Multidrug resistance (MDR) is a major factor which contributes to the failure of cancer chemotherapy, and numerous efforts have been attempted to overcome MDR. To date, none of these attempts have yielded a tolerable and effective therapy to reverse MDR; thus, identification of new agents would be useful both clinically and scientifically.To identify small molecule compounds that can reverse chemoresistance, we developed a 96-well plate high-throughput cell-based screening assay in a paclitaxel resistant ovarian cancer cell line. Coincubating cells with a sublethal concentration of paclitaxel in combination with each of 2,000 small molecule compounds from the National Cancer Institute Diversity Set Library, we identified a previously uncharacterized molecule, NSC23925, that inhibits Pgp1 and reverses MDR1 (Pgp1) but does not inhibit MRP or BCRP-mediated MDR. The cytotoxic activity of NSC23925 was further evaluated using a panel of cancer cell lines expressing Pgp1, MRP, and BCRP. We found that at a concentration of >10 µM NSC23925 moderately inhibits the proliferation of both sensitive and resistant cell lines with almost equal activity, but its inhibitory effect was not altered by co-incubation with the Pgp1 inhibitor, verapamil, suggesting that NSC23925 itself is not a substrate of Pgp1. Additionally, NSC23925 increases the intracellular accumulation of Pgp1 substrates: calcein AM, Rhodamine-123, paclitaxel, mitoxantrone, and doxorubicin. Interestingly, we further observed that, although NSC23925 directly inhibits the function of Pgp1 in a dose-dependent manner without altering the total expression level of Pgp1, NSC23925 actually stimulates ATPase activity of Pgp, a phenomenon seen in other Pgp inhibitors.The ability of NSC23925 to restore sensitivity to the cytotoxic effects of chemotherapy or to prevent resistance could significantly benefit cancer patients. Patients with advanced cancer are usually treated with chemotherapy in order to extend life and palliate symptoms. There is some improvement of responses seen in select cases by the addition of immunotherapy or biological response modifiers Pgp1 (MDR1) expression, frequently detected in both solid and hematologic malignancies, as well as in cancer stem cells, is a marker of chemoresistance and decreased survival in leukemia, lymphoma, osteosarcoma, small-cell lung cancer, ovarian cancer, breast cancer, and other malignancies A wide range of compounds that interact with Pgp1 and block drug efflux have been reported to reverse the MDR phenotype Screening of chemical libraries for biologically active compounds aimed at certain cellular targets is a powerful approach to identify small molecules regulating cellular function that can be useful as both research tools and therapeutic agents http://dtp.nci.nih.gov). Paclitaxel, doxorubicin, and cisplatin were obtained through unused residual clinical material provided by the pharmacy at the Massachusetts General Hospital. ET-743  and PM00104  were supplied by PharmaMar (Spain).The Structural Diversity Set is a library of approximately 2,000 small molecules derived from the almost 140,000 compounds available on plates through the National Cancer Institute (NCI). These compounds are thought to have anti-tumor activity but their functional data is limited. Detailed information on the selection, structures, and activities of these diversity set compounds can be found on the NCI Developmental Therapeutics Program web site , KHOS R2 cell lines The paclitaxel-resistant U-2OSThe Pgp1 monoclonal antibody was purchased from Signet . The monoclonal antibody to MRP1 and MTT reagent were purchased from Sigma-Aldrich . The monoclonal antibody to BCRP antibody was purchased from Chemicon .2-95% air atmosphere and passaged when near confluent monolayers were achieved using 2% trypsin-EDTA solution. Drug-resistant cell lines were periodically cultured in the respective drug to confirm their drug resistance characteristics.All the cell lines were cultured in RPMI 1640  supplemented with 10% fetal bovine serum, 100-units/ml penicillin and 100 µg/ml streptomycin (Invitrogen). Cells were incubated at 37°C in 5% COTR cells with chemotherapy drug paclitaxel were used for initial high-throughput screening. SKOV-3TR showed 100-fold more resistance to paclitaxel as compared with parental sensitive cells and could grow in 0.3 µM concentration of paclitaxel without showing toxicity. Screening conditions in SKOV-3TR were optimized in 96-well plates for growth conditions, small molecular compound and drug concentration and assay times prior to screening. On day 1, SKOV-3TR cells were seeded at 2×104 cells/well on 96-well plates and labeled with plate A, B, C and incubated for 24 hr at 37°C  by a SPECTRAmax® Microplate Spectrophotometer . Only the compounds at 1 µM showed no cytotoxicity (without paclitaxel) to SKOV-3TR were selected for further study.SKOV-3 at 37°C . On day TR. The experiments were conducted in two control plates (A and B) and one experimental plate (C) to permit plate-to-plate comparisons  on a SPECTRAmax® Microplate Spectrofluorometer (Molecular Devices).The Vybrant™ multi-drug resistance assay kit (Invitrogen/Molecular Probes) was used to measure drug efflux properties of different resistant cell lines. This assay utilizes the fluorogenic dye calcein acetoxymethyl ester  as a substrate for efflux activity of Pgp1 or other membrane pump ABC proteins. Calcein AM is taken up by cells and hydrolyzed by cytoplasmic esterases to fluorescent calcein. Calcein AM is well retained in the cytosol. However, multidrug resistant cells expressing high levels of Pgp1 rapidly extrude non-fluorescent calcein AM from the plasma membrane, reducing accumulation of fluorescent calcein in the cytosol. Drug sensitive and resistant cells  were cultured in 96-well plate for 24 hours. Triplicated cultures of cells were treated with NSC23925, verapamil for one hour and then incubated in calcein AM in 100 µl total volume. After 30 minutes, the cells in plate were washed and centrifuged twice with 200 µl cold RPMI1640 culture medium, and cell fluorescence was measured at a wavelength of 490 nm  equipped with a SPOT RT digital camera .The Pgp-Glo™ Assay Systems (Promega) provide the necessary reagents for performing luminescent P-glycoprotein (Pgp1) ATPase assays. PGP-ATPase assay is a valuable screening tool for determining if a drug interacts with Pgp1. The ATPase assay provides a rapid, colorimetric, compound-independent measure of the concentration-dependence of any interaction of a drug with Ppg1. If a drug does not stimulate Pgp1-ATPase activity, the assay can still determine if the drug interacts with Pgp1 as an inhibitor of the ATPase activity stimulated by a known substrate, such as verapamil. The effect of NSC23925 on the ATPase activity of Pgp1 was measured according to the manufacturer's protocol.Values shows are representative of triplicate determinations in two or more experiments. Treatment effects were evaluated using a two-sided Student's t test . Errors are SD of averaged results and P<0.05 values were accepted as a significant difference between means.TR. 0.1 µM paclitaxel was determined to be the concentration of paclitaxel that is lethal to drug sensitive SKOV-3 cells, but does not affect the growth of drug resistant SKOV-3TR cells. Cells were arrayed in 96-well plates and each well was incubated for six days in optimized culture conditions with 0.1 µM paclitaxel, 1 µM library compound, or both 0.1 µM paclitaxel and 1 µM library compound  to SKOV-3y itself . NSC770325 alone .TR and OVCAR8TR, breast cancer cell line MCF-7TR, and sarcoma cell lines MESSA/Dx5, KHOS R2, and U-2OSDR . This data collectively demonstrates that NSC23925 itself is not a substrate of Pgp1, and Pgp1 does not confer resistance to the cytotoxic effects of NSC23925 in tumor cells. Also, NSC23925 is not subject to cross resistance with classical cytotoxic agents such as paclitaxel or doxorubicin. These results are of clinical significance because they suggest that NSC23925 may be a good option for the patients with tumors that have already developed MDR.Although NSC23925 reversed Pgp1 mediated MDR in the nanomolar concentration range, NSC23925 was not cytotoxic by itself at doses up to 10 µM in both sensitive and resistant cell lines. At high concentrations, NSC23925 by itself is equally inhibitory on the proliferation of both SKOV-3 and SKOV-3t manner . IC50s w to 1 µM . Thus, NTR cells . SimilarTR and OVCAR8TR. We observed that NSC23925 inhibits the efflux of both paclitaxel and doxorubicin from MDR cells and that the inhibitory effects of NSC23925 remains even when it was washed out from the efflux medium after overnight exposure (1 µM) (TR cells (data not shown).The ability of NSC23925 to inhibit Pgp1 mediated efflux and its duration of action were investigated using two MDR substrates, paclitaxel, doxorubicin, and two MDR cell lines SKOV-3e (1 µM) . NSC2392e (1 µM) . SimilarTR and OVCAR8TR. NSC23925 was shown to increase intracellular accumulation of calcein AM in these cell lines in a dose-dependent manner as determined by both image analysis  in SKOV-3analysis  and by manalysis . NSC2392analysis . In the analysis . To confysis  with molecular weight 348. Unlike NSC77037/tetrandrine, information about NSC23925 pharmacological action is lacking. There is no prior report describing the action of NSC23925 on reversing chemoresistance in cancer. We demonstrated such reversal of MDR in cell lines derived from ovarian cancer, breast cancer, colon cancer, and sarcoma tumors. We observed reversal of MDR for paclitaxel, doxorubicin, ET-743, and other anti-cancer agents.In this study, we screened 2,000 small molecule compounds in the NCI Structural Diversity Set to identify agents capable of reversing multidrug resistance in MDR cell lines. The present study extends the previous techniques such as flow cytometric analysis, dye efflux assay, and bioinformatic approaches to find novel MDR inhibitors by directly reversing MDR in a cell-based high-throughput assay Pgp in multidrug resistant cancer cells In multidrug resistant cell lines which highly express MDR1 (Pgp1 protein), Pgp1 functions as an energy-dependent membrane transporter that rapidly extrudes chemotherapeutic drugs from cells, and therefore prevents the drugs from exerting cytotoxic effects. Many studies with multidrug resistant cells correlated resistance to reduced accumulation of drugs within the cells due to increased efflux or decreased influx by Pgp1Our core observations are that (1) the function of Pgp1 mainly functions to efflux drugs out of MDR tumor cells, (2) NSC23925 was observed to inhibit the efflux of substrates of Pgp1, such as paclitaxel and doxorubicin, (3) NSC23925 at 10 µM for 48 hours does not inhibit the expression level of Pgp1, and (4) NSC23925 stimulates Pgp ATPase activity. Taken together, these observations support the hypothesis that NSC23925 reverses MDR in tumor cells by uncoupling Pgp ATPase activity from the drug efflux activity of Pgp1.The potency was confirmed by several assays  using a panel of multidrug resistant cell lines with different degrees of drug resistance and Pgp1 expression. In these assays, full to partial reversal of resistance in several MDR cell lines to several major classes of chemotherapy drugs was achieved in the presence of 10 to 2,000 nM NSC23925. Direct comparison with several modulators such as verapamil and CsA in different assays demonstrated that NSC23925 is the most potent among the three modulators. NSC23925 was 20-fold more potent than verapamil and 50-fold more potent than CsA in the same concentration.in vitro was 10 to 20 fold lower than the concentration at which any toxicity (IC50) was observed in the cell lines. Further confirmation of selectivity of NSC23925for Pgp1 was provided by the fact that the NSC23925, even at very high concentrations (50 µM), did not inhibit MRP1 or BCRP function. These findings provide support for further development of NSC23925 or its derivates as candidate inhibitors of MDR in cancer treatment. Such efforts can further our understanding of the molecular mechanisms of drug resistance.Our study shows that reversal of MDR by NSC23925 was through selective and potent inhibition of Pgp1 function. For example, in contrast to the modulatory activity in the resistant cell lines, NSC23925 had no significant effect on cytotoxic drug activity in non-Pgp1 expressing parental cell lines nor did it affect the cytotoxicity of non-Pgp1 substrates such as cisplatin and methotrexate. Moreover, the concentration of NSC23925 required to fully reverse drug resistance


In this study we have investigated the role of thrombospondin (TSP) as a possible ligand playing a key role in human M3Da. melanoma cell interaction with platelets and in tumour growth. TSP is secreted (80 +/- 6 ng TSP 10(-6) cells) and bound to the surface of M3Da. cells via receptors different from CD36, as shown by biosynthetic labelling and immunofluorescence studies. The levels of TSP binding to M3Da. cells evaluated by binding studies, using an anti-TSP monoclonal antibody (MAb) (LYP8), shows 367,000 +/- 58,000 (mean +/- s.d.) LYP8 binding sites per cell with a dissociation constant (Kd) of 67 nM. TSP binding to M3Da. cells shows 400,000 +/- 50,000 TSP binding sites per cell with a Kd of 10 nM. The capacity of anti-TSP MAb (LYP8) to inhibit M3Da.-platelet interactions was followed on an aggregometer and evaluated by electron microscopy studies. The biological role of TSP binding to M3Da. cells was investigated by implanting subcutaneously the M3Da. cell line in nude mice and following the size and time of in vivo tumour growth. Reducing the availability or the functional level of TSP by using an anti-TSP MAb (LYP8) resulted in a significant decrease in platelet aggregates interacting with M3Da. melanoma cells. Using an enzyme-linked immunosorbent assay, purified alpha nu beta 3 was shown to bind TSP. Moreover, LYP8-coated M3Da. cells showed a reduced capacity to form tumours in vivo. M3Da. cells were observed to attach and spread on human platelet TSP-coated plastic wells. This attachment by M3Da. cells was inhibited in a similar way by LYP8 and an anti-alpha nu beta 3 MAb (LYP18). The results obtained in this study show that TSP secreted and bound to the surface of a human melanoma cell line (M3Da.) acts as a link between aggregated platelets and the M3Da. cell surface. Moreover, these results shows that TSP can modulate tumour growth in vivo. Reagents such as MAbs directed against TSP and peptides derived from TSP could not only be used as a new therapeutic approach in the control of tumour metastasis of melanoma, but may also contribute to elucidation of the role of TSP in cancer biology.


Con A acceptor glycoproteins from the human Molt 4 (T cell leukaemia) and HeLa  cell lines were purified by affinity chromatography and used for the preparation of rat antisera. Cross-absorption analysis showed that each antiserum contained antibodies which recognised cell surface antigens preferentially expressed by the donor cell line. Molt 4-associated antigens were fully expressed on T cell tumour lines and normal thymocytes, but not on non T cell tumour lines, peripheral blood lymphocytes or other blood cells. Immunofluorescence studies showed that the antigens were preferentially expressed on a sub-population of immature thymocytes. HeLa-associated antigens were only fully expressed on one other epithelial tumour cell in a panel of 17 cell lines. Immunofluorescence studies showed that the HeLa-associated antigens were expressed on normal endocervical adenoepithelium but not on ectocervical, endometrial or intestinal epithelia. Thus purified Con A acceptor glycoproteins of cultured tumour cell lines are potent immunogens for the generation of antibodies recognising lineage-associated differentiation antigens. These antigens should be useful in tumour classification and in the study of normal differentiation.


Saos-2 cells, derived from a primary human osteosarcoma, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma. Saos-2 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin but unaffected by apyrase. The cell suspension shortened the plasma recalcification times of normal, factor VIII-deficient and factor IX-deficient human plasmas in a dose-dependent manner. However, the cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, a monoclonal antibody (MAb) against human tissue factor completely abolished TCIPA. Flow cytometric analysis using anti-integrin MAbs as the primary binding ligands demonstrated that the integrin receptors alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1 were present of Saos-2 cells, which might mediate tumour cell adhesion to extracellular matrix. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide which antagonises the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented Saos-2 TCIPA as well as tumour cell adhesion to vitronectin, fibronectin and collagen type I. Likewise, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) showed a similar effect. On a molar basis, rhodostomin was about 18,000 and 1000 times, respectively, more potent than GRGDS in inhibiting TCIPA and tumour cell adhesion.


Although it is generally accepted that proteolytic degradation is an important mechanism used by malignant cells in the process of metastasis, comparatively little is known about the regulation of molecules responsible for proteolysis and how they become de-regulated during human tumour progression. Using a genetically related pair of human melanoma cell lines, derived from the same patient at different stages of disease, we analysed differences in the cytokine-mediated regulation of gelatinase B (MMP-9), an enzyme thought to play an important role in cellular invasiveness, and TIMP-1, a physiological inhibitor of this enzyme. Whereas the advanced stage (i.e. metastatic) partner of this pair (WM 239) could produce gelatinase B upon induction with interleukin (IL)-1β or tumour necrosis factor alpha (TNF-α), the early stage (i.e. primary) partner (WM 115) could not. In sharp contrast, we found that TIMP-1 displayed an opposite pattern of induction in these cell lines. Specifically, the early stage cell line, WM 115, demonstrated a marked increase in the production of TIMP-1 when treated with IL-1β or TNF-α whereas the advanced cell line, WM 239, showed no such increase. Treatment with the DNA demethylating agent, 2-deoxy-5-azacytidine, resulted in a marked up-regulation of both gelatinase B and TIMP-1 in both cell lines. It was further found that constitutive overexpression of gelatinase B in WM 239 cells and an additional melanoma cell line (MeWo), derived from a metastatic lesion, was able to greatly enhance lung colonization in an experimental metastasis assay while we did not observe differences in tumorigenicity. From these results we conclude that an altered responsiveness of gelatinase B and TIMP-1 to induction by similar agents is associated with disease progression in human melanoma and that this altered responsiveness can have consequences to the aggressive nature of the disease. © 1999 Cancer Research Campaign


Resistance to anti-cancer drugs has proved to be a major barrier in the clinical management of neoplastic disease. We have investigated the mechanistic basis for resistance to folate-based thymidylate synthase (TS) inhibitors using two cell lines selected for resistance to ZD1694 -N -methylamino]-2 - thenoyl)-L-glutamic acid), a drug currently in phase III clinical trial. The degree of resistance was > 20,000 for the human lymphoblastoid cell line W1L2:R and approximately 14 for the ovarian carcinoma cell line CH1:R. In both cases resistance was associated with increased TS activity. The W1L2:R cell line had an approximately 100-fold increase in TS gene copy number and mRNA levels and a 500- to 1000-fold increase in enzyme levels determined using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern and Western blotting. The CH1:R cell line had an approximately 2- to 2.5-fold increase in TS gene copy number, mRNA and protein levels. In both cell lines the fold resistance determined was significantly higher than the fold increase in target enzyme DNA, mRNA or protein levels. Small changes in TS levels may therefore translate to clinically significant alterations in drug sensitivity.


It can also induce differentiation and inhibits proliferation of cancer cells. Until now, data using PPARγ ligands in HCC have demonstrated mainly in in vitro models that its activation could be due to an antiproliferative effect. PPARγ ligand administration has also been associated with a diminution of liver fibrosis in animal models, and potentially also on tumoral cell death. Soma data show that the favorable effect of natural and synthetized PPARγ agonists could also be independent of PPARγ activation. Furthermore, in some situations, PPARγ antagonists have also an anticancer effect. Therefore, we can conclude that the link between activation of the PPARγ pathway and an anticancer activity is suggested but until now not firmly established in HCC.Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. This cancer develops mainly in cirrhotic patients. The cirrhotic liver is considered to be a preneoplastic organ, suggesting the rationale for cancer prevention. PPAR Hepatocellular carcinoma (HCC) is the third leading cause of cancer-relateddeath worldwide .Its proγ is a nuclear transcriptionfactor, member of the nuclear hormone receptor superfamily, being activated by binding its ligand,then heterodimerizing with the Retinoid X receptor, to finally bind withspecific response elements in the nucleus, called peroxisome proliferatingresponse elements. Activation of PPARγ by agonists such asthiazolidinediones (TZDs) has been shown to have anticancer effect in vitro and in vivo in many cancer types [γ agonists can have tumorpromoting effects [PPARer types . However effects .γ agonists on HCC has been studied in the last years both in cell cultures and in animal models. Here, we summarize the main findings of these works and attempt to define the role ofPPARγ and its modulation in HCC.Effect of PPARγ RNA and protein expressionhas been demonstrated in vitro in several cell lines, including Hep3B, HepG2, Huh7, and others [PPARd others –11. Its γ protein expression wasassessed by western-blot (WB) in 5 cirrhotic patients and showed no differencebetween HCC and nontumoral cirrhotic liver [γ protein in 20 HCCs and noexpression in surrounding liver, assessed by immunohistochemistry (IHC). Inthis paper, no information was given regarding the presence of cirrhosis [n = 12) and chronic hepatitis (n = 8), showed astatistically significant decrease in PPARγ expression in HCC comparedto adjacent liver [γ mRNA was less expressed in poorly differentiated HCC compared to well-differentiated ones. The latter [γ expression in HCC andcirrhosis compared to normal liver, where it is known to be low [In human HCC, reports from 3 papersshow conflicting results: in the first paper, PPARic liver . The secirrhosis .Finallynt liver . Furthero be low .γ agonist 15-deoxy-Δ12,14-prostaglandin J2, and the synthetic agonists thiazolidinediones has been tested in many humanHCC cell cultures [μMol concentrations,although some contradictory reports have been published [μMol needed to have significant growth inhibitory effect. The antiproliferative effect was shown to be due to a cell cycle arrest, with cells accumulating at the G1 phase. Koga etal. have performed the most thorough mechanistic investigations in the field [μMol, they could show onseveral cell lines that the inhibitory effect on cell cycle was linked to anincrease in the mRNA and protein expression of the Cyclin-dependent kinase inhibitor (Cki) p21waf1/cip1 and also an increase in protein expression only of p27kip1, another CKi. These proteins wereshown to bind to the Cyclin-dependent kinase CDK2, thus inhibiting its activation.Because the intracellular concentration of p27kip1 is tightlyregulated by its degradation through the ubiquitin-proteasome pathway, theauthors have analyzed and could show that indeed, troglitazone had aninhibitory effect on p27kip1 degradation via inhibition of Skp2, anF-box protein of ubiquitin-ligase complex. These results were reproduced byothers [The effect of the natural PPARcultures , 17. Oveublished . Pioglityothers , 14, 18.γ agonists. Kawaguchi et al. [kip1 inthe pioglitazone-treated group compared to control, thus reproducing some ofthe results obtained in vitro.Finally, Yu et al. published their results on the effect of troglitazone, invitro and in vivo in a nude mice model, injected subcutaneously with thehuman Huh7 HCC cell line [kip1 and adecrease of COX-2 expression in tumors compared to controls.In vivo, four papers showed an inhibition of the development of preneoplastic and neoplasticliver nodules by PPARi et al. , using pi et al. . Guo et i et al. , using ti et al. , using pell line .They coγ pathway. In othercancer types, authors have demonstrated that troglitazone and ciglitazone couldinhibit cell proliferation in PPARγ−/− cell lines, by inhibition of translation initiation [It is important to mention that none of these studies, using TZDs, has clearlydemonstrated the antiproliferative effect of these drugs to be due toactivation of PPARitiation .γ-dependant decreased activation ofhepatic stellate cells. These early results showing the importance of PPARγ activation in the pathogenesis of fibrosis were confirmed by others [P = 0.08), although patientsin the pioglitazone group showed significantly less fibrosis after the 6-monthregimen compared to before treatment (P = 0.002).An important paper published in 2002 by Galli et al.  demonstry others , 26. Thoy others  and the y others . These dy others . One of γ in apoptosis and anoikis is less clear. Several in vitro works reportinduction of apoptosis in HCC cell lines, constantly by troglitazone and not byothers TZDs [γ agonist 15-deoxy-Δ12,14-prostaglandinJ2 (15D-JG2) on apoptosis of SK-Hep1 and hepG2 cells. They indeed showed thatgiven at a dose of 50 μMol, 15D-JG2 could induceapoptosis by caspase 3 activation. Using PPARγ antisense oligodeoxynucleotides, they demonstrated that apoptosis was induced independently of PPARγ expression. They also showed that 15d-PGJ2 inhibited NF-kB activation induced by TNFα when PPARγ was normally expressed or downregulated.The role of PPARers TZDs , 30–32. γ agonists, some authors analyzedthe effect of PPARγ inhibitors on cancer cell growth. One publication reported the effectof PPARγ inhibitors on cell adhesion and anoikis [γ in human HCC. They could show that these PPARγ inhibitors increased loss of adherence in vitrofollowed by caspase-dependent apoptosis, a finding reproducedusing PPARγ siRNAs, reflecting the specificity of the PPARγ-dependent activation driving cell death. They also showed thatinhibitors were at least 5-fold more potent in reducing cell number thantroglitazone and rosiglitazone. Another study evaluated the effect of PPARγ agonists and antagonists on cell growth, migration, and invasion in fourdifferent HCC cell lines [Although, as discussed earlier in the text, the vast majority of available data dealswith PPAR anoikis . On the  anoikis , authorsll lines . Authorsγ pathway in the prevention and treatment of HCC. PPARγ mRNA and protein expression are constantly found in cell lines. In normal liver, it is known to be expressed at low levels, ten times less than in adipose tissue. We do not know its actual level of expression, neither in cirrhotic nontumoral liver, nor in HCC, with conflicting results reported. Thiazolidinediones, which are active and specific PPARγ agonists, have shown antitumoralactivity in cell lines and in animal models. This anticancer activity is due to inhibition of cell proliferation, by interfering with important cell cycle cyclins and cyclin-dependent kinase inhibitors, such as p27kip1, but also by a proapoptotic action. The link between activation of the PPARγ pathway and the anticancer activity of TZDs is, however, not established. Finally, PPARγ antagonists have shown antitumor activity, by different mechanisms, mainly involving loss of cell adherence, migration, and invasion.Several in vitro and in vivo papers suggest a role of the PPAR


N-maleimidomethyl) cyclohexane- 1-carboxylate. Fab fragments generated by papain digestion of the 1452C11 antibody  were biotinylated with NHS-Iminobiotin. MUC1-transfected BALB/c breast cancer cell lines 413BCR and 425CCR and the parental cell line (410.4) were labelled with streptavidinylated mouse anti-MUC1 mucin mAb. BALB/c effector T-cells were separately labelled with biotinylated anti-CD3 Fab fragments (1452C11) and mixed with tumour cells in different effector to target ratios. Percentage of killing was assessed using the 51Cr cytotoxicity assay. Seventy per cent lysis was measured in the case of 413BCR (high MUC1 mucin expressor) and 40% in the case of 425CCR (low expressor) cell line. No lysis was apparent in the MUC1 negative cell line. These results demonstrate that the novel T-cell redirecting approach we have developed can produce effective immune lysis of target cells in vitro. © 1999 Cancer Research CampaignTo overcome the disadvantages of bi-specific antibody methodologies in vivo, a novel antibody approach has been designed to improve tumour targeting and effector to target ratio. The technique involves biotinylated anti-CD3 Fab fragments and streptavidinylated anti-tumour monoclonal antibodies (mAbs) that can spontaneously form cross-links. We describe here a method for the direct cross-linking of sulphydryl-conjugated HMFG1 (anti-MUC1 mucin mAb) to streptavidin by sulphosuccinimidyl-4-(


The present study of the effect of heparin and dextran 1000 on the metastasis formation after i.v. tumour cell injection in dextran non-sensitive rats using a syngeneic 20-methylcholanthrene induced fibrosarcoma showed that heparin treatment decreased with formation of pulmonary metastases in animals both untreated and treated with dextran 1000. Treatment with dextran 1000 increased the formation of pulmonary metastases in animals both untreated and treated with heparin and the effect of dextran 1000 was thus not affected by heparin treatment. Heparin did not have any direct action on the tumour cells, which influenced metastasis formation. The data suggest that heparin acts as an anticoagulant with decreased microthrombus formation around lodged cells and that dextrax 1000 stimulates metastasis formation primarily by mechanisms other than intravascular coagulation.


The urokinase plasminogen activator receptor (uPAR) plays a critical role in urokinase-mediated plasminogen activation and thereby in the process leading to invasion and metastasis. Soluble urokinase receptor (suPAR) is released from tumours, and in cancer patients the blood level of soluble receptor is increased. Using an enzyme-linked, immunosorbent assay (ELISA)-specific for the human urokinase receptor, release of soluble receptor was measured in cultures of human breast carcinoma cells, in tumour extracts and in plasma from mice with xenografted human tumours. Soluble human urokinase receptor (shuPAR) was released into culture supernatant during the growth of the human breast cancer cell line MDA-MB-231 BAG, and the level of shuPAR in conditioned medium determined by ELISA was a linear function of both viable cell number and time of incubation. Western blotting showed that the form of shuPAR measured by ELISA in conditioned medium consisted virtually exclusively of the three-domain full-length protein, while uPAR in cell lysates consisted of full-length uPAR as well as the domains (2+3) cleavage product. shuPAR was also released into the plasma of nude mice during growth of MDA-MB-231 BAG, MDA-MB-435 BAG and HCT 116 cells as subcutaneously xenografted tumours. Western blotting demonstrated that the shuPAR released from the xenografted human tumours into plasma consisted of the three-domain full-length protein, despite the finding of some cleaved uPAR in detergent extracts of tumour tissue. The levels of shuPAR determined by ELISA in the plasma of host mice during the growth of xenografted cell lines were highly correlated with tumour volume. © 1999 Cancer Research Campaign


The mechanism of action of the novel heterocycle 3,5-dimethyl-pyrido-1,2,3,5-tetrazepin-4-one (PYRZ), structurally related to temozolomide, was studied in the human ovarian tumour cell line OVCAR-3. Our results showed that, despite its marked structural similarities to temozolomide, PYRZ presents properties that are atypical of 1,2,3-triazene-containing alkylating agents. In a Maxam-Gilbert DNA sequencing assay, PYRZ showed background levels of DNA alkylation, in contrast to temozolomide which strongly alkylated DNA preferentially at guanine residues. At high concentrations, PYRZ inhibited the synthesis of DNA, RNA and protein 3 h after treatment, in contrast to temozolomide which, in previous work, was found to preferentially inhibit DNA synthesis in OVCAR-3 cells. In cells exposed to PYRZ, alkaline sucrose density-gradient centrifugation showed a dose-dependent increase in DNA fragmentation only 12 and 24 h after treatment. PYRZ induced increasing accumulation of cells in late S and G2+M 6-24 h after treatment. This also contrasts with previous work that showed delayed cell cycle arrest induced by temozolomide in OVCAR-3 cells and in the murine leukaemia L1210 cells. Cell-killing kinetics by PYRZ showed a series of sigmoidal dose-response curves with 50-90% cell killing attained as early as 24 h after treatment in the 25-100 microM dose range. (IC50 clonogenic assay 18 microM). The results suggest that the mechanism of cell killing by PYRZ may be different from that of its parent drug temozolomide, and other alkyl-triazene-containing molecules of the same class.


Comparison with cisplatin demonstrated that intracellular platinum levels were marginally higher with JM216, but that platinum binding to DNA was similar for the two drugs in each of the cell lines. Each of the cell lines exhibited an ability to repair JM216 induced platinum/DNA lesions in the N-ras gene (gene-specific repair) at equitoxic concentrations of drug. However, this occurred to a greater extent in the two resistant cell lines such that by 24 h the CH1cisR and SKOV-3 had removed 72% and 67% respectively compared with approximately 32% for the CH1. Reduced gene-specific repair capacity in CH1 cells was also seen following incubation with 25 μM (or 5 μM – 2 × IC50) cisplatin, whereas the CH1cisR and SKOV-3 cell lines were repair proficient. JM216 induced apoptosis in the three cell lines following a 2h incubation with 2 × the IC50 of drug. Fluorescent microscopy of cells stained with propidium iodide showed that the detached cell population displayed typical apoptotic nuclei. Furthermore, field inversion gel electrophoresis demonstrated the presence of DNA fragments approximately 23–50 kb in size, indicative of apoptosis, in the detached cells. JM216 induced an S phase slow down in each of the three cell lines accompanied by a G2 block in the CH1 pair. Incubation with this concentration of JM216 also resulted in the induction of p53 in the CH1 and CH1cisR. These studies suggest that the relative sensitivity of the CH1 cell line to cisplatin and JM216 is at least partly attributable to a deficiency in gene-specific repair. The oral platinum drug, JM216, exerts its cytotoxic effects through the induction of apoptosis following a slow-down in S phase in both the sensitive and resistant lines. © 1999 Cancer Research CampaignJM216, an oral platinum drug entering into phase III clinical trial, exhibited comparable cytotoxicity to cisplatin in three human ovarian carcinoma cell lines: the sensitive (CH1), acquired resistant (CH1cisR) and intrinsically resistant (SKOV-3). Platinum accumulation and binding to DNA were similar in each of the three cell lines at equimolar doses, indicating that the resistant cell lines could tolerate higher intracellular platinum levels and platinum bound to DNA at IC


Immunostaining of c-Met, uPAR showed strongly positive in both subcutaneous xenografts and lung metastatic lesions; while positive in xenografts and negative in metastatic lesions for integrin α5, β1. E-cadherin and P53 was not expressed either in xenograft or in the metastatic lesions. PCR products of HBsAg and HBxAg were both positive. The cell line MHCC97 still retained some characteristic features of original tumour. Establishment of cell line MHCC97 should be beneficial to the studies of HCC metastatic mechanisms. © 1999 Cancer Research CampaignA new human hepatocellular carcinoma (HCC) cell line with a highly metastatic potential was established from subcutaneous xenograft of a metastatic model of human HCC in nude mice (LCI-D20) by means of alternating cell culture in vitro and growth in nude mice. The line, designated MHCC97, has been cultivated for 18 months and subcultured for more than 90 passages. The line was showed to be of human origin by karyotype analysis. The cells were either grown as compact colonies (in clusters) or as a monolayered sheet with about 31 h of population-doubling time, exhibited typical malignant epithelial in morphology and were positive for α-fetoprotein (AFP). Flow cytometric analysis of the cell DNA content showed an aneuploid pattern, and its index was 1.5 as compared to that of normal human peripheral blood lymphocytes. Karyotypic analyses of G- and C-banding techniques revealed that all cells presented chromosome abnormalities in number and structure. The number of cell line MHCC97 chromosome ranged from 59 to 65 with a modal number of 60 and 61. At least two common chromosome markers, i(1q) and der(4)t(4pter→q35::?), were present in all cells, and deletion of Y chromosome also occurred in all cells. The subcutaneous and intrahepatic xenografts were formed and metastatic lesions in lungs were found after the cells were inoculated into nude mice. The rate of metastasis to lungs was 100% using orthotopic inoculation. Reverse transcription polymerase chain reaction products revealed positive expressions of integrin α


Hoechst 33342 is a fluorescent dye used for cell selection from tumours based upon intratumour location. When the dye is administered i.v. to tumour-bearing animals, cellular fluorescence is directly related to the proximity of cells to blood vessels. The present study compared inherent Hoechst fluorescence between in vitro-stained EMT6/Ro (mouse mammary sarcoma) cells and host cells, to determine if these populations have different staining characteristics that may influence cell selection procedures. Tumour cell fluorescence exceeded host cell staining by 8-fold when pure cell populations  were compared, and 3-fold for tumour cell-enriched and host cell-enriched populations from solid tumours. Inherent uptake of HO 33342 appeared to be correlated with cell volume. These differences in inherent dye uptake between host and tumour cells were found to be minor in comparison to the fluorescence gradient between the 10% brightest and 10% dimmest (78-fold) cell populations from in vivo-stained tumours.


Uterine serous papillary adenocarcinoma (USPC) is a highly aggressive variant of endometrial cancer. Human immuno-conjugate molecule (hI-con1) is an antibody-like molecule targeted against tissue factor (TF), composed of two human Factor VII (fVII) as the targeting domain, fused to human immunoglobulin (Ig) G1 Fc as an effector domain. We evaluated hI-con1 potential activity against primary chemotherapy-resistant USPC cell lines expressing different levels of TF.51Cr assays were also conducted in the presence of low doses of IL-2 .A total of 16 formalin-fixed, paraffin-embedded USPC samples were evaluated by immunohistochemistry (IHC) for TF expression. Six primary USPC cell lines, half of which overexpress the epidermal growth factor type II (HER2/neu) receptor at 3+ levels, were assessed by flow cytometry and real-time PCR for TF expression. Sensitivity to hI-con1-dependent cell-mediated cytotoxicity (IDCC) was evaluated in 5-hour-chromium release assays. Finally, to investigate the effect of interleukin-2 (IL-2) on IDCC, 5-h P<0.001). Uterine serous papillary adenocarcinoma cell lines overexpressing TF, regardless of their high or low HER2/neu expression, were highly sensitive to IDCC , although negligible cytotoxicity against USPC was seen in the absence of hI-con1 or in the presence of Rituximab control antibody. The addition of low doses of IL-2 further increased the cytotoxic effect induced by hI-con1 against chemotherapy-resistant USPC.Cytoplasmic and/or membrane TF expression was observed in all 16 (100%) USPC samples tested by IHC, but not in normal endometrium. High expression of TF was found in 50% (three out of six) of the USPC cell lines tested by real-time PCR and flow cytometry when compared with normal endometrial cells (NECs; hI-con1 induces strong cytotoxicity against primary chemotherapy-resistant USPC cell lines overexpressing TF. The hI-con1 may represent a novel therapeutic agent for the treatment of patients harbouring advanced, recurrent and/or metastatic USPC refractory to standard treatment modalities. Endometrial cancer is the most common female genital tract malignancy in the United States, with an incidence of 40 100 new cases and 7470 deaths in the United States in 2009 . Type I in vivo responses to combined cisplatin-based chemotherapy in the order of 20% and of short duration  were used for antigen retrieval. The slides were then incubated overnight at 4°C with monoclonal anti-TF antibody . EnVisionTM system  was used for secondary detection and the reactions were visualised with diaminobenzidine. Appropriate positive and negative controls were used with each case. Both cytoplasmic and membranous immunoreactivity were considered positive. Immunostaining was assessed using a semi-quantitative scoring system, as follows: 0, negative (0–5% staining); 1+, weakly positive (10–20%); 2+, moderately positive (20–50%); 3+ and 4+, strongly positive , as previously described , stage II (2 patients), stage III (6 patients) and stage IV (2 patients) USPC were retrieved from the surgical pathology files at Yale University. Specimens were reviewed by a surgical pathologist (NB). The level of TF expression was then evaluated on the most representative block by standard immunohistochemical staining. For IHC, 4 c-erbB2 gene by fluorescence in situ hybridisation, expression levels of HER2/neu receptor by IHC and mRNA expression levels by quantitative real-time PCR (qRT–PCR) for these primary USPC cell lines have been recently reported  were established after sterile processing of the tumour samples from surgical biopsies as described previously . A total of 5 μl of reverse transcribed RNA samples  was amplified using the TaqMan Universal PCR Master Mix (Applied Biosystems) to produce PCR products specific for TF. The primers and probe for TF were obtained from Applied Biosystems (assay ID Hs01076032_m1). The comparative threshold cycle (CT) method (Applied Biosystems) was used to determine gene expression in each sample relative to the value observed in the lowest non-malignant endometrial epithelial-cell sample, using glyceraldehyde-3-phosphate dehydrogenase (assay ID Hs99999905_m1) RNA as internal control.RNA isolation from all the six primary USPC cell lines used in the cytotoxicity experiments were performed using TRIzol Reagent  according to the manufacturer's instructions. Quantitative PCR was performed using a 7500 Real-time PCR System with the manufacturer's recommended protocol  to evaluate expression of TF in all samples. Each reaction was run in duplicate. Briefly, 5 M HEPES, 150 mM NaCl, 5 mM CaCl2, 25 mM arginine and 0.01% Tween 80, pH 7.4 buffer, and was sterile filtered. It was provided in aliquots of 80 μl at a stock concentration of 350 μg ml−1, which were used for our flow cytometry and hI-con1-dependent cell-mediated cytotoxicity (IDCC) studies. Briefly, USPC cell lines and phytohemagglutinin (PHA)-stimulated control peripheral blood lymphocytes (PBLs) were stained with hI-con1 at a concentration of 30 μg ml−1 for 30 min on ice or with a commercially available fluorescein isothiocyanate-conjugated mouse anti-human TF Ig . After hI-con1 staining, cells were washed twice with the same buffer and secondary goat-anti-mouse antibody (IgG1-FITC (fluorescein isothiocyanate), catalogue no. F0767 Sigma Aldrich, St Louis, MO, USA) was added for further 30 min. Analysis was conducted with a FACSCalibur instrument using Cell Quest software .Clinical grade hI-con1 was produced for Iconic Therapeutics Inc.  by Laureate Pharma,  by cultivation of baby hamster kidney cells transfected with a vector containing the gene sequence originally described by 51Cr) release assay was performed to measure the cytotoxic reactivity of Ficoll–Hypaque-separated PBLs from several healthy donors in combination with hI-con1 against USPC target cell lines. The release of 51Cr from the target cells was measured, as described (μg ml−1) and different target/effector cell ratios. Controls included the incubation of target cells alone or with PBLs, or monoclonal antibody (mAb) separately. The chimeric anti-CD20 mAb Rituximab  was used as control in all bioassays. The hI-con1-dependent cell-mediated cytotoxicity was calculated as the percentage of killing of target cells observed with mAb plus effector cells as compared with 51Cr release from target cells incubated alone.A standard 5-h chromium (−1 in 96-well microtiter plates. Target cells were primary USPC cell lines exposed to hI-con1 (concentrations ranging from 1 to 80 μg ml−1), whereas controls included the incubation of target cells alone or with PBLs in the presence or absence of IL-2 or mAb, respectively. Rituximab was used as a control mAb. The hI-con1-dependent cell-mediated cytotoxicity was calculated as the percentage of killing of target cells observed with mAb plus effector PBLs, as compared with target cells incubated alone. Each experiment was performed with PBLs collected from multiple normal donors and results from a representative donor are presented.To investigate the effect of IL-2 on IDCC, effector PBLs were incubated for 5 h at 37°C, with a final concentration of IL-2  ranging from 50 to 100 IU ml2 transforming them to ΔCTs, and comparing the results via unequal variance t-test for the USPC vs NEC difference. Group means with 95% confidence limits  were calculated by computing them on the ΔCTs and then reverse transforming the results to obtain means  of relative copy numbers. Differences in TF expression by flow cytometry were analysed by the unpaired t-test. Kruskal–Wallis test and χ2-analysis was used to evaluate differences in hI-con1-induced cellular cytotoxicity levels in primary tumour cell lines. Statistical analysis was performed using SPSS version 15 . A P-value of <0.05 was considered statistically significant.For qRT–PCR data, the right-skewing was removed by taking copy-number ratios relative to the lowest expressing normal endometrial cell (NEC) sample (‘relative copy numbers’), logWe performed immunohistochemical analysis of TF protein expression on formalin-fixed tumour tissues from 16 paraffin-embedded uterine serous carcinoma specimens. As representatively shown in C-erbB2 gene by fluorescence in situ hybridisation, were tested for TF expression by qRT–PCR. P=0.01. The fold change in the mean relative copy numbers between high USPC TF expressers vs NECs was 8.7 (P=0.01). In contrast, low TF expression using qRT–PCR was detected in the remaining three USPC cell lines . High level of TF was detected in two out of three USPC cell lines harbouring the amplification of the c-erbB2 gene and in one out of three USPC cell lines showing low HER2/neu expression . Low TF expression was also observed in the multiple PHA-stimulated PBL lines used as further controls .Surface TF receptor expression was evaluated by fluorescence-activated cell sorting analysis in all six primary USPC cell lines using hI-con1 and an anti-human TF control mAb. As negative controls, several PHA-stimulated PBLs established from healthy donors or the same USPC patients, from whom the tumour cell lines had been established, were also studied. In agreement with the RT–PCR results, high reactivity against TF was found using flow cytometry in USPC-ARK-2, USPC-ARK-3 and USPC-ARK-6 cell lines stained with hI-con1 . In cont51Cr release assay. Uterine serous papillary adenocarcinoma primary cell lines were consistently found to be resistant to NK cell-mediated cytotoxicity when combined with PBLs at E : T ratios varying at 25 : 1–50 : 1 . Similarly, USPC cell lines incubated with rituximab (2.5 μg ml−1) control antibody showed no significant cytotoxicity (range from 0.1–8%). We then investigated the sensitivity of USPC cell lines expressing different levels of TF to heterologous PBLs in the presence of hI-con1 (30 μg ml−1). While negligible levels of cytotoxicity were detected against PHA-stimulated PBLs , tumour cell lines expressing high levels of TF , regardless of their high or low HER2/neu expression, were found to be highly sensitive to hI-con1-induced antibody-dependant cell death . In contrast, tumour cell lines expressing low levels of TF  were killed at significantly lower levels when compared with the high TF-expresser USPC cell lines (P<0.001). Nevertheless, in the latter experiments, low TF-expresser USPC cell lines incubated with hI-con1 were killed at significantly higher level when compared with the same tumours incubated with rituximab control mAb , or when compared with PHA-stimulated PBL control incubated with hI-con1 .All the six primary USPC cell lines available were tested for their sensitivity to NK cell cytotoxicity when challenged with heterologous PBLs, collected from several healthy donors, in a standard 5-h ll lines ; P<0.001μg ml−1) on IDCC against USPC cell lines, PBLs from healthy donors were incubated for 5 h in the presence of 50–100 IU ml−1 of IL-2. As shown in −1 of IL-2 to the effector PBLs at the start of the assay increased the cytotoxic activity against USPC cell lines overexpressing TF compared with the use of hI-con1 alone, (P< 0.01) whereas no significant increase in cytotoxicity was detected after 5-h IL-2 treatment in the absence of hI-con1 or in the presence of Rituximab control mAb (in vitro.To investigate the effect of low doses of IL-2 in combination with hI-con1 (30 1 alone, ; P< 0.01trol mAb . These rTissue factor is involved in pathological angiogenesis and is abnormally overexpressed in multiple human tumours and in tumour vascular endothelial cells but not in normal quiescent vascular endothelial cells . Althougin vitro activity of hI-con1, a previously characterized immunoconjugate molecule developed against TF (in vitro (In this study, we have evaluated TF expression using IHC in multiple formalin-fixed, paraffin-embedded USPC samples. In addition, we have studied TF expression at RNA and protein level in six primary USPC cell lines and subsequently tested the ainst TF , 2001, ain vitro . Howeverin vitro, suggesting that TF-mediated enhancement of tumour growth requires a factor present in vivo that is not present when cells are grown in vitro (in vivo leading to the activation of type-2 proteinase activated receptor-dependent signalling (in vivo.Several studies have shown that tumour cell TF has an important role in tumour metastatic process, possibly by induction of the coating of the tumour cell with fibrin that would trap the cells in the microvasculature, thereby aiding metastases (gnalling . These fet al (et al (51Cr release assays. Taken together, these in vitro results strongly suggest that TF may provide a novel target for USPC and its tumour vasculature, which should likely result in hI-con1-induced lysis of tumour cells as well as endothelial cells in vivo.The potential cytotoxic activity of hI-con1 against human melanoma and prostate tumour cells has previously been shown by Hu et al , 2001. Iin vitro as well as in vivo (in vivo (in vivo (in vitro experiments reveal a significant increase in hI-con1-induced cytotoxicity after the brief incubation of PBLs and tumour cells with IL-2 compared with the cytotoxicity induced by hI-con1 in the absence of IL-2. Interleukin-2 seems to, therefore, enhance the cytotoxic potential of the effector cells. The administration of low doses of IL-2 might therefore be a valid therapeutic option to increase IDCC in heavily pretreated endometrial cancer patients.hI-con1-mediated tumour cell lysis depends on the presence of effector cells , 2001, ain vivo use of hI-con1 in animals harbouring xenografts of human USPC has not been studied in our current report, previous studies have shown the safety and efficacy of using hI-con1 in vivo in mice carrying mouse and human tumours (in vivo and, therefore, may have great potential as a neovascular targeting agent, with broad applications for the immunotherapy of pathological angiogenesis-involved diseases in humans.Although the  tumours , 2001, hin vivo therapies (i.e., trastuzumab; In conclusion, expression of TF in chemotherapy-resistant USPC makes hI-con1 an attractive agent for immunotherapy of chemotherapy-resistant endometrial cancer. This holds particularly true for USPC patients, in whom other potentially effective targeted


Nuclear factor-κB (NF-κB) is constitutively activated in many cancers and plays a key role in promoting cell proliferation, survival, and invasion. Our understanding of NF-κB signaling in thyroid cancer, however, is limited. In this study, we have investigated the role of NF-κB signaling in thyroid cancer cell proliferation, invasion, and apoptosis using selective genetic inhibition of NF-κB in advanced thyroid cancer cell lines.Three pharmacologic inhibitors of NF-κB differentially inhibited growth in a panel of advanced thyroid cancer cell lines, suggesting that these NF-κB inhibitors may have off-target effects. We therefore used a selective genetic approach to inhibit NF-κB signaling by overexpression of a dominant-negative IκBα (mIκBα). These studies revealed decreased cell growth in only one of five thyroid cancer cell lines (8505C), which occurred through a block in the S-G2/M transition. Resistance to TNFα-induced apoptosis was observed in all cell lines, likely through an NF-κB-dependent mechanism. Inhibition of NF-κB by mIκBα sensitized a subset of cell lines to TNFα-induced apoptosis. Sensitive cell lines displayed sustained activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway, defining a potential mechanism of response. Finally, NF-κB inhibition by mIκBα expression differentially reduced thyroid cancer cell invasion in these thyroid cancer cell lines. Sensitive cell lines demonstrated approximately a two-fold decrease in invasion, which was associated with differential expression of MMP-13. MMP-9 was reduced by mIκBα expression in all cell lines tested.These data indicate that selective inhibition of NF-κB represents an attractive therapeutic target for the treatment of advanced thyroid. However, it is apparent that global regulation of thyroid cancer cell growth and invasion is not achieved by NF-κB signaling alone. Instead, our findings suggest that other important molecular processes play a critical role in defining the extent of NF-κB function within cancer cells. Thyroid cancer is the most common endocrine malignancy . FortunaThe nuclear factor-κB (NF-κB) family of transcription factors is comprised of RelA p65), RelB, c-REL, NF-κB1/p50, and NF-κB2/p52, each of which is characterized by a Rel homology domain, which facilitates DNA-binding, homo- or heterodimerization of NF-κB family members, and interaction with inhibitory IκB proteins. A role for NF-κB in oncogenic progression has been described in a number of lymphoid malignancies and carcinomas, including thyroid, ovarian, breast, and hepatocellular carcinomas 5, RelB, ,8. NF-κBWhile the two primary modes of NF-κB activation are similar in that they culminate in NF-κB-dependent gene regulation through nuclear translocation of NF-κB dimers, the pathways are distinguished by the differential requirement of the trimeric IκB kinase (IKK) complex, which is composed of two kinase subunits, IKKα and IKKβ, and a regulatory, scaffolding subunit IKKγ. The classical pathway of activation requires phosphorylation of IκB proteins by the trimeric IKK complex, resulting in proteasome-dependent degradation of the inhibitory proteins and nuclear translocation of the classical p50/p65 heterodimer. The alternative pathway involves cleavage of the NF-κB2 precursor protein into the functional p52 subunit, which may then complex with RelB. This pathway is dependent on phosphorylation of the NF-κB2 precursor by IKKα dimers .To date, several studies have employed the use of pharmacologic inhibitors of NF-κB to establish a role for NF-κB in thyroid cancer cell growth and invasion -17. HowePharmacologic inhibitors of NF-κB have been widely used to investigate the functional consequences of constitutive NF-κB activation in cancer. Many of these inhibitors prevent phosphorylation and degradation of IκBα by blocking IKK complex activity . We initA panel of papillary thyroid cancer (PTC)  and anaplastic thyroid cancer (ATC)  cell lines were used. These cell lines harbour different activating mutations in the MAPK pathway, including the HRAS G13R mutation (C643), the BRAF V600E mutation , and the RET/PTC1 rearrangement (TPC1) . TreatmeBased on the variable growth inhibition by different pharmacologic inhibitors of NF-κB, a selective genetic approach to inhibit NF-κB signaling was used. Specifically, expression of a dominant-negative IκBα (mIκBα) ,24, whicThe five thyroid cancer cell lines demonstrated different basal levels of NF-κB transcriptional activity as determined by an NF-κB-responsive luciferase reporter, with the BCPAP and 8505C cell lines exhibiting the highest levels of NF-κB activity Figure . Basal NWe next investigated the role of NF-κB in thyroid cancer cell proliferation and survival. Thyroid cancer cells were transduced with either Ad-mIκBα or Ad-GFP at an MOI of 50 or 200, and growth was assessed after 5 days by automated viable cell counting. Transduction with control Ad-GFP was performed to monitor transduction efficiency and control for the effects of adenoviral transduction on cell growth. Inhibition of NF-κB by mIκBα expression did not decrease thyroid cancer cell proliferation or survival in four of the five cell lines tested Figure , even unTo determine the mechanisms governing growth inhibition by mIκBα in the 8505C cell line, measures of apoptosis and cell cycle analysis were performed. Cleaved poly (ADP-ribose) polymerase (PARP), a measure of apoptosis, was undetectable by Western blot analysis, suggesting that NF-κB inhibition does not induce apoptosis (data not shown). However, flow cytometry revealed a significant increase in the number of cells in S-phase following mIκBα expression. This finding corresponded with a 28% decrease in the number of mIκBα-expressing cells in G2/M, indicating a block in the S-phase to G2/M transition Figure . WesternTNFα signaling is responsible for activation of numerous pro-apoptotic pathways, which can be opposed by pro-survival NF-κB signaling through activation of the IKK complex . Figure To investigate the mechanisms by which only a subset of cell lines are sensitized to the combined pro-apoptotic effects of TNFα treatment and NF-κB inhibition, we examined activation of the pro-apoptotic JNK/SAPK pathway. Activation of the JNK pathway was assessed by Western blot analysis with an antibody specific to the activated, phosphorylated (Thr183/Tyr185) forms of JNK1/2. Transient activation of JNK/SAPK pathway in control GFP-transduced cells was only observed in the TPC1 cell line Figure . InteresAn understanding of the role of NF-κB signaling in regulation of thyroid cancer cell invasion is particularly relevant given the nature of the disease and the mortality associated with locally invasive and metastatic tumors. Therefore, we assessed the role of NF-κB in thyroid cancer cell invasion using Matrigel-coated transwell assays. In these studies, cells  were transduced with either Ad-GFP or Ad-mIκBα and allowed to invade for 24 hours. Control-transduced C643 cells were the most invasive (~450 cells/field), while TPC1 cells were the least invasive (~150 cells/field). SW1736 and BCPAP cells were moderately invasive (~300 cells/field) (data not shown). Invasion by the SW1736 and TPC1 cell lines was significantly inhibited  by mIκBα expression, while BCPAP and C643 cells were resistant  -2, MMP-9, and MMP-13 Figure . Figure In this report, we have used a selective genetic inhibitor of NF-κB signaling to determine the effects of this pathway on proliferation, apoptosis, and invasion in a panel of ATC and PTC thyroid cancer cell lines. Our data indicate that NF-κB does not have one common role in the regulation of proliferation, apoptosis, or invasion in advanced thyroid cancer and that sensitivity to NF-κB inhibition does not correlate with baseline levels of NF-κB transcriptional activity. Instead, we have demonstrated that some cancer cells depend on NF-κB signaling for proliferation (8505C), while others require it for invasion and resistance to TNFα-induced apoptosis . Furthermore, some cell lines are not dependent on NF-κB signaling for these basic cancer properties .in vivo tumor formation, growth in soft-agar, and resistance to chemotherapy-induced apoptosis The authors declare that they have no competing interests.KTB designed and performed the experiments, interpreted data, assembled the figures, and drafted the manuscript. BRH conceived of the study and directed the research, with assistance from RES. RES and BRH critically reviewed the manuscript, which was approved by all authors.


In order to unravel possible mechanisms of clinical resistance to topoisomerase I inhibitors, we developed a topotecan-resistant human IGROV-1 ovarian cancer cell line, denoted IGROV(T100r), by stepwise increased exposure to topotecan (TPT). The IGROV(T100r) cell line was 29-fold resistant to TPT and strongly cross-resistant to SN-38 (51-fold). However, the IGROV(T100r) showed only threefold resistance to camptothecin (CPT). Remarkably, this cell line was 32-fold resistant to mitoxantrone, whereas no significant cross-resistance against other cytostatic drugs was observed. No differences in topoisomerase I protein levels and catalytic activity as well as topoisomerase I cleavable complex stabilization by CPT in the IGROV-1 and IGROV(T100r) cell lines were observed, indicating that resistance in the IGROV(T100r) cell line was not related to topoisomerase I-related changes. However, resistance in the resistant IGROV(T100r) cell line to TPT and SN-38 was accompanied by decreased accumulation of the drugs to approximately 15% and 36% of that obtained in IGROV-1 respectively. No reduced accumulation was observed for CPT. Notably, accumulation of TPT in the IGROV-1 cell line decreased under energy-deprived conditions, whereas the accumulation in the IGROV(T100r) cell line increased under these energy-deprived conditions. The efflux of TPT at 37 degrees C was very rapid in the IGROV-1 as well as the IGROV(T100r) cell line, resulting in 90% efflux within 20 min. Importantly, the efflux rates of TPT in the IGROV-1 and IGROV(T100r) cell lines were not significantly different and were shown to be independent on P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP). These results strongly suggest that the resistance of the IGROV(T100r) cell line to TPT and SN-38 is mainly caused by reduced accumulation. The reduced accumulation appears to be mediated by a novel mechanism, probably related to impaired energy-dependent uptake of these topoisomerase I drugs.


We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.


The survival of patients with locally advanced head and neck cancer is still poor, with 5-year survival rates of 24–35%. The identification of prognostic and predictive markers at the molecular and cellular level could make it possible to find new therapeutic targets and provide "taylor made" treatments. Established cell lines of human squamous cell carcinoma (HNSCC) are valuable models for identifying such markers.The aim of this study was to establish and characterize a series of cell lines and to compare the cisplatin sensitivity and 18F fluoro-2 deoxy 2 glucose (18F-FDG) uptake of these cell lines with other cellular characteristics, such as proliferation parameters and TP53 and CCND1 status.Explant cultures of fresh tumour tissue were cultivated, and six new permanent cell lines were established from 18 HNSCC cases. Successfully grown cell lines were analysed regarding clinical parameters, histological grade, karyotype, DNA ploidy, and index and S-phase fraction (Spf). The cell lines were further characterized with regard to their uptake of 18F-FDG, their sensitivity to cisplatin, as measured by a viability test , and their TP53 and CCND1 status, by fluorescence in situ hybridization (FISH), polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) with DNA sequencing and, for cyclin D1, by immunohistochemistry.Patients with tumours that could be cultured in vitro had shorter disease-free periods and overall survival time than those whose tumours did not grow in vitro, when analysed with the Kaplan-Meier method and the log-rank test. Their tumours also showed more complex karyotypes than tumours from which cell lines could not be established. No correlation was found between TP53 or CCND1 status and 18F-FDG uptake or cisplatin sensitivity. However, there was an inverse correlation between tumour cell doubling time and 18F-FDG uptake.In vitro growth of HNSCC cells seem to be an independent prognostic factor, with cell lines being more readily established from aggressive tumours, a phenomenon more dependent on the molecular genetic characteristics of the tumour cells than on tumour location or TNM status. In 2006, 101,600 new cases and 42,400 deaths resulting from oropharyngeal cancer were registered in Europe . There iCell lines of human squamous cell carcinoma are valuable models for identifying such markers, and for studies of tumour biology. In this study, explant cultures of fresh tumour tissue were cultivated and six new permanent cell lines were established from 18 patients with head and neck squamous cell carcinoma (HNSCC). The cell lines established in this study were used to test for cisplatin sensitivity, 18F-FDG uptake, as a measure of metabolic activity, and various other tumour characteristics.Fresh tumour samples were collected during 1995–1999 from 18 patients with HNSCC. The patients participated voluntary and with informed consent. Seventeen of the 18 patients with HNSCC were previously untreated and one patient had a residual tumour after radiotherapy. Eight tumours were located in the oral cavity, four in the larynx, two in the nasopharynx, and one each in the oropharynx, hypopharynx and in the maxillary sinus. One was an untreated lymph node metastasis of unknown primary origin. Table Fresh tumour tissue samples obtained during surgery were immersed immediately in buffered balanced saline. The tissues were washed several times, trimmed and minced into 1 to 2 mm pieces, which were placed in T25 tissue culture flasks with DMEM supplemented with 2 mM L-glutamine and 10% foetal bovine serum (FBS). The flasks were incubated at 37°C in an atmosphere containing 5% carbon dioxide. Primary tissue culture flasks were observed daily. To reduce the fibroblast growth, DMEM D-valine was added instead of L-valine. Fibroblasts were also removed mechanically and by brief exposure to trypsin (0.1%) and EDTA (0.02%). Tumour cells were subcultured when the flasks were 50% confluent. After three or four passages the cells were stored in liquid nitrogen. Low passage numbers (<30) were used for this study.The cell lines were denoted LU-HNSCC 3 to 8. All cell lines had an epitheloid appearance, and grew in a typical cobblestone pattern indicating squamous cell carcinoma origin. The cells differed in size and grew in colonies with cell-to-cell contact into confluence. All cell lines, besides number 6, grew as monolayer cultures and were easy to detach using trypsin; cells from LU-HNSCC 6 were more difficult to detach and to grew in multiple layers.in vitro tumour cell growth rate, 15,000–100,000 cells were plated in Petri dishes, and the number of cells was counted every second day in a Bürker chamber. The growth rate for each cell line was determined at least twice and the results were found to be reproducible. The mean values of 2–5 samples were estimated. The doubling times were derived from the exponential growth phase, and are given in Table To determine the in vitro, a cellsolution containing the same cell amount from each cell line were injected subcutaneously into the lateral thoracic wall of nude mice. Tumour formation was observed for all cultured cell lines. The purpose of this experiment was to confirm the malignant characteristics of the cultured cell lines and to exclude a fibroblast cell population. The tumour formations in nude mice were no further examined in this experiment. The study was approved by the Regional Ethics Board of Southern Sweden Committe.To verify the malignancy of the established cell lines Frozen samples from 16 biopsies from primary tumours were analysed, and two samples from formalin-fixed and paraffin-embedded specimens were also analysed. Flow cytometry DNA analysis was performed as previously described . BrieflyThe separated cells were then treated with ribonuclease , incubated with pepsin , and stained with propidium iodide . Human lymphocytes were processed in parallel with the tumour samples and used as an external diploid control for the fresh samples. Flow cytometric DNA analysis was performed in a FACS Caliber . Up to 20,000 nuclei were analysed from each sample. The DNA histograms obtained were automatically processed using Modfit LT 3.1™ software. The DNA index (DI) was calculated as the ratio of the modal channel values of the G0 and G1 peaks. By definition, the tumours manifesting a single DNA population were classified as diploid (i.e. DI = 1.00), and tumours manifesting two or more populations as non-diploid. The S-phase fraction (Spf) was estimated assuming that the S-phase compartment constituted a rectangular distribution between the modal values of the G0/G1 and G2 peaks.Fresh samples from all but one of the 18 primary tumours previously had been subjected to short-term culturing and G-banding analysis . All sixCCND1) expression. A commercial monoclonal antibody  was used at a dilution of 1:20. A specimen known to be strongly positive, previously collected from a patient, was used as a positive control. The IHC results were scored as follows: A-negative; B 1–5% of the tumour cells positive; C 6–50% positive; D >50% positive. The negative controls were tested without primary antibodies.Immunohistochemical (IHC) analysis was performed on paraffin-embedded specimens to detect cyclin D1 (in situ hybridization (FISH) was performed as previously described [Fluorescence escribed , with miTP53, as previously described [TP53 gene in seven or eight different fragments. The PCR products were screened for mutations using SSCP. Samples showing altered mobility shift in SSCP were further analysed with direct DNA sequencing to determine the exact location and type of mutation.Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis and DNA sequencing were used to study the occurrence of mutations in exons 4–11 of escribed . Briefly2 atmosphere at 37°C. On day two, cisplatin  was added in serum-free medium, and the cells were incubated for 1 h at concentrations ranging from 0 to 100 μM. Thereafter, the drug-containing medium was removed, and cells were allowed to grow in drug-free medium for 5 days. On day 7, the cell viability was estimated by the crystal violetThe cell lines LU-HNSCC 3–8 were harvested by trypsinization, counted and seeded  in 24-well plates, and allowed to grow for two days as monolayer cultures in DMEM medium , supplemented with 10% FBS and antibiotics , under a 5% COassay, as described previously . Briefly2 atmosphere at 37°C. On day three, 2 ml 18F-FDG solution (0.62–1.33 MBq/ml) was added. After an hour the solution was removed by aspiration. The Petri dishes with cells were rinsed three times with PBS. The cells were then harvested from the Petri dishes by trypsinization and neutralized with 4 ml medium, and collected as samples for 18F-FDG determination together with the discarded 18F-FDG solution.The established cell lines LU-HNSCC 3–8 were harvested by trypsinization, counted and seeded  on day 0. The cells were allowed to grow for two days as monolayer cultures in DMEM medium supplemented with 10% heat-inactivated FBS containing an antibiotic (GIBCO)(100 U/ml streptomycin sulphate), under a 5% COThe 18F-FDG uptake in the cells and in the washing fractions was estimated using a calibrated 3 x 3 inch NaI(TI) well counter (in house)  and all 18F-FDG values were normalized for time. Electronic cell counting was performed using a NucleoCounter™  with the NucleoView™ software. The total cell content and number of viable cells were calculated per ml and correlated to the 18F-FDG uptake corrected for decay. This experiment was repeated in a second series.Descriptive statistics, Fisher's exact test, and the chi-squared test for trend, were used to evaluate differences in clinical parameters between patients whose tumours could be cultured, and those that could not. Disease-free periods and overall survival time in these groups were examined using Kaplan-Meier graphs and log-rank tests . The degree of linear relationship between pairs of variables measured on a continuous scale was summarized using correlation (r) and a test for zero slope in a corresponding linear regression model. Kruskal-Wallis' test was used to test the null hypothesis of equal cisplatin sensitivity for the cell lines. For comparison of 18F-FDG uptake between the cell lines, the following multiple linear regression model was used:where the dependent variable 18F-FDG is 18F-FDG uptake and the independent variables are:V = Number of viable cellsfive dummy variables contrasting cell lines 2–6 to cell line 1:j = 1 if cell line = j, j = 2–6Ij = 0 otherwiseIand five interaction parameters (products):jV = V if cell line = j, j = 2–6IjV = 0 otherwiseIThis linear model has 12 parameters with the following interpretation:1: Intercept for the reference cell line (1)c1: Slope for the reference cell line (1)bj: Intercept difference between cell line j and the reference cell line, j = 2–6cj: Slope difference between cell line j and the reference cell line, j = 2–6bIn this modelling framework, an F-test was used to test the null hypothesis of equal 18F-FDG uptake for the cell lines at a fixed number of viable cells. The packages SPSS 14.0  and Stata 10.0  were used for statistical analysis.in vitro were established from 18 HNSCCs, which constitutes an overall success rate of 33%.Six new permanent squamous cell carcinoma lines in vitro, calculated from date of diagnosis, is shown in Figure in vitro (p = 0.056). No differences were observed in the two groups regarding tumour site, TNM status, stage, grade, ploidity or DNA indices (data not shown).The overall survival of the patients as a function of the propensity of their tumours to grow in vitro (seven cases), or showed an abnormal karyotype with simple changes (one case). There was no aberration in common among the tumours that yielded viable cell lines, and it was noted that none of them displayed homogeneous staining regions. Only minor changes were noted when comparing the karyotypes obtained after short-term culturing of primary tumours and in the corresponding cell lines (data not shown).Five of the six tumours that grew as cell lines had complex karyotypes, defined here as at least three different aberrations, when examined after short-term culturing. In contrast, only three of the 11 tumours from which no cell line could be established, and from which material had been sent for short-term culturing, had complex karyotypes. The remaining eight cases either failed to grow TP53 mutations, two in exon 7 and one in exon 5  adjusted for time, was strongly correlated with the number of viable cells present, as illustrated in Figure TP53 and CCND1 status or DNA ploidity.No correlation was observed between 18F-FDG uptake or gene expression and cell characteristics, such as We found an inverse correlation (r = -0.82) between cell doubling time (DT) and 18F-FDG uptake; the shorter the doubling time, the higher the 18F-FDG uptake .This inverse relationship was even stronger if the cell line LU-HNSCC 3 with no observations above 600,000 viable cells was omitted  or if the cell line LU-HNSCC 7 with no observations below 700,000 viable cells was omitted and the 18F-FDG uptake was predicted for 500,000 viable cells . The experiment was repeated with similar results.In brief, the correlations between 18F-FDG uptake and number of viable cells varied from 0.81 to 0.98 and the predicted 18F-FDG uptake at 1,000,000 viable cells varied significantly between the cell lines also in the second experiment (p < 0.0001). Also the negative correlation between 18F-FDG uptake and DT was reproduced in the second series . By combining the data from the two experiments, the p-value for the inverse correlation between 18F-FDG uptake and DT dropped to 0.004.50 for the different cell lines varied between 6 and 29 μM. The cisplatin sensitivity did not show any relationship with TP53 mutations, CCND1 amplification or overexpression, or tumour doubling time.The cisplatin sensitivity of the different cell lines is illustrated in Figure in vitro were more aggressive in their biological behaviour, with shorter patient disease-free periods and overall survival time, compared with those that did not grow in vitro. No correlation was found between ability to grow and clinical parameters such as TNM status, or tumour grade or site. In agreement with our results, Kim et al. established nine new permanent SCC cell lines, but their propensity to grow in vitro did not appear to be related to tumour site or grade [in vitro growth, in the present study seems to be an independent prognostic factor, in concordance with other authors [in vitro could be dependent on their genetic alterations. Support for this hypothesis comes from the finding that all the culturable cell lines, except for one in this study were seen to have complex karyotypes after short-term culturing. Additionally, complex karyotypes have been shown to be associated with poor prognosis [In accordance with other studies -12, we for grade . Thus, i authors -12 altho authors . The caprognosis .We also investigated possible differences between the two tumour groups regarding DNA content, index and S-phase fraction, but no statistically significant differences were found. These cellular characteristics have been widely investigated previously, since they are assumed to reflect the loss of normal cell proliferation control and the underlying genetic abnormalities. The prognostic value of DNA content is, however, more uncertain. While some studies have found a correlation with poor outcome and higher recurrence rate in aneuploid tumours ,17, the in vitro study using HNSCC lines, Minn et al.[CCND1 status and cell proliferation suggests that deregulated CCND1 could be a factor affecting 18F-FDG uptake. However, we found no correlation between cyclin D1 expression or CCND1 amplification and 18F-FDG uptake. Similar results, i.e. no correlation between CCND1 status and 18F-FDG uptake, have been reported in a clinical trial on lung cancer patients [The extent of 18F-FDG uptake has been suggested to provide a measure of tumour aggressiveness, and thus to be associated with poor prognosis in many tumour types ,20, inclnn et al. found a nn et al. found a nn et al., but no patients .TP53 mutations to be accompanied by increased glycolysis, which could be the result of reduced synthesis of proteins in the COX ∏ subunit or increased transcription of HK-2 [TP53 and increased 18F-FDG uptake.Some studies have found  of HK-2 ,31. We fCCND1 or TP53 status in HNSCC, although similar studies have been carried out on other tumours [TP53 overexpression and higher 18F-FDG uptake [TP53 and CCND1 status could be that the tumour cells in vitro have an excess of nutrients, and that they must be placed under stress to reveal a correlation. Therefore, the next experimental step will be to treat the cell lines with cisplatin, perhaps providing more insight into the complex and still enigmatic mechanisms behind the intracellular uptake and accumulation of 18F-FDG.To best of our knowledge, there are no previous reports on possible correlations between 18F-FDG uptake and  tumours ,30, demoThe six cell lines were also tested regarding cisplatin sensitivity. Cisplatin-induced cell death was measured using crystal violet staining, a method evaluated before . A statiin vitro cell line from a tumour to be an independent negative prognostic marker. Furthermore, we found it feasible to study metabolic activity with 18F-FDG uptake, and other tumour biologic characteristics, including the chemosensitivity of the cell lines. Despite the relatively small number of tumour lines, we found a statistically significant correlation between a shorter tumour doubling time and higher 18F-FDG uptake. However, no significant difference was seen between 18F-FDG uptake and other proliferation parameters, including TP53 and CCND1 status. Although, the complex metabolic interactions between host and tumour, which create the microenvironment in vivo, will not be reproducible in cultured cell lines the growing knowledge of tumour cell characteristics will provide more understanding of the clinical behaviour of HNSCC tumours and of prognosis and therapy results for HNSCC patients.The results in this present study support the value of tumour cell cultures as a model for prognostic and predictive studies. We found the successful establishment of an The authors declare that they have no competing interests.EH participated in the experiments in vitro, interpretation of the study and drafted the manuscript. EK conceived of the study, and participated in its design and interpretation. BB performed the flowcytometry analysis and the interpretation. PB performed the statistically analyses and interpretation. AB analysed the PCR-SSCP and DNA sequencing and interpretation. EB participated in the design of the study and revising the manuscript. FM evaluated and analysed the cytogenetic results. TO performed the FDG uptake measurements and interpretation. KR performed the FISH method and evaluation. JW participated in its design and coordination. PW conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Neoplastic events are marked by uncontrolled cell proliferation. One major focus of cancer research has been to identify treatments that reduce or inhibit cell growth. Over the years, various compounds, both naturally occurring and chemically synthesized, have been used to inhibit neoplastic cell proliferation. Two such oncostatic agents, melatonin and retinoic acid, have been shown to suppress the growth of hormone-responsive breast cancer. Currently, separate clinical protocols exist for the administration of retinoids and melatonin as adjuvant therapies for cancer. Using the oestrogen receptor (ER)-positive MCF-7 human breast tumour cell line, our laboratory has studied the effects of a sequential treatment regimen of melatonin followed by all-trans retinoic acid (atRA) on breast tumour cell proliferation in vitro. Incubation of hormonally responsive MCF-7 and T47D cells with melatonin (10(-9) M) followed 24 h later by atRA (10(-9) M) resulted in the complete cessation of cell growth as well as a reduction in the number of cells to below the initial plating density. This cytocidal effect is in contrast to the growth-suppressive effects seen with either hormone alone. This regimen of melatonin followed by atRA induced cytocidal effects on MCF-7 cells by activating pathways leading to apoptosis (programmed cell death) as evidenced by decreased ER and Bcl-2 and increased Bax and transforming growth factor beta 1 (TGF-beta1) expression. Apoptosis was reflected morphologically by an increase in the number of lysosomal bodies and perinuclear chromatin condensation, cytoplasmic blebbing and the presence of apoptotic bodies. The apoptotic effect of this sequential treatment with melatonin and atRA appears to be both cell and regimen specific as (a) ER-negative MDA-MB-231 and BT-20 breast tumour cells were unaffected, and (b) the simultaneous administration of melatonin and atRA was not associated with apoptosis in any of the breast cancer cell lines studied. Taken together, the results suggest that use of an appropriate regimen of melatonin and atRA should be considered for preclinical and clinical evaluation against ER-positive human breast cancer.


The syndecans, a family of cell-surface heparan sulphate proteoglycans, have been proposed to mediate cellular interactions with extracellular effector molecules, such as growth factors and components of the extracellular matrix, during critical phases of development. Transcripts of all four syndecans are expressed at varying levels in the developing rat intestine and in a series of immature rat intestinal epithelial cell lines. In addition, we report the novel finding that, in the intestinal epithelial cell lines, expression of syndecan-1 transcript is up-regulated by transformation with activated H-ras. This is in contrast to other cell lines in which ras transformation is associated with a decrease in syndecan-1 levels. The observed increase in the syndecan-1 occurs as a result of increased transcription and can be correlated with the degree of transformation of the IEC-18 cells. Transformation is also associated with a decrease in apparent molecular weight and increased shedding of the proteoglycan into the culture medium. Increased shedding of syndecan-1 into the culture medium after transformation with H-ras may contribute to the disruption of proteoglycan interactions with the extracellular matrix, leading to alterations in cell adhesion and organization.


It is suggested that the observed variations in uptake of label with changes in pH, cell concentration and the inorganic phosphate concentration of the medium indicate that the “priming” stage is critically dependent on the conditions of the experiment.In a previous paper, the exact conditions under which the radioactive drug 2-methyl-6,7-ditritio-1,4-naphthaquinol bis disodium phosphate could be selectively incorporated into HEp/2 cells were reported. This work has now been extended and suggests that the selective property associated with two human tumour cell lines established in culture, HEp/2 and HeLa, and two forms of mouse ascites tumour cells propagated Work with the non-radioactive analogue, Synkavit, indicates that under conditions where the drug is incorporated selectively into cells, incubations in excess of 20 minutes cause a large percentage of the population to lose its reproductive integrity.