Cell lines constitute a powerful model to study cancer, and here we describe three new epithelial ovarian cancer (EOC) cell lines derived from poorly differentiated serous solid tumors , as well as the matched ascites for one case (OV-1946).In addition to growth parameters, the cell lines were characterized for anchorage independent growth, migration and invasion potential, ability to form spheroids and xenografts in SCID mice.TP53 mutations were found in all cell lines, with TOV-1946 and OV-1946 harboring the same mutation, and none harbored the commonly observed somatic mutations in BRAF, KRAS or germline BRCA1/2 mutations found to recur in the French Canadian population. Conventional cytogenetics and spectral karyotype (SKY) analyses revealed complex karyotypes often observed in ovarian disease.While all cell lines were capable of anchorage independent growth, only the TOV-1946 and OV-1946 cell lines were able to form spheroid and produce tumors. Profiling of keratins, p53 and Her2 protein expression was assessed by immunohistochemistry and western blot analyses. Somatic This is the first report of the establishment of matched EOC cell lines derived from both solid tumor and ascites of the same patient. Epithelial ovarian cancer (EOC) is often described as the silent killer or the disease that whispers mainly due to absence of symptoms. This combined with the lack of specific/sensitive markers and/or techniques of screening leads to the diagnosis at late stages of the disease in more than 70% of patients. Unfortunately, the five year survival rate at this point of the disease is less than 30% . AlthougEOC is a complex disease stratified according to histopathological and morphological criteria. The majority of EOCs are thought to arise from the ovarian surface epithelium (OSE) that is derived from the coelomic epithelium. OSE is composed of multipotent cells that can differentiate and give rise to tumors of different histopathology types ,2. The lOver the past years several laboratories, including ours , have esIn this study, we describe three new serous EOC cell lines that were derived in our laboratory from either solid tumors or ascites of two chemotherapy-naïve patients. This is the first report characterizing cell lines derived from both solid tumor and ascites of the same patient. Moreover, the molecular and growth characteristics of the three cell lines present some unique features thereby providing the research community with new tools in the study of different aspects of serous EOC.Tumor samples were collected and banked following surgeries performed within the Division of Gynecologic Oncology at the Centre hospitalier de l'Université de Montréal . The study was approved by the CHUM institutional ethics committee and written consent was obtained from patients prior to sample collection. Stage was determined at the time of surgery. Histopathology and tumor grade were assigned by a pathologist according to the International Federation of Gynecology and Obstetrics (FIGO) criteria .2 and 5% O2.All primary cultures and cell lines were cultured in OSE medium  supplemented with 10% fetal bovine serum (FBS), 2.5 μg/ml amphotericin B and 50 μg/ml gentamicin. Cells were incubated in 5% COThe TOV-1946 cell line was established using the previously described scrape method on tumor tissue from patient 1946 ,43. BrieThe OV-1946 cell line was established from a mass of cells from the ascites of patient 1946. The mass was macro-dissected into small pieces, which were kept in a 100 mm petri dish for 27 days at which point adherent cells reached 80% confluence. Pieces of tissue were then discarded. Cells were divided in a proportion of 2:3 every week for the first 15 passages and then 1:2 twice a week until passage 70. Cells were then maintained and divided in a proportion of 1:5 twice a week.The TOV-2223 cell line was established from patient 2223 tumor tissue using the collagenase method. Briefly, tumor tissue was macro-dissected onto a 100 mm petri dish containing serum free OSE medium supplemented with 1000 U of collagenase . After 3–4 hours at 37°C, cells were resuspended into 8 ml of medium and the remaining tumor tissue pieces were discarded. The medium containing cells was then divided into four 60 mm petri dishes and 10% FBS was added. Cells were divided 1:2 once a week until passage 19 and then twice a week until passage 70. Subsequently, cells were maintained and divided in a proportion of 1:3 twice a week.For immunohistochemistry and western blot analyses, the following antibodies were used: beta actin AC-15 , p53 (D0-1) , anti-c-ErbB2/c-Neu , Keratin 19 Ab-1 , Keratin 7 Ab-2 (MS-1352-P0) and Keratin 8 Ab-4 .2O2 treatment was used to eliminate endogenous peroxidase activity. The sections were blocked with a protein blocking serum-free reagent  and incubated with different antibodies for 60 min at room temperature.Formalin fixed paraffin embedded tumors were sectioned at 4 μm and the slides were stained using the immunoperoxidase method. Briefly, tissue sections were heated at 60°C for 30 minutes, deparaffinized in toluene and rehydrated in an ethanol gradient. Slides were submerged in boiling citrate buffer (0.01 M citric acid adjusted to pH 6.0) and microwaved for 10 min to unmask antigens. A 3% H2O2 as a peroxidase substrate. Nuclei were counterstained with hematoxylin and all sections were observed by light microscopy at 400× magnification. Substitution of the primary antibody with phosphate buffered saline served as a negative control.The optimal concentration for each primary antibody was determined by serial dilutions. Tissues were incubated with either a secondary biotinylated antibody  or a rabbit anti-goat biotin-conjugated antibody (1:300)  for 20 min followed by incubation with a streptavidin-peroxidase complex  for 20 min at room temperature. Reaction products were developed using diaminobenzidine containing 0.3% H5 cells were seeded onto 60 mm petri dishes. On day 1, 3, 5, 7, 9, 11 and 13 the cells were trypsinized, resuspended in medium and counted using a hemacytometer. Each experiment was performed in triplicate for each harvest and repeated once. Saturation density was defined as the mean maximum number of cells at confluence counted from two independent experiments performed with triplicates and the doubling time was calculated according to the slope of the linear portion of the growth curve.Growth rates were assessed as previously described . On day 4 cells in agarose (0.33 g/100 ml OSE complete medium for the upper layer and 0.66 g/100 ml OSE complete medium for the base layer) dATP ; 1× PCR buffer ; 2.5 nmol each dCTP, dGTP and dTTP; 0.3 nmol dATP; 1.5 mM MgCl2; 15 pmol of each primer [h primer ; and 0.5BRCA1 and 8765delAG, 6085G>T and 3398delAAAAG in BRCA2 were investigated in DNA from patient matched peripheral blood lymphocytes as described [The common French Canadian founder mutations 4446C>T and 2953delGTAinsC in escribed .® system (ASI) mounted on a Zeiss Axioplan II microscope and analyzed using the SkyView version 1.6.1 software (ASI).Metaphase preparation and cytogenetic analyses with a trypsin-Giemsa banding technique of the TOV-2223, TOV-1946 and OV-1946 ovarian cancer cell lines were performed according to standard cytogenetic procedures. Clonal chromosomal abnormalities and GTG-banded karyotypes were described according to the International System for Human Cytogenetic Nomenclature . MetaphaCell lines were derived from both solid tumors (TOV-1946 and TOV-2223G) and ascites (OV-1946) of two chemotherapy naive patients. Patients 1946 and 2223 from which the cell lines were derived both presented poorly differentiated (grade 3) serous papillary cystadenocarcinoma at stage IIIC. Based on residual tumor present following surgery, both patients were considered to be suboptimaly debulked. Patient 2223 received a palliative treatment only and survived for 18 months while patient 1946 died from post-operative complications. Patient 1946 did not have any known familial history of cancer. However, two sisters of patient 2223 were diagnosed with ovarian cancer (70 and 80 years old) and numerous colon cancers were diagnosed within the family. Clinical data for both patients are summarized in Table At initial passage all three cell lines appeared more (TOV-1946 and TOV-2223) or less (OV-1946) heterogenous with populations of cells with obvious fibroblastic contaminants Figure . With suHematoxylin-Eosin stained tumor tissue sections of patient 1946 and 2223 Figure . This reOne of the hallmarks of cancer cells is their ability to grow in the absence of exogenous growth factors, which can be verified by culturing cancer cells in low serum conditions. All cell lines were able to grow in a medium containing only 1% of FBS. In general however, growth rates were slower in low serum conditions than those observed in a typical 10% FBS environments (data not shown).in vivo in the original tumor  analysis was limited to the recurrent mutations found in women of French-Canadian descent as cell lines were derived from patients of this ancestral origin [TP53 were identified in the DNA from all three EOC cell lines  [TP53 mutation analysis revealed no evidence of heterozygosity, it is likely that no functional p53 is encoded in these cell lines. No mutations in the other genes studied were found in the cell lines.In order to further characterize the cell lines, we performed somatic mutation analysis of database ,58. The s Figure  and 2I. atabase) . As TP53The cytogenetic alterations of the three cell lines were assessed by G-banded karyotyping and by spectral karyotyping (SKY). Twenty-five metaphases in G-banding and thirty-two metaphases in SKY (TOV-2223), twenty-two metaphases in G-banding and thirty-nine metaphases in SKY TOV-1946) and nineteen metaphases in G-banding and fifteen metaphases in SKY (OV-1946) were analyzed. The G-banded analysis of the three cell lines revealed a modal number of 51 to 71 chromosomes with complex karyotypes that are described as a composite karyotype containing the clonal chromosomal abnormalities . The homogeneity of cells observed at passage 0 of OV-1946 could reflect the fact that these cells came from a mass in the patient's ascites that is more homogenous then a tumor mass often composed of, among others, epithelial tumor cells, stromal cells and endothelial cells.Cell lines were also evaluated on their capacity to survive and grow in low serum conditions. It is worth noticing that although in normal growth conditions both TOV and OV-1946 cell lines present similar growth rates, in low serum conditions the later is almost two times slower. This could be in line with the fact that the OV-1946 cell line comes from ascites which is rich in growth factors and in culture can replace FBS . This ceKRT7, KRT8 and KRT18. In order to determine if our cell lines presented these EOC markers, we monitored protein expression of Krt7, Krt8 and Krt18 by western blot. When antibodies were appropriate for immunohistochemistry on paraffin embedded tissues, we also compared keratins expression in the original tumor tissues. It is also well described that monitoring expression of Krt7 and Krt20 could distinguish between ovarian and gastro-intestinal tract tumors . Both tHER2 gene leading to the overexpression of the protein Her2 . Theiewed in -67. In Eiewed in -63). AllDifferences in growth rates, migration, invasion and spheroid formation between the TOV-1946 and TOV-2223 underline the diversity of phenotypes that can be observed within the same type of serous EOC disease. The results from the comparison of the TOV-1946 and OV-1946 growth characteristics, demonstrate that these cell lines present with unique phenotypes. Indeed, TOV-1946 cells had a better capability to invade and migrate although their capacity to form spheroid is reduced compared to the OV-1946 cells. One might speculate that cells derived from solid tumor conserve their migration and invasion property but in ascites, these characteristics are less vital. It has been previously shown that cells present in ascites form spheres or aggregates that can adhere to different extracellular matrices as well as to normal human mesothelial cells  but are in vivo tumorigenicity can usually be predicted by the ability of the cells to grow in soft agar [in vivo [As both the TOV-1946 and OV-1946 injected mice developed ascites we hypothesize that the ability for cell lines to induce ascites formation in mice is intrinsic to the given cell line and independent of their origin (solid tumor versus ascites). It has been previously shown that the oft agar . The OV-[in vivo . ConsistThe unique features of the described cell lines provide complementary models for different aspects of the disease. For example, while TOV-2223 and the TOV-1946 cell lines are both derived from solid tumors of the same type of serous EOC disease, they vary in important aspects including their aggressiveness and karyotype. They may prove useful for comparative studies to uncover molecular events that distinguish very aggressive from more indolent serous disease in ovarian cancer. Although the TOV-1946 and OV-1946 lines were derived from the same patient, they were collected from the solid tumor and the corresponding ascites respectively. Although common genome modification are shared between these two cell lines  , the wound-healing assays, mice assays as well as writing and editing of the manuscript according to all authors revisions. LP derived and established the new cell lines from tumor samples and performed the immunohistochemistry on the primary cultures and cell lines. JM performed the immunohistochemistry on tumor tissue. JL performed the Western blot.M–LP performed the invasion assays. SLA and ZS performed mutations analyses. JH analyzed the SKY and G-banding assays. PNT, DMP and A–MM–M contributed to the conception and design of the study as well as analysis and interpretation of the data. All authors revised the manuscript and gave final verbal approval.The pre-publication history for this paper can be accessed here:A) G-banded metaphases from the TOV-2223 cell line (cells 15 and 36 respectively). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 45 and 46 respectively from the TOV 2223 cell line with identification of some marker chromosomes.Click here for fileA) G-banded metaphases from the TOV-2223 cell line (cells 15 and 36 respectively). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 45 and 46 respectively from the TOV 2223 cell line with identification of some marker chromosomes.Click here for fileA) G-banded metaphases from the TOV-1946 cell line (cells 6 and 43 respectively). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 44 and 36 respectively from the TOV-1946 cell line with identification of some marker chromosomes.Click here for fileA) G-banded metaphases from the TOV-1946 cell line (cells 6 and 43 respectively). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 44 and 36 respectively from the TOV-1946 cell line with identification of some marker chromosomes.Click here for fileA) G-banded metaphase from the OV-1946 cell line (cell 24). Arrows indicate the abnormal chromosomes, mar: marker chromosome. B) Combined inverted-DAPI and SKY image of cell 11 from the OV-1946 cell line with identification of some marker chromosomes.Click here for file


The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at http://www.path.cam.ac.uk/~pawefish. © 2000 Cancer Research Campaign


The most cytotoxic species is Cp(CpCO-NH-C6H4-(CH2)2CH3)TiCl2 with an IC50 of 24(2) μM, followed by Cp(CpCO-NH-C6H4-Br)TiCl2, IC50 of 46(4) μM and Cp(CpCO-NH-C6H4-OCF3)TiCl2, IC50 of 49(6) μM. There is no correlation between the nature of the para substituent on the phenyl ring and the cytotoxic properties on MCF-7 cell line.A new titanocenyl amide containing flavone as pendant group has been synthesized by reaction of titanocenyl carboxylic acid chloride and 7-Aminoflavone and structurally characterized by spectroscopic methods. This species and eight previously synthesized titanocenyl amide complexes have been tested in breast adenocarcinoma cancer cell line, MCF-7. The functionalization of titanocene dichloride with amides enhances the cytotoxic activity in MCF-7. Two sets of titanocenyl amides can be identified, with IC The fact that it possesses antitumor properties in cancer cell lines that are insensitive to cisplatin as well as lower toxic effects than cisplatin, has motivated the scientific community to continue investigating this species 2TiCl2 · 2HCl, on MCF-7 with an IC50 of 62 μM . Other types of functionalized titanocenes have been reported with enhanced cytotoxic properties and ICar range –22. For of 62 μM . Our mor7), with a propyl group on the para position of the phenyl ring, exhibited the highest cytotoxic activity. Finally, other strategies are currently being pursued such as replacement of chlorides by fluorides as ancillary ligands on Titanocene Y and derivatives [Beckhove and co-workers have reported anticancer properties of bis-[p-methoxybenzyl)cyclopentadienyl] titanium dichloride (Titanocene Y). This is active against a wide variety of cancer cell lines. Moreover, Titanocene Y has been tested in explanted human breast tumor cells and in xenografted MCF-7 tumors in mice with promising results in terms of doses, low toxicity, and reduction of tumor volume . Also itivatives . It has ivatives .The functionalization of titanocene dichloride with amides (phenyl amides) increases the cytotoxic activity of the resulting titanocenyl amide complexes. One possible explanation could be the Ti-O(amide) bond which provides stability in aqueous environment and makes the complex more resistant to hydrolysis. Although this is highly speculative, based on the structural features of these titanocenyl amides and the hypothesis that albumin is the carrier protein of titanocene into the target place inside the cell , 25, we Comparative Table of Titanocenyl Amides Complexes Between Colon Cancer HT-29 and Breast Cancer MCF-7 Cell Lines.Click here for additional data file.


The single-crystal X-ray structure of complex [SnPh2(L)(DMSO)] (5) shows that the ligand is doubly deprotonated and is coordinated as tridentate ligand.The six coordination number is completed by two carbon atoms of phenyl groups. There are two similar monomers 5a (Sn1) and 5b (Sn51) in the asymmetric unit. The monomers 5a and 5b are linked through intermolecular hydrogen bonds of N–H–O and C–H–S type. C–H → π, intermolecular interactions, intra- and intermolecular hydrogen bonds stabilize this structure and leads to aggregation and asupramolecular assembly. The IR and NMR  spectroscopic data of the complexes are reported. The in vitro cytotoxic activity has been evaluated against the cells of three human cancer cell lines: MCF-7(human breast cancer cell line), T-24 (bladder cancer cell line), A-549  and a mouse L-929 (a fibroblast-like cell line cloned from strain L). Compounds 1, 3, and 4 were found active against all four cell lines. Selectivity was observed for complexes 3 and 4 which were found especially active against MCF-7 and T-24 cancer cell lines.The synthesis and spectral characterization of novel diorganotin complexes with 3-hydroxypyridine-2-carbaldehyde thiosemicarbazone, H Organotin(IV) compounds find wide applications as catalysts and stabilizers, and certain derivatives are used as biocides, as antifouling agents and for wood preservation. It has been observed that several diorganotin adducts show potential as antineoplastic and antituberculosis agents –4. 1) is a member of the so-called α-(N)-heterocyclic carbaldehyde thiosemicarbazones (HCTs), which are the most potent known inhibitors of ribonucleoside diphosphate reductase. Compounds (1) and 5-hydroxypyridine-2-carbaldehyde thiosemicarbazone have shown high anticancer activity in animal models but were found to be readily glucuronidated and rapidly excreted  have been studied by us  (5). IR and NMR spectroscopic data are reported. The results of the cytotoxic activity of diorganotin complexes have been evaluated for antiproliferative activity in vitro against human cancer cell lines: MCF-7, T-24, A-549, and a mouse L-929.The present paper includes the interaction of SnPh−1) and nujol mulls dispersed between polyethylene disks (400–40 cm−1). The free ligand was dissolved in (CD3)2SO and 1H, 1H–1H COSY and 13C spectra were acquired on a BRUKER 300 MHz spectrometer. Compounds 2–4 were dissolved in CDCl3 and were spectroscopically characterized by the use of 1D and 2D NMR spectroscopy on a Varian 600 MHz spectrometer. Experimental data were processed using VNMR and WinNMR routines. Chemical shifts (δ) are reported in ppm while spectra were referenced by the standard experimental setup. 119Sn NMR spectra were acquired on the Varian 600 MHz and tin spectra were referenced by the use of external solution of Me4Sn in C6D6. The splitting of proton resonances in the reported 1H-NMR spectra is defined as s = singlet, d = doublet, t = triplet, and m = multiplet. The chemical shifts are reported in ppm for 1H and 13C NMR. Elemental analyses were carried out by the microanalytical service of the University of Ioannina, Greece.All reagents were commercially available (Aldrich or Merck) and used as supplied. Solvents were purified and dried according to standard procedures. Melting points (m.p.) were determined in open capillaries and are uncorrected. IR and far-IR spectra were recorded on a Perkin Elmer Spectrum GX Fourier transform spectrophotometer using KBr pellets (4000–400 cm−1): 3555 s, 3451 m v(OH); 3291 m, 3194 m, v; 1639 s, v(C=N); 1229 s, v(C–O); 1098 s, v(NN); 827 s, v(C=S). 1H-NMR: 11.50 ; 9.62 ; 8.12 , 3J = 4.2 Hz); 7.26  3J = 8.5, 4J = 4.2 Hz); 7.30  3J = 8.4, 4J = 1.4 Hz); 8.34 ); 8.00, 8.24 . 13C-NMR: 178.0 C(7); 153.1 C(4); 144.4 C(6); 141.1 C(1); 137.6 C(5); 125.3 C(2); 124.2 C(3). Anal. calc. for C7H8N4OS (196.04): C, 42.8; H, 4.1; N, 28.6; S, 16.3; found: C, 42.6; H, 3.9; N, 28.2; S, 16.0%.The ligand was synthesized according to a published procedure . The whi−1): 3296 m, v(NH2); 1580 s, v(C=N); 1176 s, v(C–O); 1111 s, v(NN); 804 w, v(C=S); 583 m, 564 v(Sn–C); 431 m, v(Sn–N); 231 m, v(Sn–O); 376s, ν(Sn–S). 1H-NMR: 8.09 , 3J = 4.2, 4J = 1.4 Hz); 7.18  3J = 8.5, 4J = 4.2 Hz); 7.08  3J = 8.5, 4J = 1.4 Hz); 8.76 ); 5.09 ; 0.89  = 35.7 Hz). 13C-NMR: 168.8 C(7); 163.4 C(4); 161.5 C(6); 140.2 C(1); 135.4 C(5); 127.6 C(2); 128.9 C(3); 6.22  = 309 Hz). 119Sn NMR: δ = −94.5. Anal. calc. for C9H12N4OSSn (343.0): C, 31.5; H, 3.5; N, 16.3; S, 9.3; found: C, 31.5; H, 3.7; N, 16.0;, S, 9.1 %.Dimethyltin(IV) oxide  and 3-hydroxypyridine-2-carbaldehyde Thiosemicarbazone  in benzene (20 mL) were stirred and were refluxed for 12 hours under azeotropic removal of water (Dean-Stark trap). The resulting clear solution was rotary evaporated under vacuum to a small volume (2 mL), chilled and triturated with diethyl ether to give a white solid. The powder was recrystallized from distilled diethyl ether and dried in vacuo over silica gel to give yellow solid; mp. 228–230°C, Yield 35%. IR (cm−1): 3292 m, v(NH2); 1577 s, v(C=N); 1173 s, ν(C–O); 1114 s, v(NN); 805 w, ν(C=S); 578 ms, 560 sh ν(Sn–C); 418 m, ν(Sn–N); 247 m, ν(Sn–O); 394 ms, ν(Sn–S). 1H-NMR: 8.05 , 3J = 4.2, 4J = 1.3 Hz); 7.16  3J = 8.5, 4J = 4.2 Hz); 7.07  3J = 8.5, 4J = 1.3 Hz); 8.82 ); 5.22 ; 0.87 ; 1.32 ; 1.54, 1.64 . 13C-NMR: 167.7 C(7); 162.5 C(4); 160.0 C(6); 138.7 C(1); 134.5 C(5); 126.8 C(2); 128.0 C(3); 26.9  = 265 Hz); 28.2 (Cβ); 27.3 (Cγ); 14.6 (Cδ). 119Sn NMR: δ = −194.4. Anal. calc. for C16H18N4OSSn (537.2): C, 42.2; H, 5.7; N, 13.1; S, 7.5; found: C, 42.0; H, 5.9; N, 13.2; S, 7.4 %.Dibutylltin(IV) oxide  and 3-hydroxypyridine-2-carbaldehyde Thiosemicarbazone  in benzene (20 mL) were stirred and were refluxed for 12 hours under azeotropic removal of water (Dean-Stark trap). The resulting clear solution was rotary evaporated under vacuum to a small volume (2 mL), chilled and triturated with diethyl ether to give a white solid. The powder was recrystallized from distilled diethyl ether and dried in vacuo over silica gel to give yellow solid; mp. 126–128°C, Yield 41%. IR (cm−1): 3269 m, ν(NH2); 1589 s, ν(C=N); 1185 s, v(C–O); 1118 s, v(NN); 808 m, v(C=S); 322 ms, 303 sh ν(Sn–C); 419 m, ν(Sn–N); 248 m, ν(Sn–O); 370 ms, ν(Sn–S). 1H-NMR: 8.02, 3J = 4.2 Hz); 7.18, 3J = 8.5, 4J = 4.2 Hz); 7.29 , 3J = 8.5 Hz); 8.88 ); 5.37 ; 7.82 ; 7.34–7.30 . 13C-NMR: 167.4 C(7); 164.0 C(4); 161.5 C(6); 140.2 C(1); 135.4 C(5); 127.8 C(2); 129.3 C(3); 142.1  = 424 Hz); 135.8  = 28 Hz); 128.8  = 37 Hz); 130.2  = 8.7 Hz); 119Sn NMR: δ = −227.2. Anal. calc. for C19H16N4OSSn (663.1): C, 48.9; H, 3.5; N, 12.0; S, 6.9; found: C, 48.6; H, 3.5; N, 10.7; S, 6.8%.Diphenylltin(IV) oxide  and 3-hydroxypyridine-2-carbaldehyde thiosemicarbazone  in benzene (20 mL) were stirred and were refluxed for 12 hours under azeotropic removal of water (Dean-Stark trap). The resulting clear solution was rotary evaporated under vacuum to a small volume (2 mL), chilled and triturated with diethyl ether to give a white solid. The powder was recrystallized from distilled diethyl ether and dried in vacuo over silica gel to give yellow solid : mp. 186–188°C, Yield 34%. IR  Sp. z o.o  programs. Crystal structure was solved by direct methods (program SHELXS97) and refined by the full-matrix least-squares method on all F2 data using the SHELXL97  and [Pd(Ac4Et)2] complexes can be characterized by cellular pharmacological properties distinctly different from those of cis-platin [2(Fo4Npypipe)] and [ZnCl2(Ac4Npypipe)] where Fo4Npypipe and Ac4Npypipe are the monoion of 2-formyl pyridine N(4)-1-(2-pyridyl)-piperazinyl thiosemicarbazone and 2-acetyl pyridine N(4)-1-(2-pyridyl)-piperazinyl thiosemicarbazone have been evaluated in vitro against MCF-7, T-24, A-549, and L-929 cell lines and it was found to exhibit remarkable antiproliferative activity with mean IC50 values of 0.2–20 μM [Complexes of s-platin . The com.2–20 μM . The dip.2–20 μM .cis-platin and diorganotin(IV) oxides. Results showed that the ligand as well as the complexes 3 and 4 demonstrated excellent antiproliferative activity, IC50 values range from 0.02–2.5 μM, against all cell lines tested, while for cis-platin the IC50 values range from 0.7–41 μM. The diorganotin(IV) oxides are nontoxic against L-929 and T-24 cancer cell lines and exhibit poor cytotoxic activity in A-549 and excellent antiproliferative activity against MCF-7 cancer cell line.The antiproliferative activity of compounds is presented in 1 is more cytotoxic compared to cis-platin against the A-549, T-24 and MCF-7 cell lines and less cytotoxic against L-929 cell lines. The diorganotin complex 2 is nontoxic against T-24 cell line, less cytotoxic against A-549 and L-929, and more cytotoxic against MCF-7 cell line compared to cis-platin. The diorganotin complexes 3 and 4 are in the same μM range compared to cis-platin against L-929 and A-549 cancer cell lines and more cytotoxic against T-24 and MCF-7 cancer cell lines. The IC50 values for the ligand, 1, against MCF-7 and T-24 cell lines are 0.15 μM and 2.09 μM, respectively, and against A-549 and L-929 cell lines are 0.81 μM and 2.5 μM, respectively. Ligand is 19.8 ± 1.3 and 52.4 ± 4.6 times more active than cis-platin against T-24 and MCF-7 cell lines, respectively. The IC50 values for 3 against MCF-7 and T-24 cell lines are 1.97 × 10−2 μM and 1.1 μM, respectively, and against A-549 and L-929 cell lines are 0.77 μM and 1.05 μM, respectively. Complex 3 is 403.6 ± 20.2 times more active than cis-platin against MCF-7 cell line and 37.9 ± 3.1 times more active than cis-platin against T-24 cell line. The IC50 values for 4 against MCF-7 and T-24 cell lines are 7.28 × 10−2 μM and 0.73 μM, respectively, and against A-549 and L-929 cell lines are 0.83 μM and 1.37 μM, respectively. Complex 4 is 110.3 ± 9.9 times more active than cis-platin against MCF-7 cell line and 57.1 ± 3.9 times more active than cis-platin against T-24 cell line. Compounds 1, 3, and 4 were found active against all four cell lines. Selectivity was observed for complexes 3 and 4 which were found especially active against MCF-7 and T-24 cancer cell lines. The mentioned evident differences in the antiproliferative action of the ligand and its diorganotin(VI) complexes indicate that these complexes really exist under the condition of the biological tests. Interestingly enough, 3 and 4 were found to be more potent cytotoxic agent than the prevalent benchmark metallodrug, cis-platin, under the same experimental conditions measured by us. The superior activity of 3 and 4 assumes significance in light of the fact that cis-platin is undisputedly the most studied and widely used metallopharmaceutical for cancer therapy known to date. It is noteworthy the high selectivity against MCF-7 and T-24 cancer cell lines.The ligand


A cDNA library generated from the β-TC3 SV40 T-antigen transformed insulinoma cell line was screened for members of this receptor family potentially relevant to both beta cell development and autoimmunity. Degenerate oligonucleotide primers to a consensus region of these receptors were used and the IL-11 receptor (α chain was identified. Despite confirmation of IL-11 receptor mRNA expression, iodinated bioactive IL-11 did not bind specifically to β-TC3 cells and gp-130-dependent cytokines did not elicit signalling events in beta cell lines. This was explained by absence of gp-130 protein or mRNA in the beta cell lines tested and in primary islets. We conclude from these resuits that the previously recognised effects of IL-6 family member cytokines on pancreatic islets must be indirect


A panel of 18 well characterised human small cell lung cancer (SCLC) cell lines was assessed for the production of adrenocorticotrophin (ACTH) and its precursor peptides, pro-opiomelanocortin (POMC) and pro-ACTH. These precursor peptides were measured directly using a novel two-site immunoradiometric assay (IRMA) based on monoclonal antibodies, in conjunction with a similar IRMA for ACTH 1-39. Significant concentrations of ACTH precursors were secreted by 10 of the 18 cell lines (56%). The low levels of ACTH immunoreactivity detected in seven cell lines could be accounted for by the known cross-reactivity of precursors in the ACTH IRMA. This suggests there is little, if any, processing of ACTH precursors to ACTH. Cell pellet extracts contained undetectable or low levels of ACTH precursors and ACTH, indicating that these peptides are not stored intracellularly. During the growth of the SCLC cells in vitro ACTH precursors accumulated progressively in the culture medium. Thus the combination of a direct assay for the ACTH precursors and the panel of SCLC cell lines provides a valuable in vitro model for the expression of POMC in human tumours.


The effects of single doses of ICRF 159 (Razoxane) on the cell-cycle kinetics of lines of BKH 21S cells in vitro were studied by means of flow micro-fluorometry (FMF). A characteristic accumulation of cells in the 4n DNA region after 6 h in the presence of ICRF 159 was evident in cells which were sensitive to the cytotoxic effects of the agents, as judged by colony-forming assays. The subsequent accumulation of 8n cells as the major population within 24 h indicates that this 6 h effect is associated with the induction of tetraploidy through abnormal mitosis, rather than reflecting a G2 block of potential value in combination therapy. In ICRF 159-resistant sublines, the cell-cycle distributions were similarly affected, but only the doses of the drug high enough to reduce their surviving fraction. Somatic cell hybrids from crosses between sensitive and resistant cells demonstrated intermediate responses in ICRF 159, both in terms of cell-cycle kinetics and impairment of reproductive integrity. These data suggest a relationship between the 2 manifestations of the cellular action of ICRF 159, and also a possible predictive role for FMF in the assessment of response to this particular agent.


Br J Cancer74: 999–1004). To define regulation of TN further and investigate its function in ovarian cancer, a range of cell line models were studied. Concentrations of secreted TN in media from cultures of ovarian fibroblast cell lines were at least 100-fold greater than from carcinoma cell lines. Evidence for paracrine regulation of TN secretion was obtained by co-culture of carcinoma cells with fibroblast cells wherein secretion into the media was greater than from fibroblasts alone. Transforming growth factor (TGF)-β1, insulin-like growth factor (IGF)-II and progesterone all stimulated TN secretion while human choriogonadotropin (hCG), follicle-stimulating hormone (FSH) and γ-interferon inhibited secretion. TGF-β1 produced the greatest stimulation of TN in cultured fibroblasts and its co-expression with TN was examined in primary ovarian tumours. There was a significant association between the presence of moderate–strong expression of TN and TGF-β1. Evidence for TN having a functional role in ovarian carcinoma was obtained from adhesion and migration assays. The PE01, PE04, SKOV-3 and 59M cell lines all demonstrated marked adhesion to plastic coated with TN relative to the control protein bovine serum albumin (BSA) and expressed α2β1 and α3β1 integrins. The SKOV-3 cell line migrated more rapidly through TN than through BSA indicating that TN can facilitate migration of ovarian carcinoma cells. © 1999 Cancer Research CampaignThe extracellular matrix glycoprotein tenascin-C (TN) is overexpressed in the stroma of malignant ovarian tumours particularly at the interface between epithelia and stroma leading to suggestions that it may be involved in the process of invasion


Cell fusion has been used to analyse the genetic determinants of metastasis at the cellular level. Highly metastatic mouse melanoma cells were fused with diploid mouse lymphocytes and a range of hybrid clones isolated and tested for tumorigenicity and metastatic potential by s.c. injection into newborn, histocompatible, sublethally-irradiated mice. Although almost all clones tested were tumorigenic, most had considerably reduced metastatic potential. This suggests that tumorigenicity and metastasis are determined by different genetic elements. Histological examination of primary tumours produced by metastatic and non-metastatic hybrid cell lines showed that an essential step in the production of metastases is the separation of tumour cells from the main tumour mass and their movement into the surrounding tissues. The primary tumours of a metastatic hybrid cell line showed local invasiveness whereas those of a non-metastatic cell line did not.


Despite substantial improvements in childhood cancer survival, drug resistance remains problematic for several paediatric tumour types. The urgent need to access novel agents to treat drug-resistant disease should be expedited by pre-clinical evaluation of paediatric tumour models during the early stages of drug development in adult cancer patients.50 values ranged from 1-200 nM and drug potency correlated both with DTD levels and drug-induced apoptosis. However, synergy between RH1 and cisplatin or doxorubicin was only seen in low DTD expressing cell lines. In clonogenic assays RH1 IC50 values ranged from 1.5–7.5 nM and drug potency did not correlate with DTD level. In A673 Ewing's sarcoma and 791T osteosarcoma tumour xenografts in mice RH1 induced apoptosis 24 h after a single bolus injection (0.4 mg/kg) and daily dosing for 5 days delayed tumour growth relative to control.The novel cytotoxic RH1  is activated by the obligate two-electron reductase DT-diaphorase  to a potent DNA interstrand cross-linker. In acute viability assays against neuroblastoma, osteosarcoma, and Ewing′s sarcoma cell lines RH1 ICThe demonstration of RH1 efficacy against paediatric tumour cell lines, which was performed concurrently with the adult Phase 1 Trial, suggests that this agent may have clinical usefulness in childhood cancer. Average 5–year survival rates for childhood cancers have improved dramatically over the past few decades, such that 75% of children with cancer can now expect to be cured . RDES and A673 ESFT cells were kindly provided by Sue Burchill . The osteosarcoma cell lines 791T and U2OS were from the Paterson Institute cell bank. All cell lines were maintained in RPMI 1640 or DMEM F12  supplemented with 10% fetal bovine serum. Lysates of rhabdomyosarcoma cell lines were kindly provided by John Anderson .RH1 was provided by Allos Therapeutics Inc. , and dissolved in DMSO. Other cytotoxic anticancer drugs were purchased from Sigma-Aldrich .2 for 7–10 days until visible colonies (>50 cells) were present in the untreated well. All assays were performed in triplicate. Surviving fraction was calculated as number of colonies in the test condition/number of colonies in the untreated well and plotted logarithmically against drug concentration.Clonogenic assays were performed as described earlier .The sulforhodamine B (SRB) assay  was usedProtein lysates were prepared as described earlier  assay was used to measure functional DTD levels in cell pellets of both the sensitive and resistant cell lines. Diphtheria toxin-diaphorase activity was determined by following the spectrophotometric dicoumarol inhibitable fraction of DCPIP reduction ) and shape of the concentration-effect curve. The ratio of RH1 to the cytotoxic agent of interest was fixed from the IC50 values from the SRB assay. Cells were treated with RH1 and cytotoxic for 1 h. Seven drug concentrations were used covering the concentration-effect curve. Linear correlation coefficients (r) were generated for each curve to determine the applicability of the data to the method of analysis. In all experiments, r was >0.9.Combination Index (CI) methodology was used to evaluate multiple drug-effect interactions with CalcuSyn software  . This us6 A673 cells in 0.2 ml serum-free RPMI tissue culture media were implanted by a single subcutaneous (s.c.) injection into the right flank. For 791T studies, 3 × 106 cells in a 50 : 50 mixture of serum-free RPMI tissue culture media and matrigel to a total volume of 0.2 ml were implanted s.c. in the right flank. Tumours were allowed to grow to 200 mm3, at which point animals were randomly assigned to control or treatment groups. RH1 treatment consisted of intraperitoneal (i.p.) injection of 0.4 mg kg−1 daily for 5 days as described earlier (2 divided by 2) was measured daily. When tumour volume reached 1250 mm3, the mice were killed by a schedule 1 method and tumours were excised and formalin fixed and wax embedded before immunohistochemistry for cleaved caspase 3 . All procedures were carried out in accordance with UKCCCR guidelines 1999 by approved protocols (Home Office Project license no. 40-2804).Balb/c-NUDE mice were used for all experiments. For A673 studies, 5 × 10Individual profile plots of tumour volume against days from randomisation were examined. After taking base 10 logarithms the plots were well approximated with straight lines. Formal fitting was then performed using linear mixed effect models, which incorporated animal-specific random intercepts and slopes. Primary focus lay in the difference in average slope between the RH1 and control groups for each tumour type separately. A pooled estimate and test of this primary parameter was obtained over the two tumour groups using meta-analysis methods given the estimated effect sizes and their standard errors.*2) , which have high level DTD activity, and 791T osteosarcoma cells (5 nM) which showed the highest DTD activity. There was also no correlation between sensitivity to cisplatin or doxorubicin and sensitivity to RH1; U2OS osteosarcoma cells and LA-15S neuroblastoma cells had the same IC50 values to doxorubicin but varied three-fold in their sensitivity to RH1, and LA-15S and LA-155n neuroblastoma cells, with the same IC50 values to RH1 varied three-fold in their sensitivity to cisplatin. Thus, RH1 is a potent inhibitor of clonogenicity in these paediatric tumour cell lines, and potency is independent of DTD expression or activity.The efficacy of RH1 at inhibiting colony formation was assessed across the paediatric tumour cell line panel. RH1 potency was superior to the DNA damaging agent cisplatin, and the topoisomerase inhibitor and DNA intercalating agent doxorubicin, both of which are in widespread clinical use against childhood cancer. RH1 was the most potent of the three agents examined, with 1000-fold greater potency than cisplatin, and 19- to 88-fold greater potency than doxorubicin . Clonoge5–7.5 nM . There w50 values ranging from 1 nM for SH-EP1 neuroblastoma cells, to 200 nM for U2OS osteosarcoma cells  also had the only IC50 values for RH1 above 40 nM. There was no correlation between sensitivity to RH1 and sensitivity to doxorubicin. Although there was no correlation between expression levels or activity of DTD and sensitivity to RH1, there was a correlation within cell line pairs. For example, SH-EP1 neuroblastoma cells, with relatively high DTD activity, had 11-fold greater sensitivity to RH1 than SH-SY5Y neuroblastoma cells, which had no detectable DTD activity. This 10-fold difference in sensitivity to RH1 between the high and low DTD-expressing cell lines was also seen with the LA1-5S/55n neuroblastoma cells (IC50 11 vs 120 nM) and the 791T/U2OS osteosarcoma cells (IC50 26 vs 200 nM). The only cell line pair in which there was not a 10-fold difference in IC50 to RH1 was the Ewing's sarcoma cell lines, A673 and RDES, in these DTD activity was similar (83 vs 57 nM min−1 mg−1). Thus, paediatric tumour cell lines are sensitive to RH1 in an SRB assay of population kinetics, and in this assay, sensitivity to RH1 correlates with sensitivity to cisplatin, and within tumour types, with DTD activity.In keeping with the clonogenic assay results, the comparison of RH1 with cisplatin and doxorubicin in SRB assay of cell population growth showed that RH1 was again the most potent agent, with ICma cells . RH1 wasma cells . In the 50 values of 26 and 200 nM. LA-15S and LA-155n cells also underwent differential induction of apoptosis, again correlating with the differences in their SRB IC50 values. With A673 and RDES Ewing's sarcoma cell lines, there was little difference in their IC50 values by SRB assay, reflected by similar levels of apoptosis between the two cell lines . Thus, the effects of the combination of RH1 with cisplatin or doxorubicin were investigated. The efficacy of fixed ratio combinations of RH1 and either cisplatin or doxorubicin was assessed for all eight cell lines again using the SRB cell population growth assay. Calcusyn software was used to calculate synergy (low combination index (CI) values), additivity , and antagonism . Although strong synergy was seen between RH1 and cisplatin in LA1-55n neuroblastoma cells, at all three concentration levels tested, this was not a constant feature across the panel. In SH-EP1 and LA1-5S neuroblastoma cells RH1 was antagonistic with cisplatin at all three concentration levels , and in trations . In LA1-−1 RH1 was sufficient to induce apoptosis in A673 Ewing's sarcoma grown as s.c. xenografts in nude mice. A single i.p. dose of 0.4 mg kg3 before random assignment of tumour-bearing mice to treatment with i.p. injection of RH1 (0.4 mg kg−1) or DMSO control daily for 5 days. No differences were observed between test and control groups of mice in general health or weight during the course of the experiment, suggesting no general host toxicity of RH1 treatment. Analysis of individual tumour growth rates in a mixed linear effects model allowed the slope of tumour growth to be plotted for each animal (vs vehicle-treated mice across the two tumour types verged on significance (P=0.057)  .in vitro where it is more potent than either of the clinically used conventional DNA damaging agents, cisplatin and doxorubicin. RH1 was developed as a substrate for the two-electron reductase DTD, with the aim of selective activation of the agent in tumour tissue. The absolute requirement for and importance of DTD activity for the cytotoxic function of RH1 is controversial. Within modified isogenic pairs of both breast cancer and colon cancer cell lines, RH1 is clearly more effective against the cells expressing higher levels of DTD (M min−1 mg−1), two of the neuroblastoma cell lines studied had DTD activity below this threshold , yet had clonogenic IC50 values for RH1 of 2.5 nM, identical to that for LA-15S cells with DTD activity of 103 nM min−1 mg−1, well above the suggested saturation point for maximum RH1 activation  are exquisitely sensitive to this agent Earlier studies have shown rapid onset of apoptosis within 6 h of RH1 treatment, increasing up to 48 h after drug removal . Althoug50 values significantly higher than the other lines  and were also relatively resistant to cisplatin. The data suggest that there may be potential benefit in combining RH1 with other cytotoxic agents. However, these in vitro methods are merely suggestive of possible synergy in vivo and the possibility of greater toxicity when combining several DNA damaging agents needs careful consideration.According to the CI, doxorubicin and RH1 were synergistic in several cell lines, including U2OS osteosarcoma cells, and LA155n and SH-EP1 neuroblastoma cells. Synergy between RH1 and cisplatin was only observed in LA155n neuroblastoma cells. Interestingly, U2OS and LA155n cells, in which the greatest synergy between RH1 and doxorubicin was observed, were the two most resistant cell lines to RH1 in the SRB assay, with ICin vitro. This level of apoptosis was not maintained at later time points, suggesting that repeat dosing of RH1, as used in the in vivo experiments, would be required for prolonged effect on tumour growth. RH1 inhibited the growth of both A673 Ewing's sarcoma and 791T osteosarcoma cells grown as xenografts in nude mice. This combination of Ewing's sarcoma and osteosarcoma xenograft data is reflective of the clinical situation where RH1 will be initially evaluated against all relapsed childhood solid tumours.In A673 xenografts, RH1 increased the number of apoptotic cells 24 h after a single dose of RH1, in agreement with the kinetics of apoptosis observed in vitro. RH1 induced apoptosis in paediatric xenografts grown in mice, and slowed the growth of these xenografts. This data supports further investigation of the usefulness of this agent in the clinic.In conclusion, paediatric tumour cell lines reflecting tumour types with poor prognosis were sensitive to the novel bioreductive agent RH1, regardless of their expression of DTD. In selected tumour types, RH1 was synergistic with doxorubicin


The derivation of permanent cell lines from 40 resected oesophageal carcinomas has been attempted. Five long-term lines have been established from three adenocarcinomas, one mixed carcinoma and one squamous carcinoma. Molecular and cellular analyses have been carried out on the lines and clones derived from them. Karyotype analysis indicates genetic variation among the clones. HLA-A, -B and -C is expressed constitutively, but not HLA-DR. ICAM-1-expressing phenotypes may have arisen during adaptation to long-term culture. All lines are capable of response to interferon-gamma (IFN-gamma) and all produce transforming growth factor beta 1 (TGF-beta 1). Two lines are resistant to the inhibitory growth effects of the latter, possibly contributing to malignancy. It is anticipated that these lines, originating from histologically different carcinomas, will provide a valuable, continuous resource for the investigation and treatment of these aggressive tumours.


Substitutions adjacent to the amino group in the 2-phenyl ring with a halogen atom or methyl group enhance potency in sensitive breast lines . Transient biphasic dose responses were induced but rapidly eradicated after specific drug exposure periods. Two human prostate carcinoma cell lines were refractory to the growth-inhibitory properties of 2-(4-aminophenyl)benzothiazoles; IC50 values > 30 microM were obtained. Potency and selectivity were confirmed when compounds were examined in the National Cancer Institute's Developmental Therapeutics screen; the spectrum of activity included specific ovarian, renal, colon as well as breast carcinoma cell lines. Moreover, comparing 6-day and 48-h incubations, the exposure time-dependent nature of the biphasic response was corroborated. Differential perturbation of cell cycle distribution followed treatment of MCF-7 and MDA 468 cells with substituted 2-(4-aminophenyl)benzothiazoles. In MDA 468 populations only, accumulation of events in G2/M phase was observed. Two MCF-7 cell lines were established with acquired resistance to CJM 126 , which exhibit cross-resistance to substituted benzothiazoles, but equal sensitivity to tamoxifen and doxorubicin. Compared with standard anti-tumour agents evaluated in the National Cancer Institute in vitro cell panel, benzothiazoles revealed unique profiles of growth inhibition, suggesting a mode(s) of action shared with no known clinically active class of chemotherapeutic agents.


Intereferon-γ (IFN-γ) has some anti-tumour activity in human ovarian cancer. This cytokine inhibited proliferation in three of four ovarian cancer cell lines in vitro. We then compared the action of IFN-γ in two cell lines, one sensitive and one resistant to its growth inhibitory effects. IFN-γ signalling appeared normal in both cell lines, with stat1 DNA binding activity detectable at 30 min. Continuous exposure to IFN-γ for 2–3 days was necessary for an irreversible effect on cell growth and apoptosis in cells sensitive to growth inhibition. During this time there was an increase in mRNA for the CKI p21, but no alterations in mRNA levels for other members of the CKI family. Maintenance of p21 mRNA required continuous mRNA synthesis. mRNA for the transcription factor IRF-1 was also induced in growth inhibited cells with similar kinetics to those observed for p21. Maximal induction of both p21 and IRF-1 mRNA was observed after 2–3 days IFN-γ exposure as the cells became committed to cell death. There was also a rapid increase in p21 and IRF-1 mRNA in cells resistant to the growth inhibitory effects of IFN-γ, but this increase was not maintained. Thus, continuous interaction with the IFN-γ receptor, together with a sustained induction of p21 and IRF-1, is associated with growth inhibitory and apoptotic effects of IFN-γ in ovarian cancer cells. © 1999 Cancer Research Campaign


Gravimetric and histological studies of lymphoreticular tissues during growth of a syngeneic squamous cell carcinoma in Wistar rats show that the regional lymph node anergy reported previously in this system is associated with replacement of paracortical small lymphocytes by large blastoid cells. The regional node continued to gain weight throughout the period of anergy and showed no atrophy and minimal necrosis. Over the same period the spleen increased in both weight and cytotoxicity.


The expression of hepatocyte growth factor/scatter factor (HGF/SF) was studied in 12 mesothelioma cell lines characterized by either an epithelioid or a fibroblast-like phenotype. Conditioned media from these lines were analysed by bioassay and ELISA, and HGF/SF was detected in three cell lines, all with a fibroblast-like or mixed morphology. None of eight epithelioid cell lines expressed the factor. Thus, for these cell lines, the ability to secrete HGF/SF correlated with the cell phenotype. Following on from these observations, two cell lines, BR and BT, with a fibroblast-like and an epithelioid phenotype, respectively, were further investigated. Both cell lines expressed the Met receptor but only BR secreted HGF/SF. Both cell lines responded to exogenous HGF/SF treatment by a change of morphology but in different ways: BR became more elongated and bipolar, while BT formed more spread-out cell colonies. HGF/SF acted as a paracrine effector on the epithelioid BT cells and stimulated both cell-spreading and proliferation. Interestingly, BT cells spread but did not scatter in response to exogenous HGF/SF. In contrast BR cells showed only some stimulation of cell motility with HGF/SF and no increase in cell proliferation was observed. Because HGF/SF was previously found in the pleural effusion fluids of patients with malignant mesothelioma and in paraffin-embedded tumour tissues, it is concluded that HGF/SF may well stimulate the growth and spread of malignant mesothelioma in vivo by paracrine and/or autocrine mechanisms.


XLand BAK have been determined in a panel of 12 human ovarian carcinoma cell lines encompassing a wide range in sensitivity to cisplatin. Whereas BAX, BCLXLand BAK levels did not correlate with sensitivity, there was a statistically significant inverse correlation  between growth inhibition by cisplatin and BCL-2 levels. In sublines possessing acquired resistance to various platinum-based drugs or across a panel of human ovarian carcinoma xenografts, there was no consistent pattern of BCL-2 expression. Two relatively sensitive lines (A2780 and CH1) have been stably transfected with bcl-2 and bclXLrespectively and two relatively resistant lines (A2780cisR and SKOV-3) stably transfected with bax. Overexpression of BCL-2 in A2780 cells led to resistance to cisplatin compared to the vector control when assayed at 48 h post-drug incubation but a significant increase in sensitivity at 96 h. Relative rates of apoptosis at 48- and 96-h post-cisplatin exposure mirrored the growth inhibition. There was no significant difference in sensitivity of the pair of lines by clonogenic assay. No significant changes in chemosensitivity to a variety of DNA-damaging or tubulin-interactive agents were observed in the remaining transfected lines. Taken together, these results suggest that, in human ovarian carcinoma cells, high BCL-2 levels  confers a trend towards sensitivity not resistance to platinum drugs. © 2000 Cancer Research CampaignThe expression of the BCL-2 family proteins, BCL-2, BAX, BCL


The Con A acceptor glycoproteins of murine and human tumour cell lines revealed by two-dimensional fingerprinting on polyacrylamide gels fall into two main categories: constant glycoproteins expressed by all cell lines and variable glycoproteins which are only expressed by particular tumour cell lines. Since the number of variable glycoproteins on a typical fingerprint is 50, fingerprints from different cell lines are readily distinguishable. However the variable glycoproteins are not expressed idiosyncratically and cell lines derived from similar classes of tumours express similar patterns of the variable glycoproteins. For example, murine fibrosarcomas express patterns which are virtually identical with one another. Characteristic patterns are also expressed by murine macrophage tumour lines, human carcinomas and human B lymphoblastoid cells. Thus, the variable glycoproteins behave as a set of linked markers which are indicators of the type of normal pre-neoplastic precursor cell from which a tumour is derived and appear to be a new type of marker for tumour cell classification. Antibodies to these glycoproteins could prove useful in tumour localisation and diagnosis.


Characteristics of a panel of seven human ovarian tumour cell lines are presented. Positive staining with HMFG2 and ultrastructural identification of desmosomes confirmed the epithelial nature of the cell lines. The lines showed wide variations in ploidy, doubling times and clonogenicity in soft agar. Both vimentin and keratin were equally expressed in five lines, one line showed strong preferential expression of keratin and one line showed preferential expression of vimentin. Karyotypic changes associated with ovarian cancer were identified in all the lines. Four of the seven cell lines showed loss of chromosome material distal to 11p13-15. These cell lines offer considerable potential for research into the biology and genetics of ovarian cancer.


Thymidylate synthase (TS) expression has been characterized for a panel of eight human colorectal carcinoma and five human leukaemia cell lines, to relate differences in intrinsic TS activity, protein and mRNA levels to growth inhibition caused by continuous exposure to THYMITAQ, a specific non-classical antifolate TS inhibitor. Although a 20-fold variation in sensitivity to THYMITAQ was found within the colorectal cell line panel (IC50 0.12-2.7 microM), sensitivity was not related to TS activity, TS protein or TS mRNA levels. For the leukaemic cell lines, only a twofold range in sensitivity to THYMITAQ was observed (IC50 0.87-2.3 microM), and this did not correlate with TS activity, TS protein or TS mRNA levels. Across all of the cell lines, TS activity was linearly related to TS protein levels . However, for both the colorectal and leukaemia cell line panels, no relationship was found between TS mRNA/18S rRNA ratios and either TS activity or TS protein, consistent with the importance of post-transcriptional mechanisms in regulating TS activity. Two of the colorectal cell lines (BE and HCT116) and one of the human leukaemic cell lines (HL60), were intrinsically resistant to THYMITAQ (IC50 > 2 microM) in the absence of TS overexpression, suggesting that, subsequent to TS inhibition, events such as DNA repair and tolerance to apoptotic stimuli are also important determinants of sensitivity to THYMITAQ.


The influence of anaesthetics on the in vivo response of B16 melanoma to melphalan was studied using an in vitro cell-survival assay. Three anaesthetics were used, Saffan (Althesin) Sagatal  and Hypnorm. When Saffan was administered to tumour-bearing animals before melphalan there was a significant increase in tumour-cell kill. This effect was not observed with Sagatal or Hypnorm. Maximum increase in tumour-cell kill was achieved when Saffan was administered about 1 h before melphalan, and was dependent on Saffan dose. Clonogenic tumour-cell repopulation after melphalan was rapid (TD - 1 day) and the rate was similar from 2 levels of cell kill. When Saffan was combined with melphalan the repopulation rate was the same as with melphalan alone, and the increased cell kill was reflected in increased growth delay. The in vitro response of B16 melanoma cells to melphalan was unaltered by pretreatment with, or simultaneous exposure to Saffan. The results suggest that the mechanism of the enhanced cell kill in vivo is probably due to an indirect systemic effect, rather than a direct effect on the tumour cells.


Prokineticins are multifunctional secreted proteins that were originally identified as regulators of intestinal contraction but subsequently shown to affect vascular function, hyperalgesia, spermatogenesis, neuronal survival, circadian rhythm, nociception, feeding behaviour, immune responses, haematopoiesis and the development of the olfactory and gonadotropin-releasing hormone systems. Their role in the reproductive tract is still not fully elucidated, although they are reputed to increase microvascular permeability. Expression of prokineticins and their receptors has been reported in the ovary, uterus, placenta, testis and prostate. Their expression has also been reported in various pathologies of the reproductive tract, and future studies will highlight whether inhibition of prokineticin function in these pathologies would be a useful therapeutic target. Prokineticins (PKs) are factors that have been recently described and are involved in a wide array of functions in different tissues through the activation of their cognate receptors. Here, we first give a general background of the structure and functions of PKs and then focus on the role of PKs in reproduction.et al.et al.The PKs are two recently identified proteins with roles in physiological and pathological conditions. The names PK1 and PK2 were assigned to these proteins by Li Dendroaspis polylepis) and named venom protein A (VPRA) PK1 is the human orthologue of a nontoxic protein isolated from the venom of the black mamba , which is essential for the activity of these proteins. Mutations to this sequence, by insertion of a methionine preceding the N-terminal alanine, substitution of the N-terminal alanine with a methionine The PKs are the cognate ligands for two closely related G-protein-coupled receptors (GPCRs), termed PKR1 and PKR2, which share 85% amino acid identity and exhibit the greatest differences in their N-terminal sequences PKRs have been reported to couple either to Gi PKs were initially reported to be expressed in the gastrointestinal tract, where they were shown directly to stimulate contraction of the ileum longitudinal muscle of guinea pigs PKs are also expressed in steroidogenic tissues, including the testis In vitro studies suggest that PKs promote survival and differentiation of the granulocytic and monocytic lineages. Moreover, the expression of PKR1 and PKR2 in progenitor and mature blood cells A role for PKs has also been suggested in haematopoiesis and in regulation of the immune response in vitro showed PK2-induced migration of subventricular zone-derived neuronal progenitors −/− and PKR2−/− mice showed hypoplasia of the olfactory bulb −/− phenotype is strikingly similar to the Kallmann syndrome (KS) in humans, and recently PK2 and PKR2 mutations were identified in several KS patients PK2 expression has been demonstrated in the central nervous system, and a role in supporting neuronal survival has been suggested PKs are also involved in nociceptive sensitization and regulation of feeding behaviour. Administration of PK2 through several routes decreased the nociceptive threshold to thermal and mechanical pain in rats There are two levels at which PKs can modulate reproductive function. As discussed earlier, PKs have an important role in the development of the GnRH system but they can also function directly in reproductive organs.PKs and their receptors are expressed in the testis and the prostate. In the testis, PK1 is predominantly expressed in testosterone-producing Leydig cells, whereas PK2 is restricted to primary spermatocytes It has been proposed that PKs, through their cognate receptors, function as regulators of proliferation and the formation of fenestrae in the human testis vasculature The expression of PKs and their receptors has been reported in the prostate PKs and their receptors are expressed in the ovary, uterus and in various tissues of pregnancy in vitro suggest that PK1 has a role in the proliferation and survival of endothelial cells of the bovine corpus luteum Studies In the non-pregnant uterus, PK1 is expressed in the glandular epithelium, as well as in the endothelial and stromal cells of the endometrium, predominantly in the functional layer Further support for a role of PKs in reproductive function can be gleaned from PKR2 knockout mice, which show hypoplasia of the reproductive tract PK1, but not PK2, PKR1 or PKR2, expression peaks during the midluteal ‘window of implantation’, with immunolocalization to endometrial glandular epithelium, stromal and endothelial cells, and myometrial vascular endothelium as well as smooth muscle Hyperaemia and endothelial leakage at the implantation site are one of the earliest signs of implantation The expression of PK1 and PKR1 peaks in the trophoblast between 8–10 weeks of gestation, with PK1 being localized to the syncytiotrophoblast, minimal expression in the cytotrophoblast and no expression in the extravillous trophoblast It has been proposed that PK1 and PKR1 have an important role in placentation, owing to their expression peaking during the crucial hypoxic period of placentation (8th–10th week) before establishment of the haemochorial circulation et al.Several studies suggest that PKs have a role in pathological conditions of the reproductive tract. In the male, Samson A direct role for PKs in cancers of the female reproductive tract has not been ascertained. In endometrial and ovarian carcinoma, it has been shown that PK1 expression is either absent or reduced compared with normal tissue Hyperplasia and hypervascularity of the stroma are key features in the development of polycystic ovarian syndrome (PCOS). The search for factors responsible for these features has identified strong expression of VEGF and PK1 in PCOS et al.In preeclampsia, the underlying pathophysiology remains unclear but the resulting placenta is relatively hypoxic, with evidence of chronic inflammatory and vaso-occlusive lesions. A study by Chung The diverse spectrum of established and potential regulatory functions of PKs in the nervous, immune, haematopoietic, vascular, gastrointestinal and reproductive systems suggest that they will be the targets of drug development for numerous pathologies. Within the reproductive system, they are expressed in the testis, prostate, ovary, uterus, placenta and neurones regulating GnRH neuronal development. The last of these is the only established function, and substantial experimental research is needed to complement the observational studies in this emerging arena, to establish functions in reproductive tissues. The development of immunoneutralizing antibodies and small-molecule antagonist and agonist analogues of these complex proteins will greatly enhance the elucidation of their functions, with the promise of establishing therapeutic interventions in reproductive pathologies. Such molecules will also aid in the dissection of the relative contribution of each of the PKs and their receptors to reproductive physiology and pathology. Moreover, future research aimed at the development of targeted and tissue-specific knockout of the genes encoding each of these proteins in mice will help to elucidate their specific role in reproductive function.


A highly significant radiosensitising effect was seen in both cell lines (P<0.001 for MGH-U1 and S40b cell lines). This effect was independent of the concentration of the drug and the duration of exposure prior to treatment with ionising radiation. Cell cycle kinetics of both cell lines was not significantly altered with ZD1839 (0.01 μM) as a single agent. A modest induction of apoptosis was observed with ZD1839 (0.01 μM) as a single agent, but a marked induction was observed with the combination treatment of ZD1839 and ionising radiation. These results suggest a potentially important role for ZD1839 in combination with radiotherapy in the treatment of muscle invasive bladder cancer.The epidermal growth factor receptor (EGFR) is expressed in a wide variety of epithelial tumours including carcinoma of the bladder. Stimulation of the EGFR pathway is blocked by ZD1839 (Iressa), a highly selective EGFR tyrosine kinase inhibitor. Radical radiotherapy is an established organ sparing treatment option for muscle invasive bladder cancer and this study has explored the possibility for the use of ZD1839 as a radiosensitiser in this scenario. The effect of combination treatment with ZD1839 (0.01  Carcinoma of the bladder is the second most common malignancy of the genitourinary tract accounting for 13 600 new cases in the UK in 1999 .α (TGF-α). This results in receptor-associated tyrosine kinase activation, which triggers a cascade of downstream signalling pathways resulting in cell cycle progression, proliferation, angiogenesis and cell survival. A wide variety of human tumours including breast, NSCLC, bladder and SCC of the head and neck have been reported as expressing moderate to high levels of EGFR  is one of a family of four closely related transmembrane glycoprotein receptors consisting of an extracellular ligand binding domain, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor exists as an inactive monomer that undergoes homo- or heterodimerisation after activation by ligands such as epidermal growth factor (EGF) and transforming growth factor-adiation , 1997. IZD1839, a synthetic anilinoquinazoline, is an orally active, highly selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that inhibits ligand-stimulated EGFR autophosphorylation and signal transduction pathways implicated in the promotion of cancer cell growth and proliferation . TreatmeThe established human bladder cancer cell line MGH-U1 and its radiosensitive mutant clone S40b were cultured in Hams F12 media with 10% foetal calf serum, 1% glutamine and 1% penicillin and streptomycin. Both cell lines were kindly donated by Professor T McMillan  and Dr S Powell . ZD1839 (Iressa) was kindly donated by Astra Zeneca Pharmaceuticals .μM for 24 h. The drug was removed by washing twice with PBS. The cells were then trypsinised, counted using a Coulter Multisizer II Cell Counter® and plated into 60 mm dishes with 5 ml of media. The dishes were incubated for 10 days at 37°C and 5% CO2. The media were aspirated and the plates were stained with 1% crystal violet and surviving colonies with greater than 50 cells per colony were counted.Exponentially growing cells from both cell lines were treated with ZD1839 at final concentrations ranging from 0.01 to 10 μM. The drug was changed every 24 h and a further 10 ml of freshly reconstituted ZD1839 was added. On day 7, the cells were trypsinised and counted using a Coulter Multisizer II Cell Counter®.Exponentially growing cells were treated continuously for 6 days with 10 ml of ZD1839 at final concentrations ranging from 0.01 to 1000 μM for 1–6 days prior to irradiation. The drug was replenished daily. Colonies were stained and counted as before. The clonogenic survival data were fitted to a radiation survival curve using the using the linear quadratic equation and the DRFIT Radiation Survival Fitting Program  in a sealed Caesium-137 gamma-irradiator. The irradiated cells were trypsinised, counted using a Coulter Multisizer II Cell Counter® and plated in 60 mm Petri dishes and incubated as before. The process was repeated for exponentially growing cells treated with ZD1839 at concentrations ranging from 0.01 to 10  Program . DiffereThe expression of EGFR was determined using an EGFR Elisa Analysis Kit . Exponentially growing cells were treated with a detector antibody to EGFR and incubated at 37°C for 4 h. After washing with buffer supplied with the kit, horseradish peroxidase-conjugated streptavidin was added and after 1 h absorbance was measured using a Plate Reader at 450/595 nm.μM) for 24 h. The drug was washed off with media twice. For cell cycle distribution analysis at 0 h following exposure to ZD1839 for 24 h, cells were treated as described below. For cell cycle distribution analysis at 24 and 48 h following exposure to ZD1839, 10 ml of media was added and cells were incubated at 37°C for a further 24 and 48 h from the time of exposure to ZD1839. The cells were trypsinised, centrifuged at 1000 r.p.m. for 5 min, then washed with PBS and centrifuged at 2000 r.p.m. for 5 min. Cell pellets were fixed with 500 μl of ice-cold 70% ethanol and quickly vortexed. The cells were centrifuged at 2000 r.p.m. and the supernatant discarded. Ribonuclease A (50 μl of 100 μl ml−1 solution) was added and incubated for 5 min. PI stain (300 μl of 50 μg ml−1 solution) was added and incubated for 15 min. The specimens were analysed using Flow Cytometry at 620 nm.Cell cycle distribution was analysed at 0, 24 and 48 h after exposure to ZD1839 for 24 h using flow cytometry with propidium iodide (PI) staining. Cells were treated with ZD1839 (0.01–10 μM) for 24 h followed by irradiation (2–10 Gy), or received irradiation alone (2–10 Gy). The cells were trypsinised and centrifuged at 1000 r.p.m. for 5 min. The pellets were washed with 500 μl of cold PBS, centrifuged at 500 g for 5 min and 1 ml of binding buffer was added per 106 cells. A measure of 10 μl of 25 μg ml−1 Annexin V–FITC and PI stain (10 μl of 50 μg ml−1 solution) were added and samples were incubated in the dark for 15 min. The samples were analysed using flow cytometry.Apoptosis was evaluated using Annexin V–FITC conjugates and PI staining. Exponentially growing cells were treated with ZD1839 (0.01 μM). The length of continuous exposure to ZD1839 (1–6 days) at concentrations of 0.01–10 μM also had no significant effect on cell counts or on clonogenic survival after 10 days (six doubling times) (data not shown).‘The clonogenic survival of both cell lines was not significantly affected by exposure to ZD1839 for 24 h, compared to that of the controls, at the concentrations studied  and the radioresistant MGH-U1 cell line (P<0.001). For MGH-U1, the survival fraction at 2 Gy (SF2) was 0.88 and at 10 Gy (SF10) was 0.042. For S40b, the SF2 was 0.66 and the SF10 was 0.0079. The SF2 for the combination exposure to ZD1839 for 24 h preirradiation at a concentration of 0.01 μM was 0.83 and 0.42 for MGH-U1 and S40b, respectively. The SF10 for the combination exposure to ZD1839 for 24 h preirradiation at a concentration of 0.01 μM was 0.012 and 0.00146 for MGH-U1 and S40b respectively, 10 values. Prolonged exposure to ZD1839 for 3 and 6 days preirradiation with the drug being replenished daily did not improve the overall radiosensitising effect of the 24 h exposure regimen. In the MGH-U1 cell line, the SF2 for 3 and 6 days exposure was 0.80 and 0.83 and the SF10 was 0.0089 and 0.0109, respectively. In the S40b cell line, the SF2 for 3 and 6 days exposure was 0.43 and 0.456 and the SF10 was 0.00238 and 0.00165, respectively, μM having no statistically significant difference in both cell lines (P=0.8 and 0.84 in MGH-U1 and S40b), ZD1839 demonstrated a significant radiosensitising effect on both the radiosensitive S40b cell line (−1 of protein) than the radioresistant MGH-U1 cell line (16.7 fm mg−1 of protein).Both cell lines demonstrated a moderate expression of EGFR, with the radiosensitive S40b cell line expressing higher levels (21.5 fm mgμM) revealed no demonstrable alteration in the proportion of cells in different phases of the cell cycle in relation to control cells. There was no significant accumulation of cells in the G0/G1 phase of the cell cycle in the MGH-U1 cell line , or in the S40b cell line . Similarly, there was no significant alteration in the proportion of cells in the S phase of the cell cycle in either cell line at either concentration of ZD1839 (15.2% in the control sample vs 16.2 and 16.6% in the 0.1 and 10 μM samples in the MGH-U1 cell line and 28 vs 27.7 and 21.9% in the 0.1 and 10 μM samples in the S40b cell line), Analysis of cell cycle kinetics at 0, 24 and 48 h postexposure to ZD1839 (0.1 and 10 vs 5.3% in control samples for MGH-U1 cells and 17.8 vs 5.7% in the control sample for S40b cells). Following the combination of ZD1839 and radiation, there was a significant induction of apoptosis in both the radiosensitive and the radioresistant cell lines. In the radiosensitive S40b cell line, 24.3% of cells were undergoing apoptosis compared to control values of 5.7% (P=0.04), while in the MGH-U1 cell line, 20.3% of cells were undergoing apoptosis compared to control values of 5.3% (P=0.04), ZD1839 was found to have a modest proapoptotic effect on both cell lines as a single agent resulting in the induction of apoptosis in the MGH-U1 cell line . A similar effect on the S40b cell line was also noted . Treatment of both cell lines with radiation alone resulted in the induction of apoptosis in both cell lines with a greater proportionate induction in the S40b radiosensitive cell line  compared to the radioresistant MGH-U1 line (16.7 fmol mg−1 of protein), although this does not represent particularly high expression. Previous studies demonstrating an inverse correlation between the IC50 of ZD1839 and EGFR expression are in agreement with our findings  pathway  and this was similar in both cell lines. Treatment with radiation alone resulted in the induction of apoptosis in both cell lines and this was significantly more extensive for the radiosensitive S40b cell line than the parent MGH-U1 line . Combination treatment with ZD1839 and radiation resulted in a marked induction of apoptosis in both cell lines compared to control cells . The mechanism by which ZD1839 promotes apoptosis as a single agent and in combination with ionising radiation is currently under investigation  and the lack of any significant alteration in cell cycle kinetics, there is a definite and pronounced radiosensitising effect in the radioresistant MGH-U1 cell line and an even greater effect in its radiosensitive mutant clone S40b. These results, in conjunction with those of a number of other groups, suggest that ZD1839 has a significant effect on potentiating radiation-induced cell death at doses higher than 2 Gy in a wide variety of cells of varying radiosensitivity. The potentiation of cell death induced in the radioresistant and the radiosensitive cell line is an encouraging feature as many tumours are inherently heterogeneous in their radiosensitivity (The possible mechanisms for the potentiation of radiation-induced cell death by ZD1839 remain to be established and require further investigation. However, our study suggests that despite the relatively high concentrations of ZD1839 needed to achieve cytotoxicity in bladder cancer cell lines (ICsitivity . Radical


A series of monoclonal antibodies was produced by immunizing mice with fresh carcinoma of the colon and with the cell line HT-29. These antibodies could be classified into 3 different groups: (a) those which were HT-29 specific and reacted with no other cell line or fresh colon carcinoma samples; (b) antibodies which reacted with HT-29 and were also reactive with a number of other in vitro cell lines; and (c) antibodies which appeared to be specific for carcinoma of the colon cell lines in that they reacted selectively with colon carcinoma cell lines and not with carcinoma lines derived from other tissues. It was clearly shown by immunoperoxidase staining of both normal and neoplastic cells of the gastrointestinal mucosa, from stomach to colon, that these antibodies were not tumour-specific. Indeed, one antibody (250-30.6) has a remarkable specificity for secretory epithelium of any tissue whether it be found in the gastrointestinal tract, respiratory system or urinary system. We question the existence of tumour-specific antigens detected by monoclonal antisera described thus far, and comment on the remarkable specificity of some of the sera produced, emphasized by the secretory cell-specific antibody described herein.


The prostate cancer cell lines PC3 and LNCaP have been shown to lack expression of the tumour suppressor gene MMAC1/PTEN, in contrast to the immortalized non-tumorigenic epithelial lines PNT1a and PNT2. We have measured the effects of reintroduction of wild type (wt) and mutant MMAC1 genes on to these genetic backgrounds, using gene constructs expressing either wt MMAC1 or various mutants deficient in the dual specificity phosphatase domain of the protein. Over-expression of wild type PTEN protein induced cell shrinkage and rounding, but did not result in increased levels of classical apoptosis. Permanently transfected lines containing the MMAC1 gene could only be obtained from the PNT cells, as PTEN expression resulted in rapid loss of both tumour lines. In contrast, mutation of the phosphatase domain resulted in partial attenuation of the phenotypic effects of MMAC1 after transient transfection, and also allowed the derivation of permanent tumour cell lines containing the mutated MMAC1 gene. The results suggest that re-expression of wt PTEN is incompatible with survival of human prostate cancer cells in vitro, and that the full biological activity of this common tumour suppressor requires functions additional to the established protein and lipid phosphatase activities in epithelial systems. © 2000 Cancer Research Campaign


Human foetal keratinocytes were transfected with a recombinant plasmid (pSV6-1) which contained an origin defective SV40 genome. The resulting transformed cell line had many properties in common with previously described SV40-transformed keratinocytes, including expression of simple epithelial-type keratins. It was non-tumourigenic in nude mice at early passages, forming small benign cysts, however, after approximately 46 in vitro passages, these transformed keratinocytes formed invasive squamous cell carcinomas in athymic nude mice. Several in vitro changes were associated with this acquisition of tumourigenicity (a) an alteration in cellular morphology, (b) development of a cytogenetically marked clone and (c) loss of cell surface fibronectin. The loss of fibronectin was also observed in vivo; cysts formed by SV6-1 Bam/HFK produced human fibronectin whereas tumours did not, although both tumours and cysts were laminin- and keratin-positive. These results indicate that the spontaneous development of secondary events in immortalised human cells may lead to the acquisition of a malignant phenotype.


But the compound has demonstrated undesired agonist-like effect depending on cell, tissue and species studied. Using PR-transfected breast cancer cells MDA-MB-231, this report describes the similarities and differences between progesterone- and RU486-mediated effects on cell growth, cell differentiation and, at the molecular level, on the activation of p44/p42 MAP kinases (MAPK). Like progesterone, RU486 inhibited cells growth by arresting the cells in G0/G1 phase of the cell cycle. In contrast to progesterone that induced cell spreading, RU486 induced a multipolar, stellate morphology. RU486-treated cells showed no increase of stress fibers, nor was there any increase of focal adhesions as progesterone-treated cells did. Furthermore, despite of the fact that both compounds inhibited cell growth, RU486 significantly stimulated the activation of p44/p42 MAP kinases whereas progesterone markedly inhibited the activation. Nonetheless, the effects of RU486 were PR-mediated and RU486 was able to antagonize the effect of progesterone on cell growth and focal adhesion. In conclusion, RU486 can act not only as a progesterone antagonist, a progesterone agonist but also induced morphological and molecular changes that were distinct from progesterone-mediated effects in PR-transfected MDA-MB-231 cells. The non-progesterone-like effect of RU486 may be mediated through a pathway that is different from the progesterone-mediated pathway, or it is the result of a blockade of certain critical step(s) in the progesterone-mediated pathway. In any case, undesired side effects of antiprogestin may create clinical complications. PR-transfected MDA-MB-231 breast cancer cells provide a model for studying the functions of progesterone analogues.© 2001 Cancer Research Campaign


CB30865 was the most potent growth inhibitory agent . CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 , which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4–48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action. © 1999 Cancer Research CampaignThree lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC


Phototherapy in the presence of haematoporphyrin derivative has been shown to have a preferential effect on malignant tumours when compared to normal tissue. This communication presents a comparison of the sensitivity to photochemotherapy in vitro of different cell lines. Asynchronous populations of cells were exposed to light in the presence of haematoporphyrin derivative, and found to be inactivated with a comparable efficiency. The lines were of human, Chinese-hamster or mouse origin and had different abilities to form tumours after heterotransplantation into nude mice or transplantation into syngeneic, immunosuppressed mice. Synchronized cells from 4 of the lines showed a similar variation in sensitivity to light throughout the cell cycle. Cells near the middle of interphase showed the highest sensitivity, whilst cells in early G1 were found to be least sensitive towards treatment with haematoporphyrin derivative and light.


The results of the bioassay showed that these compounds possess potent antitumoractivities against the studied human carcinoma cell lines.O-Cholesteryl-O-phenyl-N-phenylphosphoramidate (


In vitro cloned lines derived from a primary nickel-induced rat rhabdomyosarcoma exhibited diverse levels of susceptibility to spontaneous NK activity. The presence of NK target structures was revealed by competition assays on all cloned cell lines, and the NK susceptibility of the tumour lines varied according to their osmotic fragility. Tumour cell lines derived from metastatic lung nodules presented similar NK susceptibilities to cells originating from the primary tumour. However, cloned cell lines differed in their capacity to form lung colonies after i.v. injection, and in their potential for invading lungs after s.c. primary tumour development. No correlation was found between lung colonization potential and NK resistance. Studies of the correlation between metastatic potential and NK sensitivity revealed that (1) all the NK resistant tumour cells were highly metastatic; (2) NK susceptible tumour cells could be either highly or weakly metastatic. Therefore, highly metastatic tumour cells could be either resistant or susceptible to NK lysis. We conclude that the property of resistance to NK contributes to a high metastatic potential. However, other properties could counterbalance and finally prevail over NK susceptibility thus enabling NK susceptible cell lines to be also highly metastatic.


EO9 [3-hydroxy-5-aziridinyl-1-methyl-2-prop-beta-en- alpha-ol] was selected for clinical trial in Europe because of its preclinical profile but also because of its distinct mechanism of bioactivation. Several studies have shown that cells rich in DT-diaphorase may be particularly sensitive to EO9. The present study examined the relationship between DT-diaphorase activity and sensitivity to EO9 in a panel of cell lines largely derived from human and rodent leukaemias/lymphoma and solid tumours. A possible relationship between chemosensitivity and enzyme activity was demonstrated (correlation coefficient 0.796). A number of the human cell lines were established as xenografts in nude mice but, with the exception of HT29, DT-diaphorase specific activity was greatly reduced compared with the corresponding cell lines. These data suggest that in vitro studies of bioactivation of drugs by specific enzymes is unlikely to be relevant for the same tumour in vivo. Except for HCLO, all xenografts failed to respond to EO9 as a single dose. HT29 tumours in vivo had similar DT-diaphorase activity  to the cell line (337) but failed to respond to a single dose or daily dose schedule. A preliminary attempt to investigate an hourly dose schedule demonstrated a modest anti-tumour effect accompanied by enhanced toxicity. Attempts to optimise EO9 exposure parameters to potentiate activity in tumours with high DT-diaphorase activity are under way, but as yet the relevance of this particular enzyme for in vivo EO9 activity requires further investigation.


The activity of diamine oxidase  which converts putrescine into gamma-aminobutyraldehyde in the degradative pathway of polyamine, was studied in 4 human melanoma cell lines, 2 of which produce tumours in greater than 80% of nude mice , whereas the other 2 induce tumours in less than 25% . The activity of DO in these cells varies with the growth rate: 24 h after seeding there is an initial increase in DO activity, followed by a steep decline during exponential growth. At 96 h, when cells reach saturation density, the activity of DO is significantly greater in the highly tumorigenic cell lines than in the poorly tumorigenic cell lines. Kinetic studies show that for the highly tumorigenic lines apparent Km values are 10.6 X 10(-6)M +/- 0.2 (M3Dau) and 14.2 X 10(-6) M +/- 0.6 (M4Beu), whereas for the poorly tumorigenic lines the values are 4.5 X 10(-6) M +/- 0.3. After transplantation into nude mice, the M1Dor cell line, which exhibits a low Km (app.) for DO of which had high Km (app.) value. Km (app.) determination of DO could be an approach for characterizing human melanoma cells differing in their tumorigenic potential in nude mice.


Drug resistance is a major obstacle to successful chemotherapy of primary liver cancer, which is associated with high expression of the multidrug resistance (MDR) gene product P-glycoprotein (Pgp), a multidrug efflux transporter. The most effective single agents in treatment of primary liver carcinoma belong to the anthracycline family, yet several anthracyclines are known to be substrates for Pgp. In the present study, we compared four anthracyclines with respect to cell growth inhibition, intracellular accumulation and cellular efflux using the HB8065/R human hepatoma cell line which is rich in Pgp, and the Pgp-poor parental line HB8065/S. The anthracyclines were also administered in conjunction with the Pgp-modifying agents verapamil and SDZ PSC 833 to assess modulation of resistance. The HB8065/R cells were sensitive to aclarubicin (ACL) and highly resistant to epirubicin (EPI), doxorubicin (DOX) and daunorubicin (DNR). SDZ PSC 833 enhanced accumulation, decreased efflux and increased cytotoxicity of EPI, DOX and DNR in the HB8065/R cells, but none of these effects was seen with ACL. In conclusion, ACL is apparently not transported by Pgp and retains its activity in a multidrug-resistant human hepatoma cell line; such properties can be exploited for clinical purposes.


The cytolethal response to treatment with prednisolone was investigated in vitro in eight human lymphoblastoid cell lines containing varying concentrations of specific cytoplasmic glucocorticoid receptors. A similar response was observed in seven of the lines irrespective of their concentration of cytoplasmic receptors and pharmacological doses of steroid, well above those required to saturate receptors in cell-free extracts, were required for a massive lethal response. One cell line derived from Burkitt's lymphoma was refractory to lethal effects even with pharmacological doses of steroid. A similar unresponsiveness to the cytolethal effect of prednisolone in vitro was observed in fresh lymphoblasts derived from patients with acute lymphoblastic leukaemia despite evidence of a satisfactory clinical response to therapy which included steroid. The resistance of human lymphoblastoid cells to treatment with glucocorticoids in vitro may result from a defect in activation subsequent to the binding of steroid to cytoplasmic receptors.


Sublines of the human small-cell lung carcinoma (SCLC) cell line GLC4 with acquired resistance to teniposide, amsacrine and mitoxantrone  were derived to study the contribution of DNA topoisomerase II alpha and -beta  to resistance for TopoII-targeting drugs. The cell lines did not overexpress P-glycoprotein or the multidrug resistance-associated protein but were cross-resistant to other TopoII drugs. GLC4/VM20x showed a major decrease in TopoII alpha protein  without reduction in TopoII beta protein; GLC4/AM3x showed only a major decrease in TopoII beta protein (to 18%) and not in TopoII alpha. In GLC4/MIT60x, the TopoII alpha and -beta protein levels were both decreased . The decrease in TopoII alpha protein in GLC4/VM20x and GLC4/MIT60x, was mediated by decreased TopoII alpha mRNA levels. Loss of TopoII alpha gene copies contributed to the mRNA decrease in these cell lines. Only in the GLC4/MIT60x cell line was an accumulation defect observed for the drug against which the cell line was made resistant. In conclusion, TopoII alpha and -beta levels were decreased differentially in the resistant cell lines, suggesting that resistance to these drugs may be mediated by a decrease in a specific isozyme.


Four human and 4 rat astrocytomata and mammalian adult brain and spinal cord were examined by indirect immunofluorescence with human serum containing smooth muscle antibody. Cryostat sections of astrocytomata showed staining of the tumour cell cytoplasm and processes while in normal adult brain and spinal cord the entire astrocyte stained. Impression film and tissue culture monolayers of astrocytomata showed staining of cell processes and a fine, filamentous network in the cell body. The reaction with astrocytoma tumour cells was stronger than that with the corresponding normal astrocytes. Specificity of the staining reaction was established by its prevention on neutralization absorptions of the serum with extracts or homogeneous of smooth muscle. The presence of smooth muscle-associated antigen in astrocytes and astrocytomata is indicative of contractile protein providing a mechanism of cell movement in vivo.