A tumour-associated antigen (TAA.62) with an apparent mol. wt. of 62 kd, identified by a human monoclonal antibody , was found to be expressed at elevated levels in the cytoplasmic compartment of malignant as compared with normal mammary epithelial cells in both tissues and cultured cells. Increased levels of cytoplasmic expression of the antigen were also observed in malignant cells of cervix, colon, kidney, lung, and stomach. The patterns of expression of TAA.62 in cultured cells mirrored those of tissues and the antigen was expressed at elevated levels in the established breast cancer lines or oncogenically transformed mammary carcinoma cell line (tumourigenic) compared with the immortalised mammary epithelial cell line (non-tumourigenic). Aliquots of TAA.62 were purified to homogeneity from the conditioned-medium of malignant and immortalised breast cells by immunoaffinity chromatography using immobilised anti-TAA.62 antibody, and gel filtration. Both preparations of TAA.62 yielded a single band with an apparent molecular weight of 62 kd under reducing condition on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and both were identical in terms of size and immunoreactivity to anti-TAA.62 antibody. However, TAA.62(T) isolated from tumourigenic cell lines itself interacted with a cell surface molecule having an apparent molecular weight of 160 kd on both the malignant and immortalised cells: TAA.62(I) isolated from immortalized cell lines, showed no comparable interaction. Scatchard analysis of the concentration-dependent binding of TAA.62(T) to 160 kd-receptor molecule revealed a 2.6 x 10(4) binding sites per cell. The association constant of such binding was determined to be approximately 16.6 nM. Finally, addition of anti-TAA.62 antibody to culture medium resulted in the inhibition of proliferation of the malignant cells, but showed no effect on the normal cells. The results suggest that TAA.62 may interact as a ligand with its 160 kd cell surface receptor with a possible growth related function.


Three human lung squamous carcinoma cell lines  demonstrate features of squamous differentiation including involucrin synthesis and competence to form cornified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation.


Cytokinetic parameters of various types of lung cancer were determined in bronchoscopy specimens after in vivo labelling with the thymidine analogue bromodeoxyuridine (BrdU). The S-phase fraction and BrdU labelling index were measured flow cytometrically, allowing calculation of the S-phase transit time and potential tumour doubling time. The methodology used was found to be feasible for obtaining cytokinetic data from 76% of the bronchial biopsy samples. Despite the difference in clinical behaviour and growth pattern between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), no significant differences were observed between the mean values of the cytokinetic parameters of SCLC and NSCLC. The estimated cell loss factor was higher in NSCLC than in SCLC. It appears that the growth of a tumour, as clinically observed, is to a considerable extent influenced by cell loss. In accord with this assumption is the fact that we have observed non-BrdU labelled S-phase cells, both in tumour biopsies and in apparently normal tissue. The presence of these so-called unlabelled S-phase cells in relation to cell loss is discussed.


We investigated the interaction between human lung cancer cells, laminin, and several differentiating agents. When grown on laminin coated substrate eight out of 11 small cell lung cancer (SCLC) cell lines exhibited attachment to laminin and three had extensive outgrowth of long neurite-like processes. Of seven non-small cell lung cancer cell lines, selected for their in vitro anchorage-independent growth, attachment was observed in only three cell lines, and process formation was far less extensive than in SCLC cell lines. Among several differentiating agents, only dcAMP, which alone induced attachment and some process formation, increased laminin-mediated attachment and process formation of two SCLC cell lines, NCI-N417 a variant cell line, and NCI-H345, a classic cell line. The expression of several neuroendocrine and neuronal markers was investigated in these two SCLC cell lines. The expression of the light subunit of neurofilaments increased in NCI-N417 within 3 to 4 days of seeding, while NCI-H345 exhibited approximately 5 fold increase in expression of the GRP gene and a 3 fold increase expression of the beta-actin gene. The expression of a number of other neuroendocrine and neuronal markers did not change following growth on laminin. The doubling times remained unchanged independent of the presence of and attachment to laminin while topoisomerase II gene expression levels in NCI-N417 cells decreased approximately 5 fold when cells were growing on laminin.


Reference cultures derived from a transplantable rat mesothelioma were obtained by cloning cells three times in soft agar. Each line, designated "CARM-Lines", was selected on the basis of their epithelial or fibroblastic phenotype, and their uniform morphology. Three epithelial lines were used for more detailed in vitro studies comparing morphological and biological criteria at early and late passages. All three lines exhibited both epithelial and fibroblastic elements after 10-14 passages in vitro, demonstrating that the dimorphic histology of these tumours could be derived from a single aberrant cell. Morphology and growth characteristics of these cells were density-dependent. Anchorage dependent and independent clonogenic assays did not correlate. Anchorage dependent colony formation was the only parameter which differed markedly from the original parent line in the assays described. In vivo evidence of chondrogenesis and attempted ossification support the concept of a multipotential cell contributing to the diverse primary tumour morphology by cellular modulation or differentiation.


A panel of six 'wild type' and three VP-16 resistant small cell lung cancer (SCLC) cell lines is used to evaluate to what extent in vitro sensitivity testing using a clonogenic assay can contribute to combine cytotoxic drugs to regimens with improved efficacy against SCLC. The resistant lines include (a) H69/DAU4, which is classical multidrug resistant (MDR) with a P-glycoprotein efflux pump (b) NYH/VM, which exhibits an altered topoisomerase II (topo II) activity and (c) H69/VP, which is cross-resistant to vincristine, exhibits a reduced drug accumulation as H69/DAU4 but is without P-glycoprotein. 19 anticancer agents were compared in the panel. The MDR lines demonstrated, as expected, cross-resistance to all topo II drugs, but also different patterns of collateral sensitivity to BCNU, cisplatin, ara-C, hydroxyurea, and to the topo I inhibitor camptothecin. The complete panel of nine cell lines clearly demonstrated diverse sensitivity patterns to drugs with different modes of action. Correlation analysis showed high correlation coefficients (CC) among drug analogues , and between drugs with similar mechanisms of action , whereas different drug classes demonstrated low or even negative CC (e.g. BCNU/VP-16 -0.21). When the CC of the 19 drug patterns to VP-16 were plotted against the CC to BCNU, clustering was observed between drugs acting on microtubules, on topo II, alkylating agents, and antimetabolites. In this plot, camptothecin and ara-C patterns were promising by virtue of their lack of cross-resistance to alkylating agents and topo II drugs. Thus, the differential cytotoxicity patterns on this panel of cells can (1) give information about drug mechanism of action, (2) enable the selection and combination of non-cross-resistant drugs, and (3) show where new drugs 'fit in' among established agents.


Two human cell lines (UACC-812 and 893), both containing significant amplification of the HER-2/neu gene, were established from biopsy specimens of breast carcinomas. One patient had Stage II breast carcinoma; the other had metastatic disease. Characterisation of these lines has revealed that both are highly aneuploid containing multiple clonal chromosome alterations, have doubling times near 100 h, and are oestrogen and progesterone receptor negative. Electron microscopy demonstrates that both lines contain numerous microvilli, cytoplasmic filaments, multivesicular bodies, and desmosomes. Immunoblot analysis for P-glycoprotein using the monoclonal antibody C219 was negative for both patient cell lines. These relatively rare cell lines may represent a useful model to investigate human breast carcinomas.


Controversy exists as to whether interferons usefully influence the growth of epithelial carcinomas. A small cell lung carcinoma (SCLC) cell line, WX322, has been derived which is greater than 1000-fold more sensitive to alpha-interferon (IFN) when grown in agar than other reported SCLC cell lines. The WX322 line has been characterised to prove its epithelial origin and its chemosensitivity compared with that of the NCI-H69 small cell line. The WX322 cell line expresses neuroendocrine and epithelial markers and possesses a morphology consistent with SCLC origin. A concentration of 5 IU ml-1 of IFN produced 50% inhibition of colony formation in agar in the WX322 line, whereas a concentration of greater than 10(5) IU ml-1 was required to produce a comparable effect with the NCI-H69 cell line. In contrast, WX322, possessed similar sensitivity to NCI-H69 cells when exposed to a range of cytotoxic agents. Analysis of the cell cycle indicated that IFN increased the percentage of cells in the G0/G1 phase for the WX322 cell line but increased the percentage in S phase for the NCI-H69 line. Growth of the xenograft, from which the cell line was derived, was also inhibited by IFN at doses greater than 10(5) IU/mouse/day. The WX322 cell line whether grown in agar or as a xenograft shows an unusually high sensitivity to IFN and provides an interesting model for studying mechanisms of IFN cytotoxicity to epithelial cells.


The present study describes a comparison of two potent immunotoxins which utilise an identical targeting component, a monoclonal antibody (SEN7) specific for small cell lung cancer (SCLC), conjugated to two different effector components, blocked ricin (bR) and Pseudomonas exotoxin A (PE). SEN7 recognises a novel epitope on the neural cell adhesion molecule (NCAM) which is highly associated with SCLC. The immunotoxins SEN7-PE and SEN7-bR were selectively and potently active against a number of SCLC cell lines, of both classic and variant morphologies, inhibiting the incorporation of [3H]leucine with IC50 values ranging between 22 pM and 85 pM and between 7 pM and 62 pM for SEN7-PE and SEN7-bR respectively. Intoxication by both immunotoxins proceeded rapidly following short 2 h lag phases; the initial rates of protein synthesis inhibition occurred with t50 values of 6.5 h for SEN7-PE and 5.5 h for SEN7-bR. Monensin drastically enhanced the cytotoxic activity of the weakly active SEN7-ricin A-chain by 2,100-fold and of SEN7-bR by 80-fold but had no effect on SEN7-PE. In limiting dilution assays, four and more than 4.5 logs of clonogenic SW2 tumour cells were selectively eliminated from the cultures during continuous exposure to the immunotoxins SEN7-PE and SEN7-bR respectively, while antigen-negative cells required up to 1,000-fold more drug for a similar cell kill. SW2 cells surviving SEN7-bR treatment in the cultures did not express NCAM and consequently were not selectively killed by SEN7 immunotoxins. SW2 cells surviving continuous exposure to SEN7-PE showed no alteration in NCAM expression but were more resistant to intoxication mediated by PE. These cells were still sensitive to SEN7-bR.


A new cell line, XH1, has been derived from an invasive focally keratinising adenosquamous carcinoma of the cervix in a 32 year old patient. It has been maintained in long term monolayer culture for 26 months, and passaged over 100 times (much greater than 300 population doublings). It is aneuploid with a mean chromosome number of 78. Examination using two minisatellite hypervariable DNA probes has shown it to be different from other cell lines maintained in this laboratory and from HeLa. Two sublines, XH1a and XH1b, show marked differences in monolayer culture, growth in soft agar, and xenograft formation. XH1 and XH1a cells readily form subcutaneous xenografts, and lung colonies can be established by their intravenous injection. Subcutaneous injection of XH1b cells results in rapid cell growth for a few days after which the tumour undergoes degeneration and then regresses completely. The XH1 karyotype has many rearranged chromosomes. Parental XH1 cells and both sublines show integration of HPV16 into the genome.


We have compared the effects of alpha and recombinant gamma interferons (IFNs) on the growth of human lung cancer cell lines in vitro. There was a diversity of response amongst the lines studied, the most sensitive being COR-L23 (a large cell anaplastic carcinoma line) and POC . In these two lines, IFN-gamma was found to be more potent than IFN-alpha. During cell growth of line POC in the presence of IFN-gamma no significant shift in cell cycle distribution occurred. When lines COR-L23 and POC were grown as xenograft tumours in nude mice, daily injection of 4 X 10(5) units per mouse per day of IFN-gamma produced no discernible retardation of tumour growth.


The novel imidazoisoquinoline SDZ 62-434, originally identified as a platelet-activating factor (PAF) antagonist, has antiproliferative activity in a range of cell lines from human solid and haematological malignancies. Using an MTT cytotoxicity assay, IC50 values of 5 microM - 111 microM were observed following a 24 h exposure. Similar results were obtained using a clonogenic assay. The HT29 colon adenocarcinoma was particularly sensitive while the MCF-7 breast carcinoma was the most resistant in our panel. Only a 2-3 fold cross-resistance was seen in the doxorubicin and cisplatin resistant variants of the A2780 ovarian carcinoma; the drug did not modulate sensitivity to doxorubicin in either parent or resistant lines. No cross-resistance to SDZ 62-434 was seen in a doxorubicin-resistant MCF-7 variant. Cytotoxicity was not due to non-specific membrane lysis. The potent PAF antagonist WEB 2086 did not modulate SDZ 62-434 cytotoxicity, indicating no role for PAF receptors. Precursor incorporation studies in A2780 cells showed that DNA synthesis was inhibited more effectively than protein synthesis while RNA synthesis was unaffected. SDZ 62-434 inhibited both bombesin and platelet-derived growth factor-induced DNA synthesis in quiescent Swiss 3T3 cells. This suggests a possible role for SDZ 62-434 as an inhibitor of signal transduction in cancer cells.


The production of insulin-like growth factor I (IGF-I) and IGF-I binding proteins (BPs) by human lung tumour cell lines in vitro has been examined and the levels of these substances in the serum of lung cancer patients investigated. While small cell lung cancer (SCLC) cell lines secreted both IGF-I and BPs, non-small cell lung cancer (NSCLC) cell lines secreted BPs only. No evidence of increased serum IGF-I levels was obtained in a cohort of 52 lung cancer patients having SCLC and NSCLC histologies. In contrast, serum levels of low molecular weight BPs were markedly elevated in the majority of lung cancer patients.


We have investigated the efficacy of a F(ab'gamma)2 bispecific antibody (BsAb) with dual specificity for the CD7 molecule in one Fab arm and for the ribosome inactivating protein (rip) saporin in the other arm, for delivering saporin to the acute T-cell leukaemia cell line HSB-2. Saporin titration experiments revealed that BsAb increased the toxicity of saporin 435-fold for HSB-2 cells, reducing the IC50 for saporin alone from 0.1 mumol to 0.23 nmol when BsAb was included. The rate of protein synthesis inactivation brought about by BsAb-mediated toxin delivery to HSB-2 cells was very similar to that described for conventional immunotoxins (IT's) with a t10 (time taken for a one log inhibition of protein synthesis compared with controls) of 46 h obtained at a saporin concentration of 1 nmol and 226 h at 0.1 nmol. BsAb titration studies demonstrated a clear dose response effect of BsAb concentration on target cell protein synthesis inhibition and cell proliferation. The absolute specificity of toxin delivery was unequivocally demonstrated by a failure of BsAb to deliver an effective dose of saporin to the CD7- cell line HL60 and by the blocking of BsAb-mediated delivery of saporin to HSB-2 cells with an excess of F(ab)2 fragments of the anti-CD7 antibody, HB2. These studies have clearly demonstrated the effectiveness of this BsAb for delivering saporin to a T-ALL cell line utilising CD7 as the target molecule on the cell surface. BsAb's would therefore appear to offer a realistic alternative to IT's for toxin delivery to tumour cells and may even offer certain advantages over conventional IT's for clinical use.


This study was undertaken to elucidate the mechanism(s) of differential sensitivity of human bladder cancer cell lines J82 and SCaBER to mitomycin C (MMC) and its analogue, BMY 25067. The IC50 values for MMC and BMY 25067 in the SCaBER cell line were respectively 5- and 4-fold higher than in J82. BMY 25282 and BMY 25067 were significantly more cytotoxic, on a molar basis, than MMC in both the cell lines. NADPH cytochrome P450 reductase and DT diaphorase activities were significantly higher in the J82 cell line than in SCaBER, suggesting that relatively lower sensitivity of the SCaBER cell line to MMC and BMY 25067 may be due to deficient drug activation. This conclusion was supported by the observation that IC50 values for BMY 25282, which has lower quinone reduction potential than MMC and BMY 25067, did not differ significantly in these cell lines. A correlation between drug sensitivity, oxyradical formation and levels of antioxidative enzymes was not observed. These results suggest that the relatively lower sensitivity of SCaBER cells to MMC or BMY 25067 may be independent of differential oxyradical formation. MMC-induced DNA interstrand cross-link (ISC) formation was markedly lower in the SCaBER cell line than in J82. However, it remains to be seen if the reduced ISC frequency in the SCaBER cell line is a consequence of deficient drug activation or results from increased repair of the damaged DNA.


We established a vindesine-resistant (x 11.6) human small-cell lung cancer cell line (H69/VDS) by stepwise exposure of parent line H69 to vindesine. H69/VDS showed cross-resistance to taxol (x 10.1), vincristine (x 6.9) and colchicine (x 3.4) but not to doxorubicin, cisplatin or etoposide. There was no significant difference in intracellular [3H]-vincristine and doxorubicin accumulation between H69 and H69/VDS cells. The human mdr1 mRNA was not detected in either of the cell lines. These results indicated that H69/VDS did not express a typical multidrug resistant phenotype. Addition of 20 microM verapamil enhanced the growth inhibitory effect of vindesine on both H69/VDS (x 12.0) and H69 cells (x 3.8). The amount of total tubulin in H69/VDS cells was lower than that in the H69 parental cells. No significant increase was observed in the amount of total and polymerised tubulins of H69 cells. In H69/VDS cells, however, verapamil increased the amount of total tubulin to the level of parental cells, but decreased the amount of polymerised tubulin. Modulation of tubulin may play a role in the resistance to vindesine.


Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance.


The biochemical basis of multidrug-resistant (MDR) phenotype has been investigated in drug-resistant sublines independently obtained in our laboratories by single step doxorubicin (DOX) selection of LoVo, DLD1, and SW948 human colon carcinoma (HCC) cell lines. All the chemoresistant sublines have been found to be cross-resistant to DOX, actinomycin-D (ACT-D) and vincristine (VCR) but not to cis-diamminedichloroplatinum (CDDP), and have exhibited an increased expression level of mdr1 mRNA and gp170 glycoprotein. Comparative analyses in drug-resistant and sensitive cells of resistance index, extracellular and intracellular equitoxic DOX concentrations, and mdr1 gene products expression have indicated that MDR phenotype is a multifactorial phenomenon due to different and possibly independent biochemical mechanisms which cooperate, in varying degrees from cell line to cell line, in conferring cellular chemoresistance.


The patterns of acidic and neutral glycosphingolipids (GSLs) were examined in a syngeneic tumour system in Balb/c mice consisting of closely related cell lines with different colonisation potentials directed to the murine lungs . GSLs were analysed by high-performance thin-layer chromatography and structurally identified by fast atom bombardment mass spectrometry combined with compositional analyses and exo-glycosidase digestion. The results suggest that highly metastatic sublines L1-LM and L1-LM12 derived by in vivo selection from mouse fibrosarcoma cells (cell line L1) exhibit a drastic increase of polar ganglioside expression and a restriction to globo-series GSLs. Contrasting with this the low metastatic mutant cells (L1-LM13WGA) express a reduced portion of acidic GSLs and exhibit a shift to less polar ganglioside components. Total cellular and plasma membrane-integrated GSLs were demonstrated to exhibit largely identical patterns. Concomitant with a significant decrease in LacCer expression a substantial reduction of GM2 and a complete lack of GM3 expression can be assigned to the highly metastatic sublines of L1-cells. On the other hand, the more polar gangliosides GM1a and, to an even greater extent, GD1a  accumulate on L1-LM and their clonal sublines. The shift to acidic GSLs of higher polarity is less pronounced on the low metastatic WGA-resistant mutant cells (L1-LM13WGA) showing a preponderance of GM1a. The portion of GD1a within the fractions of acidic GSLs does not correspond to the cellular activities of CMP-NeuAc/GM1  sialyltransferase measured for high and low metastatic cell variants. Total sialic acid content of the various cell lines differs, but is not associated with the metastatic potential. Gangliosides on L1-cells exhibit a significant substitution of N-glycolyl for N-acetylneuraminic acid (13%) compared to their metastatic sublines and to mutant cells (less than 1%). A conversion of surface exposed GD1a to GM1a on membranes of metastatic cells by in situ treatment with Vibrio cholerae sialidase is associated with a significant reduction of tumour cell colonisation directed to the murine lungs.


We selected two drug resistant variants of the MCF7 human breast cancer cell line by chronic in vitro exposure to doxorubicin (MCF7/D40 cell line) and mitoxantrone (MCF7/Mitox cell line), respectively. The cell lines are similar in growth characteristics including doubling time, DNA synthetic phase and cell size. Resistance to mitoxantrone conferred only partial resistance to doxorubicin; whereas resistance selected for doxorubicin appeared to confer complete resistance to mitoxantrone. Both agents selected for cross resistance to the Vinca alkaloids. MCF7/D40 cells display a classic-multi-drug resistance phenotype with expression of P-glycoprotein, decreased drug accumulation relative to the parental line and reversal of drug accumulation and drug resistance by verapamil. MCF7/Mitox cells likewise display resistance to multiple drugs, but in contrast to MCF7/D40 cells do not express P-glycoprotein by immunoblot or RNA blot analysis. Net drug accumulation in MCF7/Mitox cells was decreased relative to the parental cells but there was no selective modulation of drug accumulation or in vitro drug resistance by the addition of verapamil. Efflux of mitoxantrone was enhanced in both the MCF7/D40 and MCF7/Mitox cell lines relative to the MCF7/S cell line. We conclude that the two drug resistant cell lines have different mechanisms of decreased drug accumulation.


The goal of the study was to identify any normal genes that may become inactivated in malignant cells, with associated modifications or loss of gene products. Consequently, attempts were made to identify such products by generating monoclonal antibodies using an immune tolerisation-immunisation procedure. Using such a technique, a plasma membrane-associated glycoprotein with an apparent molecular weight of 92 kDa was identified. The glycoprotein was termed luminal epithelial antigen (LEA.92). The pattern of expression of LEA.92 was demonstrated by an indirect immunostaining technique. Using an in vitro model system representing various stages of breast oncogenesis, LEA.92 was detected on normal or immortalised mammary epithelial cell (MEC) lines which were dependent on epidermal growth factor (EGF) and anchorage formation for growth and non-tumorigenic in nude mice. In contrast, LEA.92 was undetectable on oncogenically transformed or established lines of mammary carcinoma cell lines which were independent of EGF or anchorage formation for growth and were highly tumorigenic. The results appear to suggest a correlation between the down-regulation of LEA.92 and the development of tumorigenicity in malignant MEC lines. Furthermore, the patterns of expression of LEA.92 on breast cells in tissue mirrored those of breast epithelial cells in cell cultures. LEA.92 was detected on the surface of normal but not malignant epithelial cells, which included breast, cervix, colon, lung, pancreas and stomach. LEA.92 appeared to be distinct from receptor for epidermal growth factor, antigens associated with milk fat globule membrane and the family of epithelium-specific keratins.


Expression of hCG and its free subunits by non-trophoblastic tumours is well recognised. Previously we reported hCG secretion by normal and malignant bladder epithelial cells in vitro. Here we examined culture medium from 83 different cell lines derived mainly from common epithelial tumours. Thirty-two of the cell lines were found to secrete hCG-like material into their culture media. Partial immunochemical characterisation showed that of these only choriocarcinoma and fetal tissue cell lines produced intact hCG and alpha subunit. The remaining 28 hCG-expressing epithelial cell lines, which are of mucosal origin, only secreted free beta subunit. Expression of free beta hCG by non-trophoblastic nonendocrine cells would appear to be especially characteristic of mucosal epithelia from the genitourinary and oral/respiratory tracts. Furthermore, this phenomenon may be characteristic of epithelium with transitional and/or squamous cell-like properties.


Multicellular tumour spheroids are cellular aggregates that can be prepared from many types of tumour cells. These three-dimensional structures provide a model for analysing the effects of cell-cell contact and intercellular microenvironments on phenomena such as autocrine regulation of growth factor synthesis. Autoregulation of the synthesis of transforming growth factor-alpha  was investigated at the message and protein levels in spheroid and monolayer cultures prepared from the A431 human squamous carcinoma cell line. The epidermal growth factor receptor (EGF-R) of these monolayer A431 cells had an average surface density of 2.2 x 10(6)/cell. Constitutive expression of TGF-alpha mRNA was an average of 3-fold greater in A431 spheroids than in monolayers, even for densely packed, confluent monolayers. This effect did not depend on hypoxic stress within the spheroids. TGF-alpha protein synthesis was enhanced in comparison with that in monolayer culture, reaching a value of up to 2-fold greater on a per cell basis. These results are discussed in the context of a TGF-alpha/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-alpha in the three-dimensional architecture of a spheroid.


Receptors for granulocyte colony-stimulating factor (G-CSFRs) have been confirmed on the cell surfaces of several non-haematopoietic cell types, including bladder cancer cells. This observation has naturally led to the hypothesis that the expression of G-CSFR on these cells may enhance their growth by G-CSF. In this study, the expression of G-CSFR was determined in both established human bladder cancer cell lines and primary bladder cancers. We studied five different human bladder cancer cell lines  and 26 newly diagnosed bladder tumours. G-CSFR mRNA expressions on cultured cell lines were determined using the reverse transcriptase polymerase chain reaction (RT-PCR) method. Furthermore, the G-CSFR binding experiments on the cultured cell lines were conducted using the Na(125)I-labelled G-CSF ligand-binding assay method. Moreover, the G-CSFR mRNA expressions on primary bladder tumour specimens were assessed using the in situ RT-PCR method. Three out of the five cultured cell lines  exhibited G-CSFR mRNA signals when the RT-PCR method was used. The G-CSFR binding experiments showed an equilibrium dissociation constant (K[d]) of 490 pM for KU-1, 340 pM for NBT-2 and 103 pM for KK cells. With in situ RT-PCR, the tumour cells of 6 out of 26 primary bladder tumour specimens (23.1%) presented positive G-CSFR mRNA signals. Thus, in this study, G-CSFR expression was frequently observed on bladder cancer cells. Therefore, the clinical use of G-CSF for patients with bladder cancer should be selected with great care.


The isobologram analysis performed at the IC 50 level revealed that the combined treatment was highly synergistic with respect to cell growth inhibition. BAL9611 and manumycin were able to inhibit the geranylgeranylation of p21rhoA and farnesylation of p21ras; both drugs inhibited p42ERK2/MAPK phosphorylation, but their combination was more effective than either drug alone. Moreover, the enhanced inhibition of cell growth in vitro by the BAL9611-manumycin combination was also observed in vivo in CD nu/nu female mice xenografted with SW620 tumours. Finally, both drugs were able to induce cell death by apoptosis in vitro and in vivo, as demonstrated by perinuclear chromatin condensation, cytoplasm budding and nuclear fragmentation, and interoligonucleosomal DNA digestion. In conclusion, the inhibition of protein farnesylation enhances the chemotherapeutic effect of BAL9611 in vitro and in vivo in a synergistic fashion, as a result of the impairment of post-translational isoprenylation of proteins and phosphorylation of p42ERK2/MAPK, whose activation is associated with post-translational geranylgeranylation and farnesylation of p21rhoA and p21ras. © 2001 Cancer ResearchCampaign http://www.bjcancer.comProteins belonging to the ras superfamily are involved in cell proliferation of normal and neoplastic tissues. To be biologically active, they require post-translational isoprenylation by farnesyl-transferase and geranylgeranyl-transferase. Enzyme inhibition by drugs may thus represent a promising approach to the treatment of cancer. Therefore, the combined effect of BAL9611, a novel inhibitor of geranylgeranylation, and manumycin, a farnesyl-transferase inhibitor, was evaluated on the SW620 human colon cancer cell line which harbours a mutated K-ras gene. BAL9611 and manumycin dose-dependently inhibited SW620 cell growth with 50% inhibitory concentration (IC


An interesting solvent dependence in the 1H spectra is rationalised in terms ofcompeting intra- and inter-molecular hydrogen bonding. An X-ray analysis of the [Ph3 PAu(2mba)]species reveals a linear P—Au—S arrangement and association in the lattice via the familar carboxylic acid dimer motif. The in Vitro cytotoxicity against seven human tumout lines is alsodescribed. The complexes display moderate to very high activity. Particularly noteworthy is their greater activity against the H226 cell line  compared with that displayedby a range of cytotoxic drugs.The preparation and full NMR (


Cyclosporin A (Sandimmune) increased the in vitro susceptibility of 'parental' and 'multidrug-resistant' (MDR) chinese hamster ovary (CHO) cell lines to three anti-tumour drugs: colchicine, daunomycin, and vincristine. Several immunosuppressive or non-immunosuppressive derivatives of cyclosporin (Cs) were compared for their ability to sensitise both parental and MDR cells to chemotherapeutic agents. Although 5-10-fold increases of sensitivity to anti-tumour drugs could be obtained for cells of the parental line with several Cs-derivatives, the largest 'gains' of sensitivity (chemosensitisation) were obtained for the cells of the MDR line and with only some of the Cs derivatives. The MDR cells employed displayed the typical MDR phenotype. However, we found no correlation between the immunosuppressive activity of Cs derivatives and their capacity to reverse MDR and all four possible combinations of these two activities could indeed be shown among the tested Cs derivatives. This study demonstrates for the first time that some immunosuppressive Cs can be devoid of chemosensitising activity.


The matrix metalloproteinases are a family of enzymes that degrade the extracellular matrix and are considered to be important in tumour invasion and metastasis. The effect of epidermal growth factor (EGF) on matrix metalloproteinase-1 (MMP1) production in two human bladder tumour cell lines, RT112 and RT4, has been investigated. In the RT112 cell line, an increase in MMP1 mRNA levels was found after a 6-h incubation with EGF, and this further increased to 20-fold that of control levels at 24- and 48-h treatment with 50 ng ml(-1) of EGF. MMP2 mRNA levels remained constant over this time period, whereas in the RT4 cells no MMP2 transcripts were detectable, but MMP1 transcripts again increased with 24- and 48-h treatment with 50 ng ml(-1) of EGF. MMP1 protein concentration in the conditioned medium from both cell lines increased with 24- and 48-h treatment of the cells and the total MMP1 was higher in the medium than the cells, demonstrating that the bladder tumour cell lines synthesize and secrete MMP1 protein after continuous stimulation with EGF. MMP1 protein was detected in urine from patients with bladder tumours, with a significant increase in concentration with increased stage and grade of tumour. MMP1 urine concentrations may therefore be a useful prognostic indicator for bladder tumour progression.


However, exact regulatory mechanisms mediating and controlling this phenomenon are still unknown. In the present study, we have investigated the effect of anti-Her-2/neu antibody on apoptosis of HER-2/neu overexpressing human breast cancer cell lines SK-BR-3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130 and HTB-131 in relation to p53 genotype and bcl-2 status. SK-BR-3, HTB-24, HTB-128 and HTB-130 cells exhibited mutant p53, whereas wild type p53 was found in HTB-25, HTB-27 and HTB-131 cells. All seven cell lines weakly expressed bcl-2 protein (10–20%). Anti-Her-2/neu antibody, irrespective of p53 and bcl-2 status, induced apoptosis in all 7 cell lines dose- and time-dependently and correlated with Her-2/neu overexpression. In addition, incubation of cell lines with anti-Her-2/neu antibody did not alter p53 or bcl-2 expression. Anti-HER-2/neu antibody did not induce apoptosis in HER-2/neu negative HBL-100 and HTB-132 cell lines. Our results indicate that within the panel of tested breast cancer cell lines, anti-Her-2/neu antibody-induced apoptosis was independent from the presence of intact p53. © 2001 Cancer Research Compaign


Five tumour cell lines have been derived from a primary squamous carcinoma of the tongue, from 2 subsequent local recurrences, and from 2 lymph-node metastases--all from the same patient. While the cell lines shared many morphological and biochemical characteristics, those derived from recurrences and metastases appeared to be less differentiated, were less well organized in culture, and displayed fewer desmosomes and tonofilaments than cells in the primary tumour line. A recurrent line showing greatest morphological divergence from the primary tumour line also demonstrated the greatest differences at the ultrastructural level, in increased production of plasminogen activator and in the composition of cell-surface glycoproteins.


First, radioligand binding studies demonstrated the presence of tachykinin NK1R on SNB-19, DBTRG-05 MG and U373 MG, but not on U138 MG and MOG-G-GCM human glioma cell lines. Second, application of SP or neurokinin A (NKA) to NK1R+glioma cell lines increased the secretion of interleukin 6 (IL-6) and potentiated IL-6 secretion induced by IL-1β. SP also up-regulated the release of transforming growth factor β1 (TGF-β1) by the U373 MG glioma cell line. Third, SP induced new DNA synthesis and enhanced the proliferation rate of NK1R+, but not of NK1R−glioma cell lines. Also, NKA stimulated the proliferation and cytokine secretion in NK1R+glioma cell lines. All the stimulant effects of SP/NKA on NK1R+glioma cell lines were completely blocked by a specific tachykinin NK1R antagonist, MEN 11467. These data support the potential use of tachykinin NK1R antagonist for controlling the proliferative rate of human gliomas. © 1999 Cancer Research CampaignThe neuropeptide substance P (SP), by stimulating tachykinin NK


Malignant cells frequently acquire a certain independency of exogenous growth factors via the coexpression of epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF)-related molecules. In the present study we investigate a possible involvement of EGF-related molecules in the growth of human lung mesothelioma. Four well-characterised cell lines are analysed for their responsiveness to exogenous EGF and transforming growth factor alpha  as well as for coexpression of EGFR and EGF/TGF-alpha. Both growth factors are able to stimulate DNA synthesis in three cell lines, although the degree of responsiveness is very variable, but neither EGF nor TGF-alpha has an effect on the cell line ZL34. In contrast, no heterogeneity is observed in the expression of EGFR, which is similarly high in all cell lines. Analysis of cell supernatants reveals that, whereas no EGF is detected, TGF-alpha is released by two cell lines. Furthermore, these two cell lines, ZL5 and ZL34, are shown to express the membrane anchored precursor pro-TGF-alpha. Thus, coexpression of EGFR and TGF-alpha is observed on two mesothelioma cell lines. The potential autocrine mitogenic role of TGF-alpha in these two cell lines was tested using neutralising antibodies against TGF-alpha and EGFR. In ZL5 cells DNA synthesis was not affected by the presence of neutralising antibodies, indicating that an external autocrine mitogenic pathway is not active in these cells. In ZL34 cells, however, the potential autocrine loop could be disrupted, as DNA synthesis was significantly reduced in the presence of neutralising antibodies. This result gives strong evidence for an autocrine role of TGF-alpha in the growth of the mesothelioma cell line ZL34.


We have investigated the anti-tumour activities of Mel-B either with or without all-trans -retinoic acid (ATRA) using the MCF-7 human breast cancer cells, as well as the PC-3 and DU 145 human prostate cancer cells both in vitro and in vivo. The antiproliferative effects of Mel-B and/or ATRA against breast and prostate cancer were tested in vitro using clonogenic assays and in vivo in triple immunodeficient mice. Furthermore, the mechanism of action of these compounds was studied by examining the cell cycle, levels of bcl-2, apoptosis and antiproliferative potency using a pulse-exposure assay. Clonogenic assays showed that the cancer cell lines were sensitive to the inhibitory effect of Mel-B  is used to treat African trypanosomiasis. Recently, another arsenical, As


All three compounds are moreactive than cis-platin, etoposide and doxorubicin against both tumour cell lines. They are as activeas mitomycin C against WiDr, but less active against MCF-7.The synthesis and spectral characterization of six novel triphenyltin compounds are described. The


Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-U1 and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines.


Human chorionic gonadotrophin (hCG) and alphafetoprotein (AFP) were measured in culture media from a panel of 29 cell lines including 9 bladder carcinomas, 5 'normal' bladder epithelia, 10 germ cell tumours, and 5 miscellaneous tumours and 'normal' cell lines. In 7 of the 9 bladder carcinomas and 4 of the 5 'normal' bladder epithelia, the media contained hCG at levels ranging from between 34 and 3,600 IU l(-1). All other cell lines, including the 10 germ cell tumour lines gave negative results for hCG. These findings indicate that in vitro secretion of hCG is a common feature of normal and neoplastic bladder transitional epithelia, and support the hypothesis that parts of the genito-urinary epithelium have a potential for hCG production.


Human primary thyroid follicular epithelial cells were transfected with a plasmid containing an origin-defective SV40 genome (SVori-) to produce several immortal cell lines. Two of the 10 cell lines analysed expressed specific features of thyroid epithelial function (iodide-trapping and thyroglobulin production). These two lines were characterised in detail and found to be growth factor-independent, capable of anchorage-independent growth at low frequency but non-tumorigenic in nude mice. These differentiated, These differentiated, partially transformed cell lines were shown to be suitable for gene transfer at high frequency using simple coprecipitation techniques.


TS protein expression correlated with TS activity (r2= 0.88 P= 0.05). Intracellular concentrations of drug following exposure to ZD9331  varied by ~2-fold and dTTP pools decreased by > 80% in all cell lines. No clear associations across the cell lines between intracellular drug concentrations, TS activity/expression, or TTP depletion could be made. dUTPase activity varied 17-fold and correlated with dUTPase protein expression (r2= 0.94 P= 0.03). There was a striking variation in the amount of dUTP formed following exposure to ZD9331 (between 1.3 and 57 pmole 10–6cells) and was in general inversely associated with dUTPase activity. A large expansion in the dUTP pool was associated with increased sensitivity to a 24-h exposure to ZD9331 in A549 cells that have low dUTPase activity/expression. dUTPase expression and activity were elevated (approximately 3-fold) in two variants of a human lymphoblastoid cell line with acquired resistance to TS inhibitors, further suggesting an important role for this enzyme in TS inhibited cells. © 2000 Cancer Research CampaignUracil DNA misincorporation and misrepair of DNA have been recognized as important events accompanying thymidylate synthase (TS) inhibition. dUTPase catalyses the hydrolysis of dUTP to dUMP, thereby maintaining low intracellular dUTP. We have addressed the relationship between dUTPase expression and cellular sensitivity to TS inhibition in four human lung tumour cell lines. Sensitivity (5-day MTT assay) to the growth inhibitory effects of the non-polyglutamatable, specific quinazoline TS inhibitor ZD9331, varied up to 20-fold (IC


Recently, a photoreceptor protein, recoverin, has been recognised as an autoantigen of cancer-associated retinopathy (CAR), a rare paraneoplastic neurological syndrome often associated with patients with small-cell lung cancer (SCLC). Although until quite recently the specific expression of recoverin in cancer cells had not been indicated, Polans et al.  demonstrated the specific expression of recoverin in lung tumour and primary cultured tumour cells from a CAR patient. We examined the expression of recoverin in human lung cancer cell lines by reverse transcription polymerase chain reaction (PCR), Northern blotting and Western immunoblotting. Recoverin was expressed in only one SCLC cell line from a patient with CAR. The sequence of recoverin cDNA from the cells was identical to the human recoverin sequence. These findings strongly support the hypothesis that the ectopic expression of wild-type recoverin in SCLC induces the cancer-retina immunological cross-reaction, leading to visual loss in CAR.


The kinetics, specificity and morphology of cytolethal responses have been studied in human glucocorticoid-sensitive and -insensitive lymphoid cell lines (HLCL) and fibroblasts following treatment with high (10(-3)M) and low (10(-6)M) doses of steroid. The high dose cytolethal response appears non-specific occurring in all cell lines with every steroid tested. By contrast, the low dose  cytolethal response requires an active glucocorticoid and a sensitive HLCL. However, both high and low concentrations of steroid induce virtually identical morphological changes in dying cells and similar changes can be induced in cells killed by deliberate feed exhaustion. Although the morphological features in each case resemble apoptosis, the "programmed" physiological form of cell death, the intracellular events leading to cytolysis seem likely to differ. The earliest morphological changes presaging cell death comprise rounding up of cells and condensation of nuclear chromatin. Nuclear changes progress rapidly thereafter and appear to result from detachment of chromatin from the nuclear matrix. The low dose cytolethal response requires the continuous presence of glucocorticoid for periods in excess of 24h, prior to which cell growth appears unaffected. The constancy of this latent interval suggests glucocorticoids may influence some replication control mechanism unrelated initially to macromolecular biosynthesis.


P< 0.05; Mann–Whitney U -test). Inhibin/activin βA- and βB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of βB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While βB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin α-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin βB-subunit mRNA or by a decrease of ActRIB mRNA levels. © 2000 Cancer Research CampaignThe possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate pithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour aterial when compared to non-malignant tissue (


O6-benzylguanine (O6-BG) and 3-aminobenzamide (3-AB) inhibit the DNA repair proteins O6-alkylguanine-DNA alkyltransferase (AGT) and poly(ADP-ribose) polymerase (PARP) respectively. The effect of O6-BG and/or 3-AB on temozolomide and 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) cytotoxicity, was assessed in seven human tumour cell lines: six with an AGT activity of > 80 fmol mg-1 protein (Mer+) and one with an AGT activity of < 3 fmol mg-1 protein (Mer-). Three of the Mer+ cell lines  were considered to exhibit resistance to methylation by a mismatch repair deficiency (MMR-), each being known to exhibit microsatellite instability, and DLD1 and HCT116 having well-characterised defects in DNA mismatch binding. Potentiation was defined as the ratio between an IC50 achieved without and with a particular inhibitor treatment. Temozolomide or BCNU cytotoxicity was not potentiated by either inhibitor in the Mer- cell line. Preincubation with O6-BG (100 microM for 1 h) was found to potentiate the cytotoxicity of temozolomide by 1.35- to 1.57-old in Mer+/MMR+ cells, but had no significant effect in Mer+/MMR- cells. In comparison, O6-BG pretreatment enhanced BCNU cytotoxicity by 1.94- to 2.57-fold in all Mer+ cell lines. Post-incubation with 3-AB  potentiated temozolomide by 1.35- to 1.59-fold in Mer+/MMR+ cells, and when combined with O6-BG pretreatment produced an effect which was at least additive, enhancing cytotoxicity by 1.97- to 2.16-fold. 3-AB treatment also produced marked potentiation (2.20- to 3.12-fold) of temozolomide cytotoxicity in Mer+/MMR- cells. In contrast, 3-AB produced marginal potentiation of BCNU cytotoxicity in only three cell lines (1.19- to 1.35-fold), and did not enhance the cytotoxicity of BCNU with O6-BG treatment in any cell line. These data suggest that the combination of an AGT and PARP inhibitor may have a therapeutic role in potentiating temozolomide activity, but that the inhibition of poly(ADP-ribosyl)ation has little effect on the cytotoxicity of BCNU.


CPT10, C6CPT50 and C6CPT100, growing respectively with 10, 50 and 100 ng ml–1camptothecin). The degree of resistance to camptothecin ranged between 15-fold (C6CPT10) and 30-fold (C6CPT50and C6CPT100). The C6CPT10cell line presented a collateral sensitivity to etoposide (3.6-fold), while the C6CPT50 and C6CPT100 cell lines were cross-resistant to etoposide (1.8-fold) The resistant lines were characterised by a two-fold reduced content and catalytic activity of topoisomerase I, and C6CPT50 and C6CPT100 presented a significant increase in topoisomerase IIα content and catalytic activity and a marked overexpression of P-glycoprotein. We explored the cytotoxicity of combinations of a topoisomerase I inhibitor (camptothecin) and a topoisomerase II inhibitor (doxorubicin or etoposide) at several molar ratios, allowing the evaluation of their synergistic or antagonistic effects on cell survival using the median effect principle. The simultaneous combination of camptothecin and doxorubicin or etoposide was additive or antagonistic in C6 cells, slightly synergistic in the C6CPT10 line and never more than additive in the C6CPT50 and C6CPT100 cell lines. The sequential combination of doxorubicin and camptothecin gave additivity in the order camptothecin → doxorubicin and antagonism in the order doxorubicin → camptothecin. Clinical protocols combining a topoisomerase I and a topoisomerase II inhibitor should be considered with caution because antagonistic effects have been observed with combinations of camptothecin and doxorubicin.© 2001 Cancer Research Campaign http://www.bjcancer.comFrom the rat C6 glioma cell line in culture, we selected camptothecin-resistant variants by growth in the presence of increasing amounts of this drug (C6


New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential.n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines.Colorectal primary tumours . Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44ex vivo three-dimensional model, potentially useful for studying metastatic process.The present colospheres provide an  Human CRC is one of the most extensively investigated tumour types, and genetic pathways involved in these malignancies have been identified . Howeverex vivo mechanical dissociation of CRC tissue. We refer to these 3D structures as colospheres. To further characterise these colospheres, we used a human colon cancer xenograft, XenoCT320, and the derived paired colon cancer cell lines, CT320X6 and CT320  and the Institut Gustave Roussy  from 203 patients observed between May 2003 and May 2006, in accordance with protocols approved by the local ethical committees and with the Helsinki Declaration of 1975, as revised in 1983.−1 HEPES, 4.5 g l−1 glucose, 1 mmol l−1 pyruvate sodium, 200 U ml−1 penicillin and 200 μg ml−1 streptomycin) and submitted to ex vivo mechanical mincing.Tumour material not required for histopathological diagnosis was placed in a ‘culture medium’ , no colosphere observed; (+), ⩽2 colospheres per field; (++), >2 colospheres per field.XenoCT320 xenograft, CT320 and CT320X6 cell lines have been previously described .Colosphere and spheroid development was monitored by time-lapse video microscopy for 65 h at a 4 min interval. Dynamic sequences were obtained on a DM IRBE stand equipped with a motorised stage  using a 37°C 8% COColospheres and spheroids were embedded using the Cytoblock method  were conducted to confirm the total gene specificity of the nucleotide sequences chosen for the primers and for the absence of DNA polymorphisms. The nucleotide sequences of the primers used were the following: Mm-TBP-U (5′-CCCTTGTACCCTTCACCAATGAC-3′) and Mm-TBP-L (5′-TCACGGTAGATACAATATTTTGAAGCTG-3′) and Total-TBP-U (5′-TGCACAGGAGCCAAGAGTGAA-3′) and Total-TBP-L (5′-CACATCACAGCTCCCCACCA-3′). The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min, and 50 cycles at 95°C for 15 s and 65°C for 1 min.Specific mouse N-fold differences in the specific murine Tbp gene expression (using Mm-TBP primers), relative to the sum of the mouse and the human TBP gene expression , termed NMm-TBP, are determined by the formula: NMm-TBP=2CtsampleΔ. The ΔCt value of the sample is determined by subtracting the Ct value of the murine TBP gene from the Ct value of the total (mouse+human) TBP gene. The NMm-TBP values of the samples are subsequently normalised such that the median of the NMm-TBP values of four mouse tissues was 100. As TBP is a ubiquitously expressed housekeeping gene, which encodes the TATA box-binding protein, a component of the DNA-binding protein complex TFIID, and shows a similar expression in our human and mouse tissues (Ct=27 for 5 ng cDNA), the final result  determines the proportion of mouse cell contamination for a given sample.Results, expressed as After harvesting, colospheres and spheroids were analysed using standard techniques for transmission electron microscopy . Injections of cells equivalent to 4 × 104 cells were administered with a 27 G needle in the subcapsular space in the right kidney. The internal diameter of the 27 G needles is 200 μm at the minimum, which would avoid any damage on spheroids and colospheres. After cell injection, the kidney was then returned to the peritoneal space and the skin was closed with surgical staples. The recipient mice were necropsied at 14 weeks after injection and kidneys, spleen, lung and liver were removed for histological examination.The tumorigenicity of XenoCT320 colospheres, CT320X6 spheroids and CT320X6 single cells was compared in a subrenal capsule assay in nude mice. Three days after xenograft tissue dissociation, colospheres of 100–150 6 viable cells in 0.2 ml of PBS. The mice were observed for 2 months for the appearance of tumour development.Tumorigenicity of the CT320X6 cell line was carried out in parallel in nude mice by the subcutaneous transplantation of 2 × 10−1)  in 24-well plates before polymerisation of the lattice. Culture medium was added to the Matrigel layer.Colospheres and spheroids were added to Matrigel solution , according to the manufacturer's instructions. Colospheres, spheroids and xenograft tissue were lysed with zymogram sample buffer and the amount of protein was estimated using the Bradford method . Twenty micrograms of protein from each sample was separated on polyacrylamide gels containing 10% gelatin. After electrophoresis, the gels were washed in renaturation buffer for 30 min, and incubated overnight at 37°C in the development buffer. Gels were stained with a Coomassie blue R250 0.5% solution and destained with 3 × destaining solution.Disaggregated colospheres, spheroids and xenografts were assessed using an Epics XL cytometre  with the following antibodies: anti-human CD133/2 (clone 293C3) phycoerythrin, anti-human EpCAM (clone HEA-125) fluorescein isothiocyanate  and anti-human CD44-FITC , at appropriate dilutions. As for xenograft tissue, human colon cancer cells were magnetically labelled and separated from mouse stroma cells using human EpCAM microbeads (Miltenyi-Biotec SAS) according to the manufacturer's instructions, before assessment by flow cytometry.4Cl (50 mmol l−1), followed by washes in PBS, colospheres were saturated overnight with 3% BSA in PBSt (0.1% Triton X-100 in PBS) at 4°C and washed in PBSt. Colospheres were incubated for 24 h at 4°C with anti-human EpCAM-FITC (clone HEA-125) in PBSt (dilution 1 : 50) and rinsed with PBSt. The DNA marker, TOPRO-3 , was then applied for 20 min at room temperature. Colospheres were mounted in glycerol/PBS (90/10: v/v). Images were recorded on a Leica TCS SP2 confocal microscope.About 30 colospheres in suspension were fixed for 3 h at 4°C in PBS containing 4% PFA, followed by washes in PBS. Colospheres were then permeabilised in 1% Triton X-100 in PBS for 1 h and washed with PBS. After a 1 h incubation in PBS/NHμm and were easily identifiable because of their smooth refringent outline. To determine whether the property to form such colospheres from patient tumours was a feature restricted to cancer tissue, the same mechanical dissociation protocol was carried out in parallel with non-tumoral colon tissue counterparts from 120 of these 203 tumour tissues. It is noteworthy that no colospheres were ever observed after dissociation of non-neoplastic colonic mucosa, showing that these spherical structures are only generated by cancer tissue. Among these 98 specimens, 28 tumours led to the generation of a large number of colospheres.Dissociation of 203 fresh colon primary tumour tissues directly harvested from human patients undergoing surgery led to the formation of spherical organoid structures, which we named colospheres , in 98 oColospheres formed by highly compacted cells were resistant to mechanical disruption. On the basis of haemalun–eosin–safran (HES) staining, only carcinoma cells were observed in colospheres, whereas no stromal cells were recognised , indicatP<0.01; Tumour collection was divided into two groups: non-disseminated tumours (AJCC stage I and II) and disseminated tumours (AJCC stage III and IV), as AJCC stage I and II tumours differ from stage III and IV tumours by the fact that the latter group displays metastases in lymph nodes and/or in distant organs. Thus, the capacity of primary tumours to form colospheres was found to be significantly correlated with tumour aggressiveness. Indeed, statistical analysis showed that the stage III and IV tumour groups gave rise more frequently to colospheres than did the stage I and II tumour groups P<0.01; .ex vivo mechanical dissociation. Furthermore, the morphological features of colospheres are close to those of spheroids generated in vitro using permanent carcinoma cell lines. This prompted us to compare XenoCT320 colospheres with spheroids derived from the CT320X6 cell line, previously established from the XenoCT320 tumour, and with spheroids derived from the CT320 cell line, established from the same original patient tumour fragment as the xenograft  were generated by remodelling of tissue fragments, whereas smaller colospheres were spontaneously formed by the aggregation of single cells. Interestingly, the rapid generation of colospheres was not because of the presence of FCS, as similar experiments performed in FCS-free medium led to the same observation (data not shown).To analyse the dynamic nature of cellular events leading to colosphere formation in only 1 day, dissociated XenoCT320 tissue was filmed as a 2D outline every 4 min for 65 h . This reTbp confirmed the absence of mouse cells in human XenoCT320 colospheres, whereas this approach always showed the presence of mouse  cells in human XenoCT320 tumour tissue (rate<10%). Although XenoCT320 colospheres mimicked adenocarcinoma, CT320X6 and CT320 spheroids resembled only carcinoma morphology. In addition, Ki-67 staining showed that colospheres were made up of viable proliferating cells. Viability assays confirmed that colospheres remained alive for up to a minimum of 3 weeks when transferred on agarose to avoid adherence to substrate -like dedifferentiation of tumour cells, a process by which epithelial cell layers lose polarity and cell–cell contact and undergo remodelling of the cytoskeleton, leading to a migratory phenotype . The natFor functional analysis, colospheres and spheroids were embedded in Matrigel in the absence of a chemoattractant. Spherical multicellular cell aggregates were then imaged during 3 days in culture. Although colospheres and spheroids were shown above to be compacted structures using SEM, it was observed that single cells succeeded in detaching from colospheres in the Matrigel assay . As for Movement into Matrigel requires extracellular matrix degradation. Two major matrix metalloproteinases (MMPs) in colon cancer, MMP-2 and MMP-9, were then studied using gelatin zymography. Although protein extracts of spheroids displayed no gelatinase activity, colospheres and xenograft exhibited both MMP-2 and MMP-9 activity .4) injected as XenoCT320 colospheres, as CT320X6 spheroids or as a cell suspension from the CT320X6 cell line (6 cells) as single cells or spheroids proved that this cell line was tumorigenic in nude mice. Tumour formation at the injection site occurred in 6 out of 15 mice injected with XenoCT320 colospheres. Conversely, CT320X6 spheroids formed no tumour (0 out of 15) and in the CT320X6 single-cell suspension, only one local tumour was formed in 1 out of 13 cases. In addition, a gross and histological examination of potential organ distant sites revealed the unique tumorigenic and metastatic phenotype of the colospheres: metastases were only observed in the case of colosphere injection .Colospheres, spheroids and carcinoma cells in xenograft highly expressed CD133. Even if the variability of the CD133This study reports the identification of colospheres, a newly characterised 3D cancer cell model directly obtained from dissociated human CRC tissue and associated with tumour aggressiveness. This model differs first from classical spheroids in that colospheres are obtained after short-term culture and not with permanent cell lines and, second, in that colospheres and spheroids clearly display distinct phenotype features.in vitro human colon cancer cell lines , able to generate colospheres after ex vivo colospheres displayed MMP-2 and MMP-9 activity, as did parental xenograft tissue, whereas paired cell line spheroids were devoid of such proteases. More importantly, as shown by a subrenal capsule assay, colospheres displayed undoubtedly a high tumorigenicity and metastatic potential, compared with the spheroid model, suggesting that these 3D structures contained more cancer-initiating cells. It could be hypothesised that the different tumorigenicity is related to a higher passage number into mice for xenografts giving rise to colospheres than for xenografts leading to the CT320X6 cell line. Nevertheless, the presumed selection of more aggressive cells through in vivo passage is not so clear. We have determined that the CT320 cell line, established directly from a patient sample without any passage into mice, and the CT320X6 cell line, derived from the xenograft XenoCT320, display a similar growth curve in nude mice (unpublished data). It is also noteworthy that tumours obtained after injection of colospheres were well-differentiated adenocarcinomas, whereas tumours derived from a cell-suspension injection were poorly differentiated carcinomas, illustrating, as largely observed , that cancer cell lines established from differentiated human tumours lead to poorly differentiated tumours, whereas xenografts directly from tissue tumour specimens maintain their differentiation level. The differences observed between colospheres and spheroids have to be put in line with the different growth conditions (ex vivo vs in vitro), which in turn involve different microenvironments. The impact of the microenvironment – such as cell–cell interactions and extracellular matrix – on cell phenotype is indeed well described (Comparison of morphological structures in colospheres and spheroids in our study model revealed that, despite their compaction, colospheres did not display tight junctions. They also displayed a disturbed expression of E-cadherin, one of the caretakers of the epithelial phenotype, compared with spheroids. Although the role of EMT in carcinoma dissemination is controversial , downreg+ (+ human cells in the xenograft and the fact that CD44+ cells were a minority sub-population of CD133+ cells are consistently in agreement with previous observations by Clarke and Coll (Owing to their round compact architecture and their direct CRC tissue origin, colospheres can be compared with colon cancer spheres, a model previously described for colon cancer stem cell expansion cultures (ex vivo three-dimensional model distinct from traditional spheroids. It is noteworthy that colospheres are formed from a random dissociation of fragments of tumours, leading to potential variability within resulting colospheres. In contrast, spheroids are obtained from cell lines using the liquid overlay technique in microwells, giving rise to a population of identical spheroids. This heterogeneity in the colosphere population could reflect inherent intratumoral heterogeneity. As a short-term culture model associated with tumour aggressiveness, it might therefore be relevant for studying metastatic processes.In conclusion, these results show that colospheres form an


A variant subline (B16-F10-B2) selected from the B16-F10 melanoma cell line, shows greater metastatic capacity and preferential growth in the brain after i.v. injection into C57BL/6 mice. Several biological properties of these two cell lines have been compared, in an effort to determine the mechanisms responsible for this non-random metastatic pattern. No differences in cell morphology, in vitro growth rates or exposed cell-surface proteins were detected. Quantitative analysis of tumour-cell arrest and distribution using 125IUdR-labelled cells indicated that, although initial arrest patterns of the cell lines were very similar, B16-F10-B2 cells survived in the lungs to a greater cell line had a higher mean number of chromosomes than the parent line, whereas the variance of chromosome distribution was less. We suggest that the selection of a brain-colonizing variant represents the emergence of a pre-existing subpopulation of cells, and provides a useful model for studying mechanisms of intracranial metastasis.


P = 0.023). Moreover, metastatic adenocarcinomas expressed more often iNOS positivity than mesotheliomas (P = 0.021). Experiments with the cell lines confirmed that malignant mesothelioma cells are capable of synthesizing iNOS. No significant association was found between iNOS expression and apoptosis or vascular density in malignant mesotheliomas. The higher expression of iNOS in the epithelial subtype of mesothelioma and pleural metastatic adenocarcinoma might be due to an increased sensitivity of these cell types to cytokine-mediated iNOS upregulation. The strong expression of iNOS suggests a putative role for NO in the growth and progression of these tumours. © 2000 Cancer Research CampaignIn this study we investigated the immunohistochemical expression of inducible nitric oxide synthase (iNOS) in a set of normal pleural mesothelial tissues, malignant mesotheliomas, mesothelioma cell lines and metastatic pleural adenocarcinomas. Furthermore, the expression of mRNA was assessed in four malignant mesothelioma cell lines in culture. Apoptosis and vascular density in malignant mesotheliomas was assessed by the TUNEL method and by immunohistochemistry with an antibody against FVIII-related antigen. Immunohistochemically mesothelial cells in non-neoplastic healthy pleural tissues were mostly negative for iNOS. Positivity for iNOS was observed in 28/38 (74%) and 24/25 (96%) of malignant mesotheliomas and metastatic pleural adenocarcinomas, respectively. Epithelial and mixed mesotheliomas expressed more often strong iNOS immunoreactivity compared to the sarcomatoid subtype (


The in vitro activity of xanafide against MCF-7, MDA-MB-231, SKBR-3 and T47D cell lines was compared to that of paclitaxel, docetaxel, gemcitabine, vinorelbine and doxorubicin. In MCF-7, xanafide demonstrated comparable total growth inhibition (TGI) concentrations to the taxanes and lower TGI values than gemcitabine, vinorelbine and doxorubicin. MCF-7 (oestrogen receptor (ER)+/p53 wild-type) was the most sensitive cell line to xanafide. MDA-MB-231 and SKBR-3 exhibited similar sensitivity to xanafide. T47 D (ER+/p53 mutated), showed no response to this agent. The in vivo activity of xanafide was further compared to that of docetaxel in MCF-7 and MDA-MB-231 cell lines using the hollow fibre assay. Xanafide was slightly more potent than docetaxel, at its highest dose in MCF-7 cell line, whereas docetaxel was more effective than xanafide in MDA-MB-231 cell line. Our results show that there is no relationship between sensitivity of these cell lines to xanafide and cellular levels of both isoforms of topoisomerase II and suggest that ER and p53 status and their crosstalk may predict the responsiveness or resistance of breast cancer patients to xanafide.Xanafide, a DNA-intercalating agent and topoisomerase II inhibitor, has previously demonstrated comparable cytotoxicity to the parent drug amonafide (NSC 308847). The current study was conducted to investigate further the anti-proliferative effects of xanafide in human breast cancer cell lines,  Breast cancer is the most common malignancy affecting women in the Western world. Systemic chemotherapy is indicated for women with metastatic breast cancer (MBC) that is oestrogen/progesterone-negative or unresponsive to hormonal therapy, or rapidly progressing and life-threatening . SeveralThe taxanes, paclitaxel and docetaxel, bind to, and stabilise microtubules, causing cell-cycle arrest and apoptosis , a novel salt formulation of amonafide, has been developed. Preliminary pharmacological and chemical equivalence studies demonstrated that xanafide had comparable pharmacokinetics and toxicity in vitro and in vivo, while having superior solubility, stability and compressibility than the parent drug, amonafide . Individualising the dose of xanafide as a function of NAT2 phenotype may optimise patient safety and therapeutic benefit. Xanafide has recently received the FDA Orphan Drug Designation for the treatment of acute myeloid leukaemia (AML) and has now entered phase II clinical trials in patients with secondary AML.Xanafide -positive and ER-negative cell lines, since it has been shown that oestrogen can enhance the effects of topoisomerase II inhibitors in ER-responsive breast cell lines  in a panel of four breast cancer cell lines: MCF-7, MDA-MB-231, SKBR-3 and T47D, ll lines .M L-glutamine at 37°C in a humidified atmosphere containing 5% CO2.MCF-7, MDA-MB-231, SKBR-3 and T47D were obtained from the American Type Culture Collection (ATCC) . Cell lines were grown in RPMI-1640 medium , supplemented with 10% fetal bovine serum, 2 min vitro use, a 1 mM stock solution of each agent was prepared by dilution in culture media. Polyvinylidene fluoride hollow fibres  were purchased from Spectrum Medical Industries .All general chemicals were purchased from Sigma Chemical Co.  unless otherwise specified. Xanafide was kindly provided by Xanthus Life Sciences. Paclitaxel, docetaxel, doxorubicin, vinorelbine and gemcitabine are commercially available. Before nu/nu mice, 5–6 weeks of age were purchased from Taconic . All studies involving these animals were conducted in accordance with National Cancer Institute (NCI) protocol and the McGill University Animal Care and Ethics guidelines.Athymic NCr 50 and TGI values, representing the 50% growth inhibition and total growth inhibition, respectively, compared to non-treated control (C) and a control at time of addition of increasing drug concentrations (Tz). On day 1, MCF-7 & SKBR-3 (5000 cells) and MDA-MB-231 & T47D (20 000 cells) were seeded into 96-well plates in a volume of 100 μl per well. On day 2, one plate of each cell line was fixed in situ with trichloroacetic acid (TCA) to establish the cell population at time of drug addition (Tz). An aliquot of 100 μl of serial dilutions of the different agents was added to the appropriate well, resulting in a series of final concentrations ranging from 0.1 nM to 100 μM. After 48 h of drug exposure, the medium in control and drug-containing wells was removed. Cells were washed with cold phosphate-buffered saline (PBS) and then precipitated with 50 μl ice-cold 50% TCA and fixed for 60 min at 4°. The supernatant was discarded, and cells were washed five times with tap water and air-dried. Fixed cells were then dyed with 50 μl of 0.4% sulphorhodamine B in 1% acetic acid solution and the plates were incubated for 10 min at room temperature. Unbound dye was removed by washing with 1% acetic acid and the plates were air-dried. Sulphorhodamine B was dissolved in 150 μl of 10 mM Tris-buffer (pH 10.5) and 540 nm optical density was measured in a Labsystems Multiskan® Multisoft apparatus. Percent net growth was calculated using the seven absorbance measurements , growth control, (C), plus the test growth at the different drug concentration levels (Ti)) as follows: ((Ti–Tz)/(C–Tz)) × 100 for concentrations for which Ti>/=Tz and ((Ti–Tz)/Tz) × 100 for concentrations for which Ti<Tz. The results are expressed as the mean of three independent experiments±s.e.m.Cytotoxicity studies were performed using the sulphorhodamine B assay  implants, a small incision was made through the skin and musculature of the dorsal abdominal wall, the fibre samples were inserted into the peritoneal cavity in a craniocaudal direction and the incision was closed with skin staples. For subcutaneous (s.c.) implants, a small skin incision was made at the nape of the neck to allow insertion of an 11-gauge tumour implant trocar. The trocar, containing the hollow fibre samples, was inserted caudally through the s.c. tissues and fibres were deposited during withdrawal of the trocar. The incision was closed with a skin staple.The n=6), xanafide and docetaxel treated groups (n=3 for each group). Xanafide was dosed at the maximum tolerated dose 30 mg kg−1, on the basis of reported in vivo studies with amonafide . Both agents, in PBS were given once daily by i.p. injection from day 3–7 after implantation as reported previously  for 4 h at room temperature on a rotation platform. Aliquots (150 μl) of extracted MTT formazan were transferred to individual wells of 96-well plates and assessed for optical density at a wavelength of 540 nm. Results are expressed as % growth inhibition compared to control±s.d.On day 8, mice were killed and fibres were retrieved. The fibres were placed into 6-well plates, with each well containing 2 ml of fresh, prewarmed culture medium and allowed to equilibrate for 30 min at 37°C. To define the viable cell mass contained within the intact hollow fibres, a 3--2,5-diphenyltetrazolium bromide (MTT) dye conversion assay was used. Briefly, 1 ml of prewarmed culture medium containing 1 mg MTT mlt-test. Two-sided P-values less than 0.05 were considered statistically significant.The comparisons between the untreated and treated groups were analyzed using the Student's A panel of four human breast cancer cell lines: MCF-7, MDA-MB-231, SKBR-3 and T47D was used in this study. Their molecular characteristics are listed in 50 value of 5 and 20 μM, respectively. Xanafide also inhibited the growth of the ER-negative SKBR-3 and MDA-MB-231 cells in a concentration-dependent manner, with an average GI50 value of 6 and 10 μM, respectively  were expressed. At doses higher than the TGIs, net cell killing was observed in the four breast cell lines tested: MCF-7, MDA-MB-231, SKBR-3 and T47D .In MCF-7 cell line, xanafide exhibited a 1.7–2.2-fold lower TGI concentration than those of docetaxel and paclitaxel, respectively. Vinorelbine and doxorubicin induced similar effects and their TGI concentrations were 10-fold higher than that of xanafide. No total growth inhibition was achieved with gemcitabine .μM, whereas paclitaxel, docetaxel and xanafide exhibited comparable TGI values: 20, 25 and 35 μM. Vinorelbine and gemcitabine were less potent as shown by their respective 2.6- to 5.7-fold higher TGI concentrations, respectively . Paclitaxel and docetaxel showed TGI concentrations of 35 and 60 μM, respectively. Xanafide did not induce any complete growth inhibition in this cell line , docetaxel dosed at 5 and 12.5 mg kg−1, i.p., or xanafide at 30 mg kg−1, i.p. on a q.d. × 5 treatment schedule, beginning on day 2 post-fibre implantation (days 3–7).On the basis of its cytotoxic activity P<0.05). Docetaxel exhibited dose-dependent growth inhibitory effects. At the lowest dose used, 5 mg kg−1, docetaxel produced a growth inhibition of 36 and 45%, in MCF-7 and MDA-MB-231, respectively (P<0.05). At 12.5 mg kg−1, the growth inhibition elicited was 39 and 51%, in MCF-7 and MDA-MB-231, respectively (P<0.05). These results show that xanafide was slightly more potent than docetaxel at its highest dose, in MCF-7. In contrast, its potency was lower than that of docetaxel in MDA-MB-231.As shown in P<0.05). Docetaxel showed dose-dependent inhibitory effects. At the lowest dose, the growth inhibition obtained was 31 and 36%, in MDA-MB-231 and MCF-7, respectively (P<0.05). At the highest dose used, 12.5 mg kg−1, the growth inhibition induced was 42 and 46%, in MCF-7 and MDA-MB-231, respectively (P<0.05).In the fibres retrieved from the s.c. sites , xanafidBody weights were recorded daily during the course of the study, and expressed as the difference observed relative to start of treatment. For the docetaxel treated mice no loss in body weight was observed while in the xanafide treated mice, body weight was reduced by 12% by day 8, which was within the acceptable range based on NCI established criteria .Amonafide, a DNA-intercalating agent and topoisomerase II inhibitor, has been used as first-line treatment for MBC. However, amonafide is extensively metabolised, including N-acetylation to an active metabolite, N-acetyl amonafide, and the extent of amonafide N-acetylation is the major determinant of myelosuppression , SF268 (glioma) and MCF-7 (breast), and in vivo in MCF-7 (breast), COLO205 (colon) and PC-3 (prostate) cell lines, using the hollow fibre assay  being the most sensitive and T47 D (ER+/p53 mutated) the most resistant. Although this cell line was responsive to low doses of the other drugs tested, the lack of activity of xanafide in the T47D could be, in part, due to its long doubling time.The aim of this study was to further investigate the antitumour effects of xanafide, in comparison with common breast drugs in a panel of breast cell lines well characterised for their oestrogen receptor, p53 status, Her-2 and topoisomerase II and T47D . Xanafidand T47D . The GI −/p53 mutated cell lines (MDA-MB-231 and SKBR-3) displayed comparable in vitro responsiveness to xanafide as demonstrated by their respective GI50 and TGI concentrations  was more resistant to xanafide, with no induced cell killing, as compared with the extent of the anti-proliferative effect observed with MCF-7 (ER+/p53 wt) (Our results showed that the two ER/p53 wt) . These fα expression could potentially provide a sufficient amount of target enzyme for optimisation of treatment with topoisomerase II inhibitors  or to apotosis  . Relatedhibitors , thus suα gene, TOP2 α, is located at chromosome band 17q12-q21, close to the ErbB-2 oncogene (HER-2/neu), which is the most commonly amplified oncogene in breast cancer. Because of the physical proximity to ErbB-2, copy number aberrations may also occur in the topoIIα gene, which may be related to the altered chemosensitivity to topoII inhibitors (α and β. Considering the TGI concentrations, MDA-MB-231 and SKBR-3 expressing low levels and high levels of both isoforms, respectively, exhibited comparable sensitivity to xanafide. Our data are in agreement with those reported by In addition, the topoisomerase II hibitors . Our resIn view of the previous findings together with our results, we speculate that p53 and ER and their signalling pathways are likely important determinants of breast tumour cells sensitivity to xanafide. Understanding these relationships may lead to strategies for xanafide-based chemotherapy optimisation and further precision targeting of tumour cells to avoid drug resistance and thereby chemotherapy failure. Furthermore, the steep response curves of xanafide activity in the four breast cell lines tested suggest that accurate individualised patient dosing is critically important for maximising clinical response while minimising neutropenic variability.


DT-diaphorase is a two-electron reducing enzyme that activates the bioreductive anti-tumour agent, mitomycin C (MMC). Cell lines having elevated levels of DT-diaphorase are generally more sensitive to MMC. We have shown that DT-diaphorase can be induced in human tumour cells by a number of compounds, including 1,2-dithiole-3-thione. In this study, we investigated whether induction of DT-diaphorase could enhance the cytotoxic activity of MMC in six human tumour cell lines representing four tumour types. DT-diaphorase was induced by many dietary inducers, including propyl gallate, dimethyl maleate, dimethyl fumarate and sulforaphane. The cytotoxicity of MMC was significantly increased in four tumour lines with the increase ranging from 1.4- to threefold. In contrast, MMC activity was not increased in SK-MEL-28 human melanoma cells and AGS human gastric cancer cells, cell lines that have high base levels of DT-diaphorase activity. Toxicity to normal human marrow cells was increased by 50% when MMC was combined with 1,2-dithiole-3-thione, but this increase was small in comparison with the threefold increase in cytotoxicity to tumour cells. This study demonstrates that induction of DT-diaphorase can increase the cytotoxic activity of MMC in human tumour cell lines, and suggests that it may be possible to use non-toxic inducers of DT-diaphorase to enhance the efficacy of bioreductive anti-tumour agents. © 1999 Cancer Research Campaign


CDKN2 gene. IN1434 cells share some cytogenetic abnormalities with Hu-O-2A/Gb1 cells, despite their apparent derivation from a different biological origin, but also have translocations involving the long and short arms of chromosome 1 and the long arm of chromosome 7, and deletion of chromosome 13 at bands 13q12–21. © 1999 Cancer Research CampaignWe have examined the genetic aberrations in two near-diploid glioblastoma multiforme cell lines that appear to have arisen from different glial lineages. One cell line, Hu-O-2A/Gb1, expresses antigens and metabolic profiles characteristic of the oligodendrocyte-type-2 astrocyte (0-2A) lineage of the rat central nervous system. This line generates, in vitro, cells with characteristics of 0-2A progenitor cells, oligodendrocytes and astrocytes. The second cell line, IN1434, is derived from an astrocyte or a precursor cell restricted to astrocytic differentiation. In Hu-O-2A/Gb1 the sole homologue of chromosome 10 is disrupted at band 10p11–12.1 by translocation with chromosomes X and 15. The translocation breakpoint is localized between genetic markers D10S2103 and . Other aberrations include a 5;14 translocation, deletion of the long and short arms of chromosome 16 and loss of one copy of the


Inherent resistance of colon cancer cells to cis-diamminedichloroplatinum(II) (CDDP) is partly attributed to reduced drug penetration through plasma membrane. Amphotericin B (AmB), a polyene antifungal antibiotic, has been shown to increase CDDP penetration and cytotoxicity on several non-digestive cancer cell lines. We demonstrated here that AmB dramatically increases the penetration of CDDP, and to a lesser extent that of carboplatin (Carbo-P) and oxaloplatin (L-OHP), in the primary resistant HT 29 human colon cancer cells when drug incubation is performed in serum-free medium. The cytotoxicity of CDDP but not that of Carbo-P and L-OHP was increased by AmB. However, AmB-induced potentiation of CDDP penetration and toxicity was almost completely abolished when cell incubation was performed in presence of human serum. We investigated whether the dilution of human serum by a high osmotic power gelatine solution (Lomol) could restore the positive effect of AmB on CDDP accumulation in HT 29 cells. Incubation of cells with CDDP and AmB in pure Lomol resulted in a 6-fold increase in platinum cellular content. However, addition of serum (25%) in Lomol solution reduced to only 2-fold the increase in platinum cellular content provoked by AmB. These disappointing results show that AmB is probably uninteresting as a modulator of CDDP resistance in clinical practice. The use of haemodilution to restore the positive AmB effect on platinum cellular accumulation cannot be warranted.


Two cell lines, one sensitive and one resistant to the cytotoxic effects of cytosine arabinoside (AraC) were studied in vitro as a drug-resistance model. The sensitivity of these cell lines, to the effects of free and liposomally trapped AraC and AraCTP as well as empty liposomes alone and mixed with free drug, was studied. This was done by following the inhibition of [3H]-dT incorporation into cellular DNA during exposure to the various drugs and liposomes. Some of the liposomal-lipid compositions inhibited [3H]-dT incorporation at very low concentrations, which made them unsuitable for further study. Liposomes composed of a 7:2:1 molar ratio of phosphatidylcholine:cholesterol:phosphatidic acid were selected as a suitable non-inhibitory carrier. Sensitivity of the two cell lines to free AraC differed by 3 logs, when compared in the [3H]-dT-incorporation assay. The resistant cell line was studied further, and was found to be up to 2 logs more sensitive to AraCTP when given in liposomes than to either the free drug alone or mixed with empty liposomes. It appears from these studies that liposomes are able to help overcome drug resistance in this cell line in vitro.


Two human primary liver cancer cell-lines, PLC/PRF/5 and Hep 3B, grown both in vitro and as xenografts in nude mice were used to evaluate the chemotherapeutic potential of a new quinazoline antifolate CB3717. Xenograft growth rate was followed both by serial serum alphafoetoprotein (AFP) measurement and direct volume estimation. A dose regime of 200 mg CB3717 kg-1 body wt day-1 for 5 days caused a significant reduction in growth rate, as measured by relative serum AFP, of both xenografts; PLC/PRF/5 derived xenograft growth was also inhibited by 125 mg CB 3717 kg-1 day-1 for 5 days. Cell culture experiments showed that the ID50 for the cell lines fell within the range of serum CB3717 concentration achieved by a dose of 300 mg m-2 given to patients. Treatment with CB3717 stimulated the incorporation of exogenous thymidine into DNA by the tumour cells, presumably because of inhibition of the de novo pathway and reduction of endogenous thymidine triphosphate pools. These results suggest that CB3717 may be a useful new therapeutic agent in human primary liver cell carcinoma and that blocking the salvage pathway may further increase efficacy.


Cytotoxic drug resistance developing after chemotherapy is thought to be the main cause of treatment failure in several human tumours, including small cell lung cancer (SCLC). Cell lines showing drug resistance following prolonged exposure to a single agent frequently acquire resistance to several functionally unrelated drugs, the phenomenon of multi-drug resistance (MDR). Classical MDR is thought to arise from changes effecting a reduction in intracellular availability of cytotoxic drugs. We describe a flow cytometry (FCM) technique to monitor the MDR phenotype in drug resistant variants of SCLC and non-SCLC cell lines. The technique is based on a multiparametric analysis of the nuclear binding of a model chemotherapeutic agent, the fluorescent dye Hoechst 33342 (Ho342), which is capable of supra-vital staining of DNA in intact, viable cells. A laboratory derived drug resistant SCLC cell line, H69/LX4, showed a significant (30%) reduction in nuclear binding compared to the parental line H69/P. Exposure to verapamil (VPL) rapidly increased (within 2 min) nuclear binding of Ho342, and the new equilibrium of nuclear staining, attained within 20 min, remained lower than the level achieved in the parental cell line, suggesting some ability of H69/LX4 to limit the effect of the drug efflux blocker. A drug resistant large cell carcinoma line showed only a small reduction (10%) in nuclear binding when compared to the parent line, and this difference was not altered by VPL. A drug resistant adenocarcinoma line showed less than 10% difference in nuclear binding compared with the parental line and neither line was significantly affected by VPL treatment. Our findings suggest that different mechanisms of resistance may occur in lung tumours of different tissue types. This technique may be extended to the rapid and direct examination of biopsy specimens of human solid tumours for evidence of multi-drug resistance.


Treatment with EGCG inhibited tumour growth (58%), microvessel density (30%), and tumour cell proliferation (27%) and increased tumour cell apoptosis (1.9-fold) and endothelial cell apoptosis (3-fold) relative to the control condition . EGCG may exert at least part of its anticancer effect by inhibiting angiogenesis through blocking the induction of VEGF. © 2001 Cancer Research Campaign http://www.bjcancer.comCatechins are key components of teas that have antiproliferative properties. We investigated the effects of green tea catechins on intracellular signalling and VEGF induction in vitro in serum-deprived HT29 human colon cancer cells and in vivo on the growth of HT29 cells in nude mice. In the in vitro studies, (-)-epigallocatechin gallate (EGCG), the most abundant catechin in green tea extract, inhibited Erk-1 and Erk-2 activation in a dose-dependent manner. However, other tea catechins such as (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epicatechin (EC) did not affect Erk-1 or 2 activation at a concentration of 30 μM. EGCG also inhibited the increase of VEGF expression and promoter activity induced by serum starvation. In the in vivo studies, athymic BALB/c nude mice were inoculated subcutaneously with HT29 cells and treated with daily intraperitoneal injections of EC (negative control) or EGCG at 1.5 mg day


Three human cell lines derived from oro-pharyngeal squamous cell carcinomas of the head were investigated for bone-resorbing activity in vitro. Culture media from all three spontaneously produced a non-dialysable osteolytic factor with activity in three in vitro assays for interleukin 1 (IL1), viz. the lymphocyte activating factor (LAF) assay, stimulation of collagenase synthesis by articular chondrocytes, and stimulation of prostaglandin E2 synthesis by fibroblasts. Addition of anti-human IL1 antibody to the culture media abolished all the bone-resorbing activity. Fractionation of the cell culture media by high performance liquid chromatography (HPLC) showed a single peak of activity in the chondrocyte assay with an apparent mol.wt of 15-17,000. This co-eluted with activity in a preparation of IL1 from rat peritoneal macrophage cultures. These results indicate that IL1 is responsible for the prostaglandin-independent bone resorbing activity synthesised by these cells in vitro, and may contribute to the bone destruction associated with the tumour.