drugs (except CDDP in 4 NHL) almost consistently increased (up to 28-fold) caspase-3/7 activity in the 5 NHL cell lines tested -Polymerase) (113 kD) into 89 kD and 24 kD fragments. In all 5 NHL and 2 BCC cell lines, drugs induced the appearance or an increase of the 89 kD fragment Figure , althougIn order to determine whether caspase-3, and upstream caspases 8 and 9 played a role in drug-induced apoptosis, caspase-3, 8 and 9-like activities were blocked in drug-treated breast carcinoma and NHL cell lines using cell permeable tetrapeptide inhibitors. None of these 3 inhibitors blocked apoptosis induced by DOX in the T47D breast carcinoma cell lines Figure , nor in CD40L is a member of the TNF family of ligand which is normally expressed by T lymphocytes and interacts with CD40 expressed on B cell and antigen presenting cells . CD40L eIn NHL cell lines, CD40 ligand expressed on adjacent non tumoral cells was found capable 1) to inhibit the apoptosis induced by five different commonly used cytotoxic agents , 2) to inhibit the activation of caspase-3/7 induced by DOX, ETO, VIN and TAX, 3) to inhibit drugs  induced PARP cleavage by apoptosis protease like YAMA/CPP32/Apopain/Caspase-3 and 4) to partially reverse the antiproliferative effect of the five cytotoxic agents in the 5 NHL cell lines tested. The inhibition of caspase-3-like and 8-like activities, but not caspase-9-like activity, by permeable tetrapeptide inhibitors also blocked the apoptosis induced by doxorubicin in NHL cell lines, suggesting that the downregulation of caspase-3-like is an essential molecular mechanism of the protective effect of CD40L.However, the modulation of caspase-3 is not the sole mechanism of the protective effect of CD40L in NHL cell lines. Raji was found to be resistant to reversion of apoptosis induction by TAX and ETO despite of modulation of caspase-3-like activation and PARP cleavage. In addition, resistance by CD40L to CDDP induced apoptosis was not associated with caspase-3/7 and PARP cleavage modulation in 4 of the 5 NHL cell lines tested. Apoptosis resistance mechanisms acting downstream of caspase-3 activation have been described in particular in the Raji cell line ,56 as weIn contrast, in CD40 expressing breast carcinoma cell lines, the protective effect of CD40L was found to be caspase-independent. Co-culture with CD40L L cells was found capable to protect breast  carcinoma cell lines against DOX induced apoptosis. Exposure of the 2 breast carcinoma cell lines tested here to doxorubicin, CDDP, Paclitaxel, Vinblastin or etoposide did not or only weakly increased caspase-3/7 activity while caspase-3/7 activity increased up to 28-fold in NHL cell lines tested after exposure to the same drugs. Finally, CD40L did not inhibit caspase-3/7 activation and PARP cleavage induced by any of the 5 cytotoxic agents in the 2 breast carcinoma cell lines tested. Actually, exposure to CD40L L cell was found to increase caspase-3/7 activity in drug-treated breast cancer cells, in marked contrast with what was observed for NHL cell lines. Consistent with these observations, cell permeable tetrapeptide inhibitors of caspase-3, 8 and 9-like activities failed to inhibit the apoptosis of breast carcinoma cell lines, in contrast to what is observed in NHL cell lines. These results indicate 1) that caspase-3 activation is not the single pathway required for the induction of apoptosis of breast cancer cell lines by the anti-cancer drugs tested considering the PARP cleavage but the weak caspase-3/7 activity modulation by drugs and 2) that the anti-apoptotic effect of CD40L on these cell lines do not involve a modulation of caspase-3/7 activity as it was shown by the absence of inhibition of caspase-3 activity and PARP cleavage.Different mechanisms for CD40L protection against drug-induced apoptosis and drug anti proliferative effect could be suggested. Some studies indicated that the antiapoptotic function of CD40 is mediated by up-regulated expression of bcl-xL gene, an antiapoptotic member of the bcl-2 family of proteins and that up-regulation of Bcl-xL could be a key event in CD40-mediated survival in both normal tonsillar B cells and the immature B-cell lymphoma WEHI-231 cells -60. MoreTaken together, these results indicate that the protective effect of CD40L against cytotoxic agents involves caspase-dependent and independent pathways in NHL, and caspase independent pathways in breast carcinoma cells lines. Of note, similar observations were obtained in the colon carcinoma HCT116 cell line treated with doxorubicin, with no significant modulation of caspase-3/7 activity nor reversal of the cytostatic effect of doxorubicin (not shown). These results strongly suggest that the anti-apoptotic effect of CD40L involves different molecular mechanisms in the lymphoma and carcinoma cell lines tested in this study.It has been reported that CD40L induces direct cytostatic effects on breast carcinoma cell lines ,39. The Ceramides have been reported to act as second messengers for the apoptosis induced by DNA damaging agents in some tumor cell lines -52. A raCD40L therefore may block the apoptosis of breast carcinoma cell lines independently of the modulation of caspase activities in these different models. Further studies on bcl-2 family protein like Bclxl/Bax, but also cell cycle, p53 and TRAF-2 would provide more information on mechanism of CD40L induced drug resistance.This study show that CD40L expressed on adjacent non tumoral cells induce a multidrug resistance to apoptosis in breast carcinoma and NHL cell lines, through both caspase dependent and independent pathways in NHL cell lines, and through a caspase independent pathway in breast carcinoma cell lines.NHL = non Hodgkin's lymphoma; BCC = breast carcinoma cell lines; DOX = doxorubicin; ETO = etoposide; CDDP = Cisplatin; VIN = vinblastin; TAX = paclitaxel; C2 = D-erythro-sphingosine, N-acetyl-C2 ceramide; C6 = D-erythro-sphingosine, N-hexanoyl-C6 ceramide; C- = C2-dihydroceramide N-acetyldihydrosphingosine; PARP = Poly (ADP-Ribose)-Polymerase.The author(s) declare that they have no competing interests.NVR carried on quantification of apoptosis by flow cytometry, proliferation assays, caspase-3/7 activity determination. LA helped for Western blotting. JYB participated in the design and coordination of the study and helped to draft manuscript. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


The cellular action of 1,25(OH)2D3 is mediated by binding to an intracellular protein, the vitamin D receptor (VDR). We have evaluated the activity of 1,25(OH)2D3 and its non-calcaemogenic analogue MC903 in the SU-DHL4 and SU-DUL5 B cell lines which carry the 14;18 translocation characteristic of follicular NHL, and also the expression of the VDR in a range of B cell NHLs. Both agents induced differentiation and had an antiproliferative effect on the SU-DHL4 and SU-DUL5 cell lines. However this occurred at a relatively high concentration (10(-7) M) which exceeds the physiological concentration of 1,25(OH)2D3 by approximately 10(3)-10(4)-fold. Expression of the VDR was low in each cell line and in the low grade lymphoma tumour samples. To account for the observed clinical response to 1 alpha OHD3  in follicular NHL a network is suggested whereby 1,25(OH)2D3 modulates the activity of CD4+T cells which have previously been shown to promote follicle centre cell proliferation. Vitamin D3 analogues may enable serum levels to be achieved which produce a direct action on follicular lymphoma cells without disturbing calcium metabolism.


Small cell bronchial carcinoma (SCC) xenografts with differing sensitivity to cyclophosphamide (CY) were investigated using a variety of techniques. Two xenografts (HX78 and HX88) were relatively sensitive to CY, one xenograft (HX72) was inherently resistant to CY and a fourth xenograft (HX78Cy) was a CY induced resistant subline of HX78 and was unstable when maintained without CY exposure. Conventional light microscopy, cytology and electron microscopy examination of the xenografts revealed appearances consistent with SCC. An antikeratin antibody demonstrated the presence of keratin in the inherent and the induced resistant xenografts (and in the unstable revertant) but not in the two sensitive xenografts; the presence of keratin suggested squamous differentiation. Monolayer culture morphology of the sensitive HX78 and the unstable revertant was anchorage independent with the cells forming floating aggregates; the induced resistant subline of this xenograft (HX78Cy) showed, by contrast, flattened, angular adherent cells. Beta HCG production was detected in the monolayer culture supernatant of sensitive HX78 cells; the level of production of beta HCG was increased in the induced resistant HX78Cy cells. Karyotype and flow cytometry studies were also performed. The morphological responses of small cell lung cancer to treatment are discussed.


Paracrine influences from fibroblasts derived from different sources of breast tissue on epithelial breast cancer cell growth in vitro were investigated. Medium conditioned (CM) by fibroblasts derived from tumours, adjacent normal breast tissue, and normal breast tissue obtained from reduction mammoplasty or from skin tissue significantly stimulated the growth of the steroid-receptor positive cell lines MCF-7 and ZR 75.1. The proliferation index (PI) on MCF-7 cells with CM from fibroblasts derived from breast tumour tissue was significantly higher than that obtained with fibroblasts derived from adjacent normal breast tissue . The PI obtained with CM from normal fibroblast cultures from reduction mammoplasty tissue, like normal tissue adjacent to the tumour, fell in the lower range of values. Skin fibroblast, like tumour tissue derived fibroblast, CM caused a high range PI. MDA-MB-231 and Evsa-T, two steroid-receptor negative cell lines, showed only a minor growth stimulatory responses with some of the fibroblast CM's. Evsa-T was occasionally inhibited by CM's. In conclusion, stromal factors play a role in the growth regulation of human breast cancer cells. The effects on cancer cell growth are, however, varying depending on the source of the stroma and the characteristics of the epithelial tumour cells.


Apigenin, a naturally occurring flavonoid, has been shown to inhibit growth in some cancer cell lines but has not been studied in pancreatic cancer. We hypothesized that apigenin would inhibit pancreatic cancer cell growth Apigenin caused both time- and concentration-dependent inhibition of DNA synthesis and cell proliferation in four pancreatic cancer cell lines. Apigenin induced G2/M phase cell cycle arrest. Apigenin reduced levels of cyclin A, cyclin B, phosphorylated forms of cdc2 and cdc25, which are all proteins required for G2/M transition.Apigenin inhibits growth of pancreatic cancer cells through suppression of cyclin B-associated cdc2 activity and G2/M arrest, and may be a valuable drug for the treatment or prevention of pancreatic cancer. Despite attempts of resection and adjuvant therapy, patients diagnosed with pancreatic cancer continue to have a poor prognosis. Many chemotherapeutic agents have been used to treat pancreatic cancer without particular success. Novel treatments to inhibit cancer cell proliferation with less toxicity are needed. Flavonoids are a class of polyphenolic compounds which display a variety of biologic activities. There has been recent interest in using flavonoid derivatives therapeutically as anticancer drugs. At pharmacological levels, various naturally occurring flavonoids have been shown to be cancer-protective in a variety of animal models. Flavonoid derivatives, such as flavopiridol and genistein, have been shown to be safe in human trials,2 and arin vitro, and further aimed to delineate the mechanism involved.Flavonoids have shown antitumor activity for a number of cancer cell types ,6. This Apigenin caused a concentration-dependent 6.25–100 μM) inhibition of thymidine incorporation in all four cell lines studied . Figure All four pancreatic cancer cell lines were treated with 100 μM apigenin for 24 hours and expression and phosphorylation of proteins involved in the G2/M transition were observed through western blotting Figure . ExpressSeveral mechanisms have been proposed for apigenin's effect on cell growth including modulation of the MAP kinase pathway ,21, inhiIn the current study, treatment of human pancreatic cancer cells with apigenin resulted in inhibition of DNA synthesis and cell proliferation through G2/M cell cycle arrest. A decrease in levels of cyclin A, cyclin B, cdc25 A and cdc25C all appear to be involved. It is not clear why levels of phosphorylated cdc2 were decreased given that reduced levels of cdc25c should have the opposite effect, however, the reduced levels of cyclin B appear to be the main mechanism involved in the arrest of cells at G2/M.Our results are congruent with previous studies in which cell cycle arrest has been shown to result from apigenin treatment in other cell lines. G2/M arrest through inhibition of cyclin B associated cdc2 has been shown to occur in epidermal and fibroblast cells ,30, breaA considerable amount of attention has been given to the role of p53 in cell cycle checkpoint control. P53 blocks cells at G2/M through direct inhibition of cdc2 kinase . All fouin vitro through G2/M phase cell cycle arrest associated with decreased cyclin B-cdc2 activity. Taken together with previously published studies, apigenin appears to exert its inhibitory effects on cell cycle progression in other cell lines as well. Cancer cells with abnormal cell-cycle machinery may benefit from extrinsic cell-cycle regulators such as apigenin.To our knowledge there is no published data concerning apigenin in pancreatic cancer. Our results demonstrate that apigenin inhibits human pancreatic cancer cell growth 3H-Thymidine was purchased from Amersham . The monoclonal mouse cyclin A, B, cdc25A, and GAPDH antibodies, and the polyclonal rabbit cdc25C antibody were purchased from Santa Cruz Biotechnology . The monoclonal rabbit cdc2 and phospho-cdc2 (Tyr15) antibodies were purchased from Cell Signaling Technology, Inc. .DMEM, trypsin-EDTA solution, and propidium iodide were purchased from Sigma . Fetal bovine serum was purchased from Atlanta Biologicals . Apigenin was purchased from Calbiochem  and dissolved in DMSO purchased from Sigma Chemicals . Methyl 2 and 5% CO2 at 37°C. The cells were regularly seeded into 75-cm2 flasks with media changes every second or third day. For experiments, cells were grown to 70% confluence, digested with trypsin-EDTA, and plated in 6-, 24-, or 48-well plates.We selected four human pancreatic cancer cell lines: AsPC-1, CD18, MIA PaCa2, and S2-013. These cell lines were purchased from American Type Culture Collection  with the exception of the S2-013 cell line, which was a generous gift from Dr. Takeshi Iwamura (Japan). Each cell line was grown in DMEM and plated as monolayers in medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 95% O3H-thymidine/well and incubating cells for another 6 hours. The cells were then washed twice with PBS, fixed with 10% trichloroacetic acid, and solubilized by adding 250 μl of 0.4 M NaOH to each well. Radioactivity, indicating incorporation of methyl 3H-thymidine into DNA, was measured by adding scintillation cocktail and counting on a scintillation counter .Cells were plated in 24-well plates at a concentration of 50,000 cells/well. After reaching 50% confluence, they were incubated in serum-free medium for 24 hours, which was then replaced with fresh serum-free medium with or without the appropriate treatments. After the required period of culture, cellular DNA synthesis was assayed by adding 0.5μCi methyl Cells were regularly seeded into three 6-well plates and incubated at 37°C for 24 hours. Cells were then cultured in serum-free medium for another 24 hours and treated in fresh serum-free medium with or without apigenin for 24, 48, and 72 hours. At the end of each time period, the cells were trypsinized to produce a single cell suspension, and the viable cell number in each well was counted using Guava Technologies' ViaCount Assay .2 flasks, serum starved for 24 hours, and then treated with apigenin. At the end of the treatment, the cells were harvested with trypsin-EDTA solution to produce a single-cell suspension. Cells were then pelleted by centrifugation and washed twice with PBS. The cell pellets were then suspended in 0.5 mL PBS and fixed in ice-cold 70% ethanol at 4°C. The fixed cells were then centrifuged at ×300 g for 10 minutes and pellets washed with PBS. After resuspension in 1 ml PBS, the cells were incubated with 10 μL Rnase I (10 mg/ml) and 100 μL propidium iodide (400 g/mL) and shaken for 1 hour at 37°C in the dark. The samples were then analyzed by flow cytometry.Cells were grown to 50% confluence in T75-cm2 flasks and grown to 60% confluence for 24 hours. The cells were serum starved for 24 hours. The cells were then placed in serum-free medium with or without 100 μM apigenin for 24 hours. The cells were lysed and protein concentrations determined with BSA as standard. Equal amounts of cell protein lysate were separated on 15% SDS-polyacrylamide gel electrophoresis (PAGE) and the protein transferred onto nitrocellulose membranes. After blocking with dried milk, membranes were incubated with the appropriate dilution of primary antibody. Membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody. Proteins were detected using the enhanced chemiluminescence detection system.Cells were seeded in T25-cmData were analyzed by ANOVA with Dunnett's or Bonferoni's corrections for multiple comparisons, as appropriate. This analysis was performed with the Prism software package . Data were expressed as mean ± SEM.MU carried out the cell culture work, thymidine incorporation, cell proliferation assays, flow cytometry, statistical analyses, and wrote the manuscript. XD participated in the conception of the study, its design and coordination, and helped draft the manuscript. MS carried out the western blotting. LG assisted with cell proliferation assays and western blotting. BM assisted with cell proliferation assays and western blotting. DB helped draft and prepare the manuscript. MT and RB participated in the design and coordination of the study. TA participated in the conception and design of the study, participated in the statistical analysis, participated in the coordination of the study and edited the manuscript.


We have established a subline (EMT6/VRP) of the mouse tumour cell line EMT6/P with acquired resistance to the calcium transport blocker verapamil (VRP). The subline was 4-fold resistant to the cytoxicity of VRP alone compared with the parent line but of similar sensitivity to adriamycin, vincristine or colchicine. EMT6/VRP cells growing in 75 micrograms ml-1 VRP were morphologically different from and larger in diameter than EMT6/P cells, but these two parameters reverted almost to normal within 3 days of VRP removal, although resistance was retained. Expression of an mRNA coding for P-glycoprotein was similar in EMT6/VRP and the parent cell line, although considerable hyperexpression was seen in a multidrug resistant subline, EMT6/AR1.0. Cellular accumulation of both 3H-daunorubicin and 3H-VRP were greater in EMT6/VRP than in the parent line. Sensitisation to adriamycin by 3.3 micrograms ml-1 VRP was, however, somewhat reduced in EMT6/VRP (i.e. to 6.1-fold) compared with the 11-fold sensitisation seen in the parent line. It is clear that resistance to VRP seen in this cell line occurs via a different mechanism from the resistance to drugs such as adriamycin, vincristine and colchicine seen in multidrug resistant cell lines.


Acquired resistance to cisplatin (cis-diamminedichloroplatinum (II)) has been generated in vitro in the 41M human ovarian carcinoma cell line, established from a previously untreated patient. Three cisplatin-resistant variants were selected at approximately 2, 4 and 6-fold resistance (in terms of 50% inhibitory concentrations), in order to study the underlying mechanisms of acquired cisplatin resistance. Compared to the parent line, platinum accumulation following exposure to equimolar concentrations of cisplatin was on average (across the entire concentration range) 2.9, 3.6 and 4.8-fold lower in the 41McisR2, 41McisR4 and 41McisR6 cell lines, respectively. Thus the difference in uptake corresponded closely with their resistance factor in the three resistant variants. Moreover, a significant reduction in platinum accumulation was observed as early as 5 min after exposure to cisplatin in the 41M vs 41McisR6 cell lines. Platinum accumulation was similar in all cell lines following exposure to equitoxic concentrations (2 h IC50) of cisplatin. Enhanced efflux of drug was not observed between the 41M and 41McisR6 cells. In addition, there was no difference in intracellular glutathione (GSH) levels. Our previous studies have shown no indication of metallothionein involvement and the decrease in cisplatin uptake in the 41McisR6 cells was reflected by a similar reduction in DNA interstrand cross-links (ISC) formation. These results suggest that the mechanism of acquired resistance to cisplatin in the 41McisR6 cell line may be predominantly due to reduced drug uptake. The 41McisR6 cells were not found to be cross-resistant to ouabain, a postulated specific inhibitor of sodium-potassium adenosine triphosphatase -ATPase), suggesting that decreased cisplatin accumulation in these cells is probably not regulated by alterations in their Na+, K(+)-ATPase levels, and Na+ potential across the plasma membrane. Cellular accumulation of a novel class of platinum (IV) ammine/cyclohexylamine dicarboxylates, which exhibit enhanced cytotoxicity over cisplatin and completely circumvent resistance to cisplatin in the 41McisR line, was also examined. The data suggests that increased accumulation of these compounds, as a result of their enhanced lipophilicity, could account for the dramatic increase in their potency over cisplatin.


The tumour cell lines U937A and L929 form large, loosely packed colonies in vitro and can be killed by the cytokine tumour necrosis factor (TNF). In contrast, their TNF-resistant mutants U937A/R and L929/R form tightly packed colonies. Since cells which form loose colonies have increased metastatic potential it is important to understand the factors governing colonial morphology. To this end, we have compared the extracellular matrices (ECMs) of the 'loose' lines, U937A and L929 with their 'tight' mutants. By immunofluorescence, a polyvalent anti-U937A serum revealed a fibrillar network in the ECMs of the 'loose' lines which was absent in the 'tight'. On Western blotting of ECMs the antiserum detected an additional 300 kDa protein in the 'loose' lines which was subsequently shown to be cellular fibronectin. The four lines secreted comparable amounts of fibronectin and this was qualitatively indistinguishable between 'loose' and 'tight' cells by peptide mapping or lectin binding. It is concluded that the differences in colonial morphology are due to the 'tight' mutants' inability to incorporate fibronectin into the ECM.


We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the α7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the α7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of α7 mRNA and protein expression in the human lung.Experiments were done using reverse transcription polymerase chain reaction (RT-PCR), a nuclease protection assay and western blotting using membrane proteins.We detected mRNA for the neuronal nicotinic acetylcholine receptor α7 receptor in seven small cell lung cancer (SCLC) cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC), one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of α7 in the NCI-H82 SCLC cell line while α7 was not detected in SAEC, suggesting that α7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the α7 nicotinic receptor, protein expression of α7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the α7 receptor. In the non-SCLC cells and normal cells that express the α7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the α7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the α7 receptor was increased at 60 and 150 min.Expression of mRNA for the neuronal nicotinic acetylcholine receptor α7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441) as well as normal lung cells. The α7 nicotinic receptor protein is expressed in fewer cell lines, and the tobacco carcinogen NNK increases α7 nicotinic receptor protein levels. A probe for 28S ribosomal RNA was also transcribed and used as an internal control. These reactions were incubated for 1 hour at 37°C. After this incubation, 1 μl of DNase I was added, and the mixture was further incubated for 15 min. at 37°C. The probes were gel purified.The nuclease protection assay was used to determine differences in expression levels in a representative SCLC cell line (NCI-H82), and in SAE cells. The Lig'nScribe kit  was used to add a T7 RNA polymerase promoter to the PCR fragment amplified by the gene specific α7 primer, and then this T7-α7 fragment was PCR amplified, and the resulting fragment was used directly in a transcription reaction using the MAXIscript in vitro transcription kit (Ambion). This transcription reaction consisted of the PCR fragment, 10X transcription buffer, 10 mM ATP, UTP and GTP, T7 polymerase, [α-The RPA III kit (Ambion) was used. A molar excess of labeled probes (28S and gene specific) were added to 20 μg total RNA or Yeast RNA (Ambion), and the RNA samples and probes were co-precipitated and resuspended in hybridization buffer. After incubation at 95°C for 4 min., and incubation overnight at 42°C, RNase digestion buffer containing RNase A/RNase T1 was added. After digestion for 30 min. at 37°C, RNase inactivation/precipitation solution was added. After precipitation the pellets were resuspended in gel loading buffer, heated at 95°C for 4 min. and loaded onto a 5% polyacrylamide, 8 M urea gel . In addition RNA Century Markers Plus templates (Ambion) were transcribed and used as markers. After electrophoresis, the gel was transferred to blotting paper, dried, and exposed to Kodak XAR film.Cell pellets were collected and membrane protein was isolated with the ReadyPrep protein extraction kit  (Biorad). Protein levels were determined using the RCDC kit (Biorad). Aliquots of 20–30 μg protein were boiled in 3x loading buffer  for 2 minutes, then loaded onto 12% Tris-glycine-polyacrylamide gels , and transferred electrophoretically to nictrocellulose 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 . Cells for some blots were incubated with 100 pM NNK  for various times.The RT-PCR assay demonstrated expression of the α7 nAChR in all seven cultured human SCLC cell lines Figure  and in STo verify the RT-PCR results, RT-PCR products from one SCLC cell line (NCI-H69) and the adenocarcinoma cell line NCI-H322 were sequenced using the forward primer used for RT-PCR amplification of the samples (data not shown). The sequences from the PCR products of both NCI-H69 and NCI-H322 were compared to the sequence of the α7 nicotinic acetylcholine receptor  and were found to be 100% homologous.Although we have shown mRNA expression for the α7 nicotinic acetylcholine receptor, it was necessary to show if expression of the α7 nicotinic acetylcholine receptor protein in seen in these cell lines. Using a specific antibody for the α7 nicotinic acetylcholine receptor (Abcam), membrane protein from the cell lines were assessed by western blotting. Expression of α7 protein was seen in 6 of the 7 SCLC cell lines Figure . ExpressAdditional non-small cell lung cancer cell lines were screened for the presence of α7 nAChR mRNA expression. Expression of α7 nAChR was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines Figure .The non-SCLC cell lines and normal cells were also screened for expression of α7 nicotinic acetylcholine receptor protein. SAEC, the adenocarcinoma cell line A549 and the squamous cell line NCI-H226 express protein for α7 , and human SAE cells from a non-smoker (information provided by Clonetics), a riboprobe was transcribed from the PCR fragment amplified by the gene specific α7 primer used for RT-PCR and was used in a nuclease protection assay. Expression of mRNA for the α7 nicotinic acetylcholine receptor was demonstrated in the SCLC cell line NCI-H82, however no protected band was detected in the normal small airway epithelial cells , the adenocarcinoma cell line A549 and the squamous cell line NCI-H226 express protein for α7, whereas the adenocarcinoma cell line NCI-H322, the carcinoid cell line NCI-H727, the squamous cell lines NCI-H520 and NCI-H2170 did not express protein for α7. However, our data are in contrast to another recent study. Carlisle et al.  found thPrevious research from our laboratories has indicated that NNK binds with high affinity to alpha-bungarotoxin (agonist for α7 and α8 ) sensitiUsing a nuclease protection assay that allows for direct comparisons between samples, we found higher levels of α7 nAChR mRNA receptor in NCI-H82 than in SAEC. The RT-PCR assay showing expression of α7 nAChR receptor in SAE cells is a much more sensitive assay, but does not allow for the comparisons seen in the nuclease protection assay. The increase in α7 nAChR receptor in the SCLC cell line NCI-H82 compared to the normal SAE cells from a non-smoker is consistent with other data from our laboratory and others. In addition, it has been shown that the NCI-H82 cell line synthesized the highest levels of acetylcholine, and addition of nicotinic antagonists slowed growth of the NCI-H82 cells . Acetylcth day of gestation [Treatment of pregnant hamsters with the tobacco carcinogen, NNK, led to an increase in the α7 nAChR in PNEC isolated from fetal hamsters on the 15estation . As indiestation ,10. In aestation ,10. Thesestation ,21-23. Oestation . TogetheExpression of mRNA for the neuronal nicotinic acetylcholine receptor α7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441) as well as normal lung cells. The α7 nicotinic receptor protein is expressed in fewer cell lines, and the tobacco carcinogen NNK increases α7 nicotinic receptor protein levels. The α7 nAChR may play an important role in the development of SCLC and other lung cancers in which smoking is involved.The author(s) declare that they have no competing interests.HP carried out all experiments with the exception of the western blots, participated in the design of the study, and helped draft the manuscript. MD performed the western blot experiments. HS conceived of the study and helped draft the manuscript.


The intracellular pH of a number of multidrug resistant cell lines was compared with that of their parental lines using the fluorescent probe bis-carboxyethylcarboxyfluorescein. In four different cases, cells having 5-fold resistance or more exhibited an intracellular pH which was 0.10-0.17 units higher than that of the parental cell line. A CHO cell line, AB1, and its 180-fold resistant counterpart, CHRC5, were further investigated with regard to the role of Na+/H+ antiport. The Na+/H+ antiport activity was greater at any intracellular pH for the CHRC5 cells than the AB1 cells. To investigate the possible role of higher intracellular pH in multidrug resistance, the effect of several agents which are either known to reverse multidrug resistance or inhibit Na+/H+ antiport activity were examined. Verapamil was found to reverse multidrug resistance but had no effect on intracellular pH while amiloride, which acidifies the cytoxol by blocking Na+/H+ antiport activity, did not cause reversal of drug resistance. In contrast to verapamil, treatment of CHRC5 cells with cyclosporin A had a parallel effect on reversal of their drug resistant phenotype and a lowering of their intracellular pH to that of the sensitive cell level. However, cyclosporin was ineffective in either lowering the intracellular pH or reversing drug resistance in DC3F/ADX cells. Therefore, except for the effect of cyclosporin A on the CHRC5 line, the effects of other agents on reversal of multidrug resistance and intracellular pH did not correlate with each other.


The effects of N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on the growth of P388 and its multidrug-resistant (MDR) variants were examined with the objective of assessing the possible changes in cyclic nucleotide-dependent protein kinases and protein kinase C-mediated pathways associated with MDR. H-8, an inhibitor of cyclic nucleotide-dependent protein kinases, inhibited the growth of the parental P388 murine leukaemic cells, but not that of MDR variants up to 200 microM. However the growth of both drug-sensitive and resistant cell lines were uniformly inhibited by H-7. Both the cytotoxic and cytokinetic results revealed that the growth-inhibition by H-8 of P388 cells is mainly due to a blockade of cell-cycle progression rather than due to a killing of cells. The degree of resistance to H-8 was directly proportional to their extent of resistance to vincristine, adriamycin, and 4'-demethylepipodophyllotoxin-9--beta-D-gluco pyr anoside (VP-16) and to that of the expression of P-glycoprotein. These findings raised the possibility that P-glycoprotein might play a role in the cross-resistance to H-8. To test the hypothesis, we examined the effect of H-8 on the binding of 3H-vincristine to membrane fraction isolated from P388/VCR-600 cells and on the enhancement of cytotoxicity to anticancer drugs in MDR cells. H-8 did not have any influences on these reactions. Thus, the cross-resistance to H-8 may be mediated through a mechanism different from an overexpression of P-glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)


Some malignant and transformed cell lines are unable to proliferate in vitro in a L-methionine-depleted medium supplemented with L-homocysteine. To investigate the utilization of preformed and endogenously synthesized methionine 4 cell lines have been chosen with a range of abilities to proliferate under such nutritional conditions. The order of the ability of these cell lines to proliferate in an L-methionine-depleted medium containing 0.1 mM L-homocysteine parallels the minimal concentration of L-methionine required for optimal growth; L-methionine auxotrophs having a greater minimal requirement. In the presence of 0.1 mM L-homocysteine all of the cell lines synthesize macromolecules from [5-14C]methyltetrahydrofolic acid during a 24 h period, and the cell line with the highest methionine requirement shows the most extensive incorporation of radiolabel into DNA and RNA, both in depleted medium and in medium containing 6.7 microM L-methionine. Double-label experiments using [5-14C]methyltetrahydrofolic acid and L-(methyl-3H) methionine show preferential incorporation of preformed over endogenously synthesized methionine by methionine auxotrophs. There is no alteration in the intracellular level of S-adenosyl-L-homocysteine (SAH) or SAH hydrolase activity in cells incubated for 24 h in methionine-depleted medium supplemented with 0.1 mM L-homocysteine. These results suggest that certain cell lines are unable to effectively use endogenously synthesized methionine.


The summer of 2006 was a heady time for neurologist Douglas Kerr. As Director of the Johns Hopkins Transverse Myelitis Center in Maryland, Kerr studies the mechanisms of neurodegenerative diseases in the hope of developing therapies to treat them. He sees patients with transverse myelitis, amyotrophic lateral sclerosis, and spinal muscular atrophy (SMA), an inherited disorder that in its most severe form renders newborns limp and “floppy,” unable to suck, swallow, or breathe. Kerr's voice tightens as he describes the fate of these babies, many of whom will die before their second birthday. He's convinced that embryonic stem cells will one day help people with progressive, motor-neuron-destroying disorders recover control of their movements, and their lives.For six long years, Kerr's team pursued the elusive elixir that would restore mobility to the paralyzed adult rats he uses to model neurodegeneration in humans. The researchers had cleared two major technical hurdles early on: they managed to derive spinal motor neurons from mouse embryonic stem cells in sufficient numbers to transplant in the rats' spinal cords, and they ensured the transplanted neurons' survival. But they struggled for years to prod the spinal motor neurons to send their axons out of the spinal cord and form functional neuromuscular junctions with the lame muscle.http://www.hopkinsmedicine.org/Press_releases/2006/Mousevideo.html.)Finally, in 2005, they hit the mark. Growth factors injected into the spinal cord induced the transplanted motor neurons to form connections with resident neurons. A second set of growth factors overcame inhibitors in myelin , allowing the motor neurons to send their axons out of the spinal cord toward skeletal muscle. And yet another growth factor injected into the muscle stimulated functional connections between the neurons and muscle. Kerr watched his rats—immobilized with a motor-neuron-destroying virus—move hind limbs that had been paralyzed for nearly four months. , which funded part of the research, called the work a “remarkable advance” demonstrating the power of stem cells to treat neurodegenerative diseases. All those years of frustration had finally paid off. But would the technique work in humans?When Kerr and his colleagues reported their results in the June 2006 online version of To find out, Kerr must use motor neurons derived from human embryonic stem cells (hESCs) and show that they can establish functional connections with skeletal muscle over the longer distances found in a larger animal. (He's settled on pigs.) He must also show that the treatments are safe. If the pig experiments generate the necessary safety and efficacy data, he will submit his results to the US Food and Drug Administration, seeking approval for a clinical trial to use the hESC-derived motor neurons in babies with fatal SMA.Kerr chose babies with SMA for the first clinical trials, he explains, because infants have less myelin to inhibit axon growth, so the chance of re-innervation is greater. Their neurons need travel just a short distance compared to adults, and the developmental cues that guide axon growth toward their appropriate targets are still in place. And because no treatment or cure exists for these babies, an experimental treatment represents their only hope. Kerr had planned to use federally approved hESCs until he found out that the federal lines could not reliably yield motor neurons with anywhere near the efficiency of newer lines generated with private funds. Still, Kerr watched hopefully as a bipartisan bill authorizing expanded federal financing of hESC research passed the US House of Representatives in 2005 and then the Senate last year. In addition to allowing federally funded researchers to derive new hESC lines from embryos awaiting destruction in fertility clinics, the Stem Cell Research Enhancement Act would lift the ban on lines derived after August 2001. But President George W. Bush vetoed the bill in July 2006, and “put a real chill on things,” Kerr says. Now Kerr is worried that if he acquires the pigs and prepares them for the stem cell therapy, he'll run into a brick wall when the time comes to get the hESCs he needs for the transplantation experiments.Even though Maryland passed a measure in 2006 to spend $15 million on hESC research, Kerr says that's just a one-time appropriation. Working with live animals costs several million dollars. “What am I going to do next year when I've got all those animals?” he asks.Kerr won't qualify for federal funding if he uses non-approved lines, but he's not willing to risk the potential problems with the federally sanctioned lines. And adult stem cells aren't an option. He tried to generate spinal motor neurons from adult stem cells and cells isolated from umbilical cord blood, but decided that programming a blank slate—hESCs—is far more efficient than deprogramming specialized cells and redirecting them toward a different fate. He put everything on hold, pending the outcome of the November midterm elections.The use of federal funds to create or destroy human embryos for research was outlawed in the US by Congress in 1997. On August 9, 2001, when President Bush announced his decision to allow federal funding to support hESC research only on lines already derived—because “the life and death decision has already been made”—scientists were just learning what the cells needed to thrive and maintain their “stemness,” the ability to self-renew and differentiate into any cell type of the body . Methods used to derive these early hESC lines were technically demanding and labor-intensive, requiring the artful touch of a highly skilled technician to tease apart cells with a glass needle to propagate the cell lines . The lines were also grown on mouse fibroblasts—cells that act as “feeder” layers to maintain the stem cells in an undifferentiated state—increasing the risk that the human cells would absorb mouse molecules and trigger rejection by the immune system if used in clinical trials.Scientists have since figured out how to wean the human cells off the mouse feeder cells, but the process is time-consuming. A team of researchers working in Miodrag Stojkovic's lab at the University of Newcastle, UK, developed a method of deriving hESCs that eliminates the risk of contamination from both mice and human donors. In their “autogenic” feeder system, a parental hESC line gives rise to a subset of differentiated fibroblasts that sustain the parental line. While Stojkovic's lines were grown on medium containing animal products, he says that this approach demonstrates how researchers can generate clinical-grade hESC lines that would meet the Federal Drug Administration's Good Manufacturing Practices safety requirements. Last July, the Singapore biotech company ESI announced that it had derived four safe lines of hESCs for clinical use. And the Hadassah Medical Organization in Israel recently developed animal-free methods for isolating pluripotent stem cells from human embryos (obtained from in vitro fertilization clinics) and deriving new clinical-grade hESC lines. These are just the types of cells Kerr could use for his pig experiments—if he were free to use any lines he wanted.Kerr had planned to use federally approved hESCs until he found out that the federal lines could not reliably yield motor neurons with anywhere near the efficiency of newer lines generated with private funds.Carol Ware, Director of the Human Embryonic Stem Cell Core Laboratory at the University of Washington School of Medicine, has been working to characterize the available NIH-approved hESC lines. So far, Ware and her colleagues have tested 14 of the 22 available lines (a 15th line arrived at the lab contaminated with mycoplasma) for growth efficiency, genomic stability, appropriate gene expression during self-renewal and differentiation, and other NIH criteria.The team found considerable variation among the lines. Some lines had a propensity to develop chromosomal abnormalities over time, and others were hard to grow. “Certain lines are very difficult to thaw,” Ware says, “so you may only get one or two cells.” Not a great return for cell lines that cost between $2,500 and $6,000.Complicating matters further, the cells seem to prefer the culture conditions in which they were derived. All of the lines were originally derived through mechanical passage. And though some were eventually converted to enzymatic passage—a speedy, less onerous technique that has become the standard method for expanding hESC lines—it's not possible to predict which lines can convert to enzymatic passage, further compromising their utility.Mechanically passaging lines is “a real pain in the neck,” says Larry Goldstein, Director of the University of San Diego Stem Cell Program, likening the technique to early versions of a software program that still need debugging. Goldstein's lab has been exploring the properties of some of the federal lines, and still uses one of the approved lines in experiments. “It's been okay in some areas, and a little tougher in others,” he says. “Others use it and are happy with it.”The lines “certainly aren't useless,” he adds, but he's found them clumsy to handle. “For our experiments, we need cell lines that grow well for more than just the most-skilled tissue person. If only one person in the lab is sufficiently skilled to grow them, you're not going to get much done.” Goldstein plans to investigate non-approved lines with private or state funding.Stem Cells. Either way, they concluded, it underscores the need to derive and study additional hESC lines.Ware found that lines also varied in their ability to form different tissues, suggesting that each line may possess unique capabilities. This variable behavior may arise from differences in the way the lines were derived or in the inherent properties of the cells themselves, the team reported online on August 17, 2006, in Harvard researcher Doug Melton came to the same conclusion even before the Bush administration restrictions were put in place, when he began looking for hESCs for his work on type I diabetes in the late 1990s. Disappointed with what he found, he decided to generate his own lines, and in 2004, Melton and his team announced that they had derived 17 new hESC lines, using funding from the Howard Hughes Medical Institute and the Juvenile Diabetes Research Foundation. Melton's team used enzymatic passaging to make the lines more user-friendly, allowing far more labs to handle the cells, provided they find state or private funding. The Harvard lines, as they're known, are also available free of charge.Ware believes the federal lines will become historical lines, as technological advances have already made the older lines seem outdated. “We're understanding culture techniques so much better now, and as you understand more and more what the cells want, you're going to get better lines.”US advocates of stem cell research read the 2006 midterm election results as a sign that embryonic stem cell research has gained widespread bipartisan support. They point to Missouri as the bellwether state. For the past five years, Missouri lawmakers tried to pass a measure to criminalize stem cell research in the state. But last November, voters not only approved a state constitutional amendment protecting stem cell research, but ousted incumbent US Senator Jim Talent, who called stem cell research “morally reprehensible,” in favor of Claire McCaskill, a vocal supporter of hESC research. Stem cell research figured prominently in six US Senate races; in each case, the candidate who supported stem cells won.“The political dynamic in the 110th Congress is going to be a lot different than it was in the 109th,” says Sean Tipton, President of the Coalition for the Advancement of Medical Research, a stem cell advocacy group. Tipton thinks the new Congress will be even more supportive than the 109th, which passed the Stem Cell Research Enhancement Act with strong bipartisan support.Douglas Kerr goes even further. He's confident the bill will pass this year with a veto-proof majority, and that “we'll have federal funding in 2007.”The Chronicle of Higher Education found that if the stem cell act were reintroduced, votes in the House would fall short of a veto-proof majority. And few doubt that Bush would exercise his veto prerogative.But what the next year holds is unclear. Nancy Pelosi, who will preside over the House of Representatives as Speaker when the new Congress convenes in January, has pledged to “promote stem cell research to offer real hope to the millions of American families who suffer from devastating diseases” in Congress's first 100 hours. But an analysis based on the stated stem cell positions of the newly constituted House and Senate by While the federal prospects remain uncertain, states are increasingly filling the void. A 2006 Congressional Research Service report to Congress lists 12 states as actively encouraging or funding stem cell research, with Wisconsin and California leading the way. The California Institute of Regenerative Medicine (CIRM), created to oversee the $3 billion stem cell research program authorized by voters in 2004, awarded $12 million in training grants last April and expects to award over 55 research grants totaling over $100 million in early 2007. Grant allocations were initially stalled by lawsuits filed by pro-life and anti-tax groups, until Governor Arnold Schwarzenegger stepped in with a $150 million state loan, and private donors and foundations pledged $45 million in loans against the bond to get the ball rolling.All this support from states and private donors puts more scientists to work, says Dale Carlson, Chief Communications Officer for CIRM. With the uncertainty at the federal level, he says, it's important that the states and private donors are stepping in, “instead of scientists stepping back and waiting till the policy changes.”While senior scientists acknowledge the difficulty of recruiting the best young minds to a field so mired in controversy, those hot on the trail of potential cures using stem cells are not about to sit idly by while Washington fiddles. Kerr is hoping for the best in 2007, but he and his California collaborator, Hans Keirstead, are pursuing nonfederal funding “while we await changes in D. C.” Both are appealing to private philanthropy groups for bridge funding to make sure their work continues.Ware and her colleagues have tested 14 of the 22 available NIH lines for growth efficiency, genomic stability, appropriate gene expression during self-renewal and differentiation, and other NIH criteria, and found considerable variation among the lines.In November, CIRM received 70 applications for comprehensive research grants totaling $80 million. University of California San Diego's Goldstein was among the applicants, voicing frustration with the limitations on scientific freedom imposed by the federal restrictions. “Bush's policy hasn't spared any frozen embryos as far as I know,” he says. “The biggest destruction of human blastocysts happens in IVF clinics and that hasn't changed.”Meanwhile the research moves ahead without the centralized control and oversight of the federal government. The lack of federal support means that US researchers—who led the way in setting standards for genetic testing and genome sequencing—cannot do the same for embryonic stem cell research. Although CIRM and the National Academy of Sciences are setting research guidelines for state-funded researchers, if US researchers aren't at the forefront of the field, they can't lead by example.But scarce research dollars, some researchers believe, is an even bigger problem. In an era of shrinking NIH budgets and heightened competition for federal grants, restricting grantees to the less tractable NIH-approved lines means the federal government is spending less money on stem cell research, and spending it less efficiently. “As a scientist you want to have the maximum number of tools available because the research problems are hard enough, even when the lines behave well,” Goldstein says. “And these aren't just theoretical problems. We're trying to figure out what's gone wrong with these terrible diseases that afflict large numbers of people.” The federal restrictions, he says, have forced the community to work with one hand tied behind its back.US advocates of stem cell research read the 2006 midterm election results as a sign that embryonic stem cell research has gained widespread bipartisan support.That means that if a US researcher who has access to ten or 15 federally approved hESC lines is working on the same question as a researcher in Singapore, for example, who may have access to 100 lines, the US researcher can't hope to compete. With reports that some lines appear genetically predisposed to behave one way or another, therapeutic applications may require creating stem cell lines that are genetically identical to the patient, to prevent immune rejection. That's a question that researchers like Douglas Kerr can't ask if they're restricted to the limited diversity of ten hard-to-grow stem cell lines.With the right cells in hand, Kerr would first seek proof of principle that his neuroregenerative stem cell therapy can work in pigs, and then move on to see if it can help babies with SMA—a path he's charting in a grant right now. If the results showed that the researchers were on the right track, they would move on to test this type of therapy in patients with the more complex lesions found in transverse myelitis, amyotrophic lateral sclerosis, and traumatic spinal cord injury. Aside from a political sea change that lifts the federal restriction on stem cell research—a shift that may have arrived with the 110th Congress, or may still come up a few votes short—what does Kerr's group need to move forward? “If we got ten good lines that were genetically normal, and had not come into contact with other species, and could become motor neurons,” he says, “we'd be set.”


Carcinoembryonic antigen (CEA) is an oncofetal antigen whose function in the progression of colorectal carcinoma remains unclear although recent studies suggest it participates in homotypic cellular adhesion. We have previously shown that 40 micrograms of CEA injected intravenously into athymic nude mice enhances experimental metastasis in liver and lung by two human colorectal carcinoma cell lines that are injected intrasplenically 30 min later. The metastatic potential of another three moderately to highly metastatic colorectal carcinoma cell lines and of one weakly metastatic line has now been analysed in this model. CEA pretreatment only enhanced colony formation by cell lines that were weakly metastatic in untreated nude mice; it did not affect experimental metastasis by highly metastatic lines. CEA pretreatment enhanced the retention of 125I Idudr-labelled weakly metastatic tumour cells within the liver and lungs 4 h after intrasplenic injection but not the retention of highly metastatic tumour cells or inert latex beads. A significant correlation existed between the formation of experimental metastases and the early retention of tumour cells within the liver after intrasplenic injection. Aggregation did not appear to be important for retention in liver because CEA did not aggregate colorectal carcinoma cells in vitro. Also CEA did not alter natural host effector cell function in a cytolysis assay in vitro. We suggest that CEA facilitates liver colonisation by three of eight human colorectal carcinomas in athymic nude mice by increasing the hepatic retention of tumour cells. The potential mechanisms by which CEA may increase the retention of tumour cells in the liver are discussed.


Menogaril is an anthracycline presently in Phase II clinical trials. Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril. P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo. Resistance to menogaril was stable for at least 2 months in the absence of the drug. The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype. It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin. Uptake and efflux of menogaril was similar in sensitive and resistant cell lines. Also, resistance was not reversed by verapamil. No major karyotypic difference was noted between P388/O and P388/MEN. There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O. In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype. Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity. Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells.


The effects of an aromatic retinoid, etretinate and a vinca alkaloid, vindesine were investigated by culture of malignant melanoma cells in vitro with these two agents; either separately or in combination. Etretinate inhibited growth of a murine melanoma but only minimal effects were recorded with two human melanomas. Vindesine however, was inhibitory for all of the cell lines and this effect was enhanced in the presence of the retinoid. Entry of 3H labelled vindesine or etretinate into drug free cells was followed in the absence or presence of unlabelled drug. It was found that etretinate enhanced cellular uptake of vindesine in two of the cell lines and this may be responsible for the enhanced toxicity of vindesine in the presence of etretinate. The human melanoma which did not exhibit retinoid stimulated vindesine uptake, appeared to be intrinsically sensitive to the vinca alkaloid. No effect on cellular retinoid uptake by vindesine was recorded in any of the melanomas. The results indicate that the intracellular concentrations combined with the intrinsic sensitivities of each cell line to etretinate and vindesine determines the toxic response.


This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.


The control of cell proliferation by gastrin has been investigated in a rat pancreatic tumour cell line, AR4-2J. Exogenous gastrin, 10(-12) to 10(-8) M, stimulated cell growth of thymidine-synchronised AR4-2J cells cultured over 48 h in serum-free medium. Cell lysates of AR4-2J cells contained an average of 4.5 and 3.5 pg gastrin per 10(6) cells, when grown in serum-supplemented or serum-free media, respectively, as revealed by radioimmunoassay. In serum-free medium, AR4-2J secrete 34 ng 1(-1) 10(-6) cells of gastrin over 48 h. Addition of an anti-gastrin immunoglobulin preparation, but not control immunoglobulins, caused a maximum 52% reduction in cell growth. These data are consistent with an autocrine role for gastrin in the control of AR4-2J cell growth. These results were supported by studies with gastrin/CCK receptor antagonists. Six non-peptide gastrin/CCK receptor antagonists inhibited AR4-2J cell growth in a concentration-related manner. The concentration required for 50% inhibition (IC50) of cell growth by the amino acid-derived antagonists proglumide (3.5 x 10(-3) M), benzotript (1.8 x 10(-3) M), loxiglumide (1.1 x 10(-4) M) and lorglumide (6.7 x 10(-5) M) were of the same order and significantly correlated with their IC50 for inhibition of 125I-gastrin binding to AR4-2J cells. Inhibition of cell growth by these antagonists was partially reversed by the addition of exogenous gastrin. In contrast, the IC50 for inhibition of cell growth with two benzodiazepine-derived antagonists, the CCK-B receptor antagonist L-365,260 (4.6 x 10(-5) M) and the CCK-A receptor antagonist devazepide (1.7 x 10(-5) M) were two-three orders of magnitude greater than those required to inhibit gastrin binding (10(-8)-10(-7) M). The growth inhibitory effects of L-365,260 and devazepide were not reversed by exogenous gastrin suggesting these benzodiazepine-derived antagonists do not inhibit cell growth by interaction with gastrin receptors. The results are consistent with gastrin being an autocrine growth factor in AR4-2J cells, and that stimulation of cell growth is due to stimulation of the gastrin, rather than CCK-B, receptor sub-type. This study highlights that gastrin receptor antagonists warrant further investigation as agents to control growth of tumours, such as those from the gastrointestinal tract, which express gastrin receptors.


We have investigated the possibility of a direct regulatory effect of gonadotrophin releasing hormone (GnRH) analogues on prostatic cancer cell growth. Here we report high affinity binding (Kd = 50 nM) of a GnRH analogue resulting in biphasic growth modulation of the human androgen-sensitive prostatic cancer cell line LNCaP. In contrast, the human androgen-insensitive prostatic cancer cell line DU145 showed low-affinity (Kd = 10 microM) binding without any biological response to the GnRH analogue. A GnRH-specific radioimmunoassay demonstrated GnRH-like immunoreactivity in the concentrated culture medium from both cell lines. Seventy-six human benign and malignant tumours were assayed following surgical resection. Nineteen of 22 (86%) malignant tumours and 49 of 54 (91%) benign tumours, exhibited high affinity GnRH-analogue binding. Fourteen of 19 (74%) malignant tumours and 17 of 49 (35%) benign tumours exhibiting high affinity binding contained GnRH-like immunoreactivity, suggesting that this system may be involved in prostatic epithelial cell growth in vivo.


We have studied the ability of cyclosporin A (CsA) and a non-immunosuppressive analogue, O-acetyl cyclosporin A  to inhibit the growth of human lung cancer cells in vitro. Using continuous drug exposure and the MTT colorimetric assay to determine cell growth we found that CsA produced partial growth inhibition at doses ranging from 0.5 to 3.0 micrograms ml-1 (0.4-2.4 microM). At progressively higher doses, complete growth inhibition and in situ cell lysis were seen. The P-glycoprotein expressing multidrug resistant (MDR) variant H69/LX4 of the small cell line H69/P was less sensitive to cyclosporins than the parent line, but this was not true of the non-P-glycoprotein expressing MDR variants of large cell line COR-L23 or adenocarcinoma line MOR. Sensitivity to OACsA was approximately 2-fold higher than that to CsA in most of the lines although not in the most sensitive line, COR-L88. Even in COR-L88, exposed to CsA or OACsA for 24 h, clonogenic cell survival was reduced only to 50%. There was no reduction in polyamine content of COR-L23 or COR-L88 cells following 48 h of exposure to CsA or OACsA. The effects on cell growth could not be inhibited by the addition of exogenous putrescine, nor could they be enhanced by the addition of alpha-difluoromethylorthinine. It does not appear therefore that inhibition of polyamine synthesis is the basis of the observed growth inhibition.


Monoclonal antibodies (Mabs) from the Second International Workshop on Small Cell Lung Cancer (SCLC) Antigens that recognise the cluster 2 SCLC-associated antigen mediated potent and selective cytotoxic effects in an indirect assay of immunotoxin cytotoxicity. In this assay, the NCI-H69 cell line was treated with each Mab at 4 degrees C, washed to remove unbound Mab, and then incubated at 37 degrees C in the presence of a fixed concentration, 1 x 10(-8) M, of the screening agent, sheep anti-mouse IgG-ricin A chain. The use of a fixed high concentration of screening agent led to a 300-fold overestimate of the potency of a cluster 2-directed immunotoxin, MOC-31-ricin A chain. In contrast, when the concentration of the screening agent was identical to the Mab concentration, a precise match to immunotoxin potency was obtained. MOC-31-ricin A chain selectivity inhibited the incorporation of [3H]leucine by the NCI-H69, SW2 and GLC-8 SCLC cell lines by 50% at a concentration between 3 x 10(-11) M and 3 x 10(-10) M, and by the NCI-H125 lung adenocarcinoma cell line at 7 x 10(-11) M, but exerted no selective toxic effects upon human lung and non-lung tumour cell lines lacking surface expression of the cluster 2 antigen.


Following EMS mutagenesis, three estramustine (EM) resistant DU 145 human prostatic carcinoma cell lines were clonally selected by exposure to incrementally increasing concentrations of the drug. Although only low levels of resistance (approximately 3-fold) were attainable, this resistance was stable in the absence of continuous drug exposure. These EM-resistant clones  did not exhibit cross resistance to vinblastine, taxol, or adriamycin, and had collateral sensitivity to cytochalasin B. None of the lines had elevated expression of P-glycoprotein mRNA or glutathione S-transferase activity, suggesting a phenotype distinct from the classic multi-drug resistance phenotype. This conclusion was supported further by the observation that two MDR cell lines (FLC mouse erythroleukaemic and SKOV3 human ovarian carcinoma cells) showed sensitivity to EM. Fluorescent activated cell sorting analysis of the effects of EM on cell cycle traverse revealed that at EM concentrations up to 20 microM an increasing percentage of wild type cells were blocked in G2/M; no such effect occurred in EMR lines. Differential interference contrast microscopy was employed to study EM's effect on mitosis. EMR lines were able to form functional, albeit smaller, spindles at EM concentrations that resulted in chromosomal disorganisation and inhibition of mitotic progression in wild type cells. EMR lines were able to progress through mitosis and cytokinesis at the same rate as untreated cells. Tritiated EM was used to evaluate potential drug uptake/efflux mutations in ERM clones. EMR 4 and 9 incorporate less EM than wild type cells; however, they have significantly decreased cellular volumes. The initial efflux rate constants for EMR clones were greater than for wild type cells. Within 5 min greater than 70% of the drug was lost from resistant cells compared to a 50% loss by the wild type. Although the specific mechanisms of resistance have yet to be defined, the lack of collateral resistance to other MDR/anti-microtubule agents could serve as the basis for the clinical use of EM in combination chemotherapy.


Tissue samples from 59 patients with lung cancer have been used to establish cell lines in culture. The primary diagnosis was small cell carcinoma in all except four. Most of the samples were of bone marrow but pleural effusions, lymph node biopsies and skin metastases were also included. The samples were usually split between HITES serum-free medium and HITES plus 2.5% foetal calf serum. A total of 19 cell lines were established and characterised. One line is large cell anaplastic lung carcinoma, four are B-lymphoblastoid and fourteen are small cell lung cancer. Considerable heterogeneity in gross morphology, neuroendocrine differentiation (by electron microscopy) and content of the enzyme L-dopa decarboxylase was seen. The use of HITES plus 2.5% foetal calf serum resulted in better establishment of cultures than did serum-free HITES.


A simple colorimetric test, the MTT assay, has been adapted for chemosensitivity testing of human small cell lung cancer cell lines, and fresh tumour samples. Optimal conditions for clinical chemosensitivity testing were determined using established SCLC lines. Nineteen different chemotherapeutic agents were tested, and sixteen of them were found to be cytotoxic in this assay system. The drug sensitivity of a panel of 16 SCLC cell lines was measured and compared. There was very little intraexperiment variation, but the interexperiment variation was significant. Cell lines which were derived from patients who had not received chemotherapy at the time the cell line was established were more sensitive  than lines derived from treated patients, and the differences were statistically significant for two of the drugs. One cell line, NCI-H209, which was derived from an untreated patient, stood out as being the most sensitive or among the most sensitive to all of the drugs tested. Another cell line, H69AR, which is a multidrug resistant subline of the cell line NCI-H69, was the most resistant to many of the natural product drugs tested. Multiple drug chemosensitivity testing was performed on eight fresh tumour samples from SCLC patients . It was possible to perform chemosensitivity testing on all of the clinical samples in which sufficient tumour cells were available. The drug sensitivity of the clinical samples was, in most cases, within the same range as for the cell lines. Since this assay is very rapid and simple to perform, it may have practical applications in clinical drug sensitivity testing of human tumours.


Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells.A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated.RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells.Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis and further intervention in cisplatin resistance. Head and neck squamous cell carcinoma (HNSCC) is a major public problem, frequently associated with devastating functional and cosmetic consequences for patients. More than 500,000 new cases are estimated to occur worldwide every year  and two Cisplatin resistance in Tca/cisplatin, a variant cell line derived from Tca8113, was developed by exposure to cisplatin for 24 months, starting at 1 μM and ending at 10 μM. Despite massive cell death among the sensitive Tca8113 cells under treatment, the cultures were maintained by regular changes of medium and intermittently increasing the cisplatin concentration until the surviving cells recovered a normal growth pattern. Before testing, the Tca/cisplatin cells were continuously maintained in cisplatin free RPMI-1640 medium  supplemented with 10% fetal bovine serum  for two years.3 cells/well (200 μl/well) for 24 h before use. The culture medium was replaced with fresh medium containing different concentrations of cisplatin ranging from 0 to 160 μM for 48 h. Water-soluble tetrazolium MTT  was added (20 μl). After a further 4 h incubation, the supernatant was discarded and the purple crystals were re-suspended in 200 μl of DMSO. The absorbance of each well was read at 570 nm on an ELISA XL . Growth rate was calculated as the ratio of the absorbance of the experimental well to that of a blank well. The IC50  was calculated as previously described [Growth inhibition was determined by a MTT assay repeated six times. In brief, cells were seeded in 96-well plates at a density of 2 × 10Apoptosis induced by treatment with different concentrations of cisplatin for 48 h was assayed using an Annexin V-FITC/PI Apoptosis Detection Kit  following the manufacturer's protocol; assays were repeated three times. The results were analyzed using Cell Quest  and flow cytometry  was used to distinguish the cells as viable (Annexin V-/PI-), early apoptotic (Annexin V+/PI-) or late apoptotic (Annexin V+/PI+).For cisplatin accumulation experiments, cells were exposed to 200 μM cisplatin for 2 h and then harvested. The drug exposure was relatively brief, so that the contribution of net platination of DNA could be isolated and the effect of DNA repair reduced, as discussed previously . Total DTotal RNA was extracted and analyzed on HG-U95Av2 Affymetrix oligonucleotide arrays  containing 12,626 probe sets for human genes, as described previously [MAP2K6, RECQL, CCND1, CCND3, ABCB1, ABCC2 and GST-Pi were selected to validate the results of microarray analysis, as described previously [Several interesting genes including eviously . Total RWhole-cell extracts were prepared in cell lysis buffer . The first antibodies were: mouse monoclonal anti-CCND1 , rabbit polyclonal anti-CCND3 , mouse monoclonal anti-ABCB1  and mouse monoclonal anti-GST-Pi . Signals were visualized using Supersignal West Pico Chemiluminescent Substrate (Pierce). Mouse monoclonal anti-β-actin  was used throughout as loading control.2 steel mesh to prepare tumor cell suspensions. Each suspension was cultured with or without 10 μM CDDP for 72 h. Growth inhibition was evaluated using a modified MTT assay to assign each sample to the cisplatin-resistant or the cisplatin-sensitive group [Fresh specimens of primary oral squamous cell carcinoma were collected aseptically from the Department of Oral & Maxillofacial Surgery, Ninth People's Hospital, between June 2003 and July 2005. The specimens were minced with scissors and passed through a 200 pores/cmve group ,12. Immu4 cells were analyzed by flow cytometry . Results were expressed as the percentage of cells in each phase of the cell cycle (Mean ± SD).Cells in exponential growth were cultured in 10 μM cisplatin for 6, 12 or 24 h, then washed twice in PBS and fixed in 70% cold ethanol overnight. Cell pellets were resuspended in 1 mg/ml RNase  at 37°C for 30 min, followed by staining with 50 μg/ml propidium iodide . For each sample, 10t test was used to determine the statistical differences between these two cell lines in cisplatin accumulation and Real-time PCR results. Immunohistochemical staining results were analyzed by a Mann-Whitney U test. P < 0.05 was considered statistically significant. A significance analysis of microarrays algorithm (SAM) was used to identify significant differences in gene expression between these two cell lines (triplicate results), with the estimated false discovery rate (FDR) setting as 1 and the response type setting as two class unpaired.An ANOVA test was used to examine the statistical differences in growth inhibition and apoptosis rates between Tca/cisplatin and Tca8113 cells. Student's The Tca/cisplatin cell line was obtained by stepwise selection from its sensitive parent cell line over a period of two years. At the beginning of induction, cell growth was strongly suppressed. However, at the end of induction, the derived cells proliferated fairly rapidly. After further maintenance for up to 60 passages in cisplatin-free RPMI1640 supplemented with 10% fetal bovine serum, the resistant cells exhibited stable biological characteristics: the population doubling time was 29.93 h, while that of the parent cell line was 38.82 h. The drug-resistant cells were stable for two years in cisplatin-free culture medium.Cells were treated with different concentrations of cisplatin for 48 h and dose-response curves were plotted as shown in Figures Intracellular accumulation of cisplatin was compared between Tca/cisplatin and Tca8113 cells by determining the amount of platinum in DNA. Time-dependent accumulation was observed Figure , and aftCCND1, ASNS), DNA repair (RECQL), small molecule non-selective transport (ITPR1), protein synthesis (GARs), transcription  and oncogenes . The up-regulated genes could result in cell cycle acceleration, increased proliferation, enhanced DNA metabolism and synthesis, altered molecular transport and elevated transcriptional activity; Tca/cisplatin had a shorter population doubling time (29.93 h) than the parental cell line (38.82 h).The Affymatrix HG-U95Av2 microarray contains 12626 probe sets, most of which have previously been characterized in term of function or disease association. In order to distinguish valid signals from background noise, three replicates were performed using independently isolated RNA samples for each cell line, and a total of 6 hybridizations were analyzed. Scatter plots of the normalized signals were constructed and the average of the correlation coefficients for replicates was 0.951, suggesting good consistency. SAM was used to identify genes that were significantly differently expressed between these two cell lines, with FDR setting = 1. As shown in Table MAP2K6 in the MAP pathway, the DNA repair-related gene RECQL, the cell cycle-related genes CCND1 and CCND3, ATP-binding cassette family members ABCB1 and ABCC2, and the cellular detoxification-related gene GST-Pi – were selected for further verification of the microarray results by Real-time PCR. Also, the expression of CCND1, CCND3, ABCB1 and GST-Pi proteins was further tested by Western blotting, exploiting their functional linkages to biochemical mechanisms that could be related to cisplatin resistance. As shown in Figure Seven genes – CCND1 and CCND3 were differently expressed in Tca/cisplatin and Tca8113 cells. We therefore analyzed the cell cycle distribution further following cisplatin exposure. As shown in Figure Analysis of the gene expression profiles suggested that the cell cycle-related genes P = 0.021) and CCND3 (P = 0.013) between these two groups.Clinical samples were classified into cisplatin-sensitive and resistant groups on the basis of growth inhibition as indicated by the MTT assay . The cutCCND1, ID1, ID3, GCA, etc.), and also some novel ones such as TRIM29, N33, GRAS and GPI [Here we established an isogenic cisplatin-resistant variant from the oral squamous cell carcinoma cell line Tca8113 so that we could compare their gene expression profiles directly. It is clear from our results that the cisplatin-resistant cell line, Tca/cisplatin, has a genome-wide expression profile that differs from the Tca8113 line. Our microarray analysis results revealed 63 differently expressed genes, including some previously reported genes that have been related to cisplatin resistance  declare that they have no competing interests.PZ carried out the molecular genetic and cellular phenotype studies, performed the statistical analysis and drafted the manuscript. ZYZ conceived of the study and participated in its design and coordination. ZXJ participated in establishing cell lines and carried out the immunoassays. WLQ participated in the study design. FAC carried out the microarray testing and performed the statistical analysis. WTC conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In order to examine in detail the sensitivity to chemotherapy of tumour cells at various organ sites and at various stages of metastasis, we have used a series of cell lines, all selected from sister subpopulations derived from a single mouse mammary tumour, which can be distinguished and quantitated from normal cells and from each other through growth in selective medium. For the studies described here, we used two lines, 4T07 and 66FAR, which will form colonies in vitro in medium containing 60 microM 6-thioguanine or 330 microM 2,6-diaminopurine, respectively. Both cell lines have similar sensitivity to the test chemotherapeutic agent, melphalan, in vitro. These two tumour cell lines were treated with melphalan in vivo, when growing either in lungs as experimental metastases at various times after cell injection or as palpable tumours growing subcutaneously. Responses to various doses of melphalan were measured by removing lungs or subcutaneous tumours and performing colony-forming assays in selective medium. The data indicate marked shifts in sensitivity as a function of metastatic stage. Analyses of dose-response curves show that both cell lines were similarly sensitive to melphalan at early times (45 min) after cell injection i.v. but became less sensitive at an intermediate time after injection (3 days). Differences between the two lines became apparent at later times after i.v. injection (by day 8 or 9) and in subcutaneous tumours, where a marked reduction in the shoulder of the dose response curve was seen in line 4T07, resulting in sensitivity equal to or greater than the of early times, whereas the dose response parameters of 66FAR remained at those of the intermediate time point. These results show that, in heterogeneous tumours, individual subpopulations of tumour cells may respond differently to chemotherapeutic agents at various disease stages. In vitro measures of tumour sensitivity do not predict these changes in in vivo sensitivity. Model systems similar to the one described here may yield information which will eventually be useful in maximising the efficacy of clinically relevant adjuvant chemotherapy regimens.


We investigated the effect of the flavonoid quercetin (Q) on the proliferation of the ovarian cancer cell line OVCA 433. Growth experiments demonstrated that Q exerted a reversible dose-dependent inhibition of cell proliferation in the range of concentrations between 10 nM and 10 microM. Two other flavonoids tested, rutin and hesperidin, were ineffective in inhibiting cell growth. Cell cycle analysis showed that the growth inhibitory effect of Q was due to a blocking effect in the GO/G1 phase. Using a whole cell assay with (6.7-3H) oestradiol (3H-E2) as tracer we demonstrated that OVCA 433 cells contain type II oestrogen binding sites (type II EBS). Competition analysis showed that Q competed for 3H-E2 binding to type II EBS while both rutin and hesperidin did not. Appreciable amounts of type II EBS were also detected in seven primary ovarian tumours. Our results suggest that Q may regulate ovarian cancer cell growth through a mechanism involving a binding interaction with type II EBS. This mechanism could also be active in vivo since primary ovarian tumours contain type II EBS.


Tumour cell adhesion, detachment and aggregation seem to play an important part in tumour invasion and metastasis, and numerous cell adhesion molecules are expressed by tumour cells. Several families of cell-cell adhesion molecules have been described, of which two groups are particularly well characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were examined. All tumours but one expressed both cadherin and NCAM. The tumours expressed one, two or rarely three cadherin bands, and different combinations of two major isoforms of NCAM with M(r)'s of approximately 190,000 and 135,000. Polysialylation of NCAM, a feature characteristic of NCAM during embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression.


Microcolonies were obtained by culturing cells of two human osteosarcoma lines (OHS and KPDX) and one human melanoma line (WIX-c) for either 24 or 72 h. The microcolonies were treated with either alpha-particle radiation emitted by the 211At-labelled monoclonal antibody (MAb) TP-3 or external beam X-rays. Survival of microcolonies was assayed by colony formation. Therapeutic gain factor (TGF) values were calculated for two survival levels, 50% and 20% microcolony regeneration (i.e. at least one cell in 50% or 20% of the colonies survived the treatments). The TGF values were affected by the specific activity of the 211At-MAb conjugate, the antigen expression of the cells and the size and growth pattern of the microcolonies. Treatment with 211At-TP-3 gave TGF values that varied from 1.3 +/- 0.4 to 4.5 +/- 0.7 (mean +/- s.e.). The antigen-rich OHS cell line had on average 1.6 times higher TGF than the antigen-poor KPDX cell line. The TGF increased significantly with colony size for the densely packed colonies of the KPDX cell line but not for the OHS cell line, which had colonies with cells growing in a more scattered pattern. Control experiments with the two non-specific 211At forms, free 211At and 211At-labelled bovine serum albumin, gave TGF values from 0.6 +/- 0.1 to 1.0 +/- 0.3. This study suggests that in vivo evaluation of 211At-MAbs using relevant tumour models is desirable.