Thiazoles, oxazole and their corresponding reduced derivatives, thiazolines and oxazolines, are found in marine sources exhibiting significant biological activities. The isolation, synthetic, and biological studies of these natural products, covering literature from January 2007 to June 2010, are summarized. The role of natural products in drug discovery has undergone many changes over the past two decades, from a decline in participation by pharmaceutical companies in the 1990s, to a renaissance in recent years. In addition, the expectations from combinatorial libraries in drug screening have not been fulfilled and more than 60% of prescription drugs are of natural product origin. As a consequence, the natural product-inspired drug discovery and development has received renewed attention in recent years.A large number of natural products, in particular from the marine environment, contain thiazole, oxazole, thiazolines or oxazolines heterocycles. In many cases, promising anti-tumor, antibacterial, anti-viral, anti-malaria and anthelmintic activities have been identified for these compounds. This review focusses on the isolation and structural determination, biological activities and synthetic studies of these marine products covering literature from January 2007 to June 2010.1) and two new analogues, bacillamides B (2) and C (3), Bacillus endophyticus obtained from a Bahamian hypersaline microbial mat batrachotoxin binding to voltage-gated sodium channels (IC50 = 92.8 nM) and activated sodium influx (EC50 = 2.31 μM) in mouse neocortical neurons.From an environmental assemblage of the marine cyanobacteria 17), GL582 (18), GL614A (19), GL614B (20), GL546A (21), GL546B (22), GL628 (23), GL614C (24), Mycrocystis sp. water-bloom collected in Gelbea reservoir, Valley of Armagedon, Israel [18) displayed weak cytotoxicity (20% grow inhibition) against Molt-4 cell line at 10 μM/mL.Eight novel thiazole and oxazole containing cyclic peptides, microcyclamides, GL616 , d-amino acid content, two thiazolines, and one β-amino acid [50 values of 2.2, 1.0, 1.5, and 4.2 μM, respectively.A collection of the cyanobacterium ino acid . The str26), Lyngbya sp. [d-amino acids and N-methylated amino acids along with several other modified amino acid residues. Bisebromoamide exhibited antiproliferative activity at nanomolar levels and potent kinase inhibition.A new thiazoline metabolite, bisebromoamide (gbya sp. . Its pla27), 28) and the formation of the α-ketoamide linkage by the use of a highly activated α,β-ketonitrile.The synthesis of the C1-N15 fragment of the originally proposed scleritodermin A (27) along with two isomers (29) and (30) were achieved [1H and 13C NMR spectra of molecule 27 did not match those reported for natural scleritodermin. In contrast, the spectra of compound 29 and those reported for the isolated marine product were identical. Thus, compound 29 was proposed as the revised structure of scleritodermin A.In 2008, the first total synthesis of the originally proposed scleritodermin A (achieved . The app31), 27 (LC50 = 9.6 μM). The presence, in some obtained compounds, of α,β-ketoamide function improves the cytotoxic activity on HCT-15 cells.Recently, the synthesis of key fragments and open analogs of scleritodermin A and their biological evaluation as cytotoxic and anthelmintic were reported. Open and simplified analogs (32), 33 and protected aminoacids using Burgess reagent to obtain the oxazolines rings. Macrocyclization was performed using PyBOP, DMAP, DIEA in CH2Cl2/DMF [Pattellamide A (2Cl2/DMF .15), The first total synthesis of Largazole  [During 2008 seven more syntheses of largazole were reported –28. Some0.07 nM) .During 2009, six reports related to the synthesis of largazole and/or analogs were published –34. A sitrans-alkene are potent for the antiproliferative effect, those with cis-alkene are completely inactive. In addition, replacement of valine by tyrosine increased selectivity toward human cancer cells over human normal cells more than 100-fold [Recently, new analogs of largazole were synthesized ,36. Stru100-fold .d-alanine, bacillamide C (3), was obtained in 22% overall yield [35, Starting from ll yield . The key4), A thiazole Ugi multicomponent reaction and an aminolisis reaction were used to obtain racemic Bacillamide C, 36), Recently, the enantioselective total synthesis of trichloroleucine-derived marine natural product neodysidenin (37), 38) and 34-epiapratoxin A (39) were obtained [40 through a stereoselective route based in three asymmetric reactions. Thiazoline formation was successfully accomplished from a Cys amide using Ph3PO/Tf2O and macrolactamization was performed between N-methylisoleucine and proline residues using HATU.The cytotoxic thiazoline, apratoxin A (obtained . The syn26), R-configuration of the stereogenic center at the thiazoline ring of the proposed structure (41), 41 were identical to those of the natural product leading to a revision of the reported stereochemistry of bisebromoamide.Recently, the first total synthesis and a revised configurational assignment of bisebromoamide were reported . The aut42), Neopeltidae. Its structure was elucidated on the basis of spectroscopic data interpretation. Neopeltolide is a potent inhibitor of the in vitro proliferation of the A-549 human lung adenocarcinoma, the NCI-ADR-RES human ovarian sarcoma, and the P388 murine leukemia cell lines, with IC50 of 1.2, 5.1, and 0.56 nM, respectively. 42 also inhibited the growth of the fungal pathogen Candida albicans with a minimum inhibitory concentration of 0.62 μg/mL [A new marine-derived macrolide designated as neopeltolide , Neopelt43) and B (44), Rapidithrix sp. [Ariakemicins A  and B , B (46), and E (47), Jaspis species. This is the first time that bengazoles were isolated from another genus. Those compounds showed growth inhibitory activity to seven murine and human cancer cell lines [Bioassay-guided fractionation of extract of the sponge ll lines .49), Fascaplysinopsis sp. with other closely related no oxazole containing metabolite taumycine B. The two compounds have the same 12-membered oxodepsipeptide ring system in common. Both were toxic to brine shrimp larvae, and taumycin A (1 μM), but not taumycin B, inhibited growth of the human UT-7 leukemic cell line. The structure of the two compounds, likely to be derived from microorganisms, was established by MS and 1D and 2D NMR data [An oxazole lipodepsipeptide, taumycins A (NMR data .50), Fascaplysinopsis sp. The structure of the compound was elucidated by interpretation of MS and 1D and 2D NMR spectra. Salarin C (50) is closely related to salarin A and is considered to be the precursor of salarins A and B. Air oxidation was found to transform 50 to salarin A. 50 was found to inhibit cell proliferation of human leukeamic cell lines UT-7 and K562, and the murine pro-B cell line Ba/F3 at concentrations of 0.0005–0.5 μg/mL [A novel oxazole macrolide, salarin C (.5 μg/mL .51) and I (52), 50. All compounds were evaluated for their cytotoxicity against K562 and UT-7 human leukemia cells. While salarins D, E, H, and J displayed dose- and time-dependent inhibition of proliferation, 51 and 52 were not active in these assays [Recently, seven new nitrogenous macrolides, designated salarins D-J, closely related to salarins A-C, were isolated from the same source. Salarin F (e assays .O-desmethylkabiramide B (53) and 33-methyltetrahydrohalichondramide (54), Hexabranchus sanguineus, a nudibranch from the Indo-Pacific. The structure elucidation of these very minor metabolites was preformed using a 1 mm 1H NMR high-temperature superconducting microcryoprobe with unmatched sensitivity [Two new trisoxazole macrolides, 9-sitivity .55–57), Diazona sp. collected in Indonesia. The cytotoxic activity of the new compounds and diazonamide A was evaluated against a panel of three human tumor cell lines, including lung (A549), colon (HT29), and breast (MDA-MB-231). Moderate cytotoxicity with GI50 values in the micromolar range was found for the new compounds; whereas the potent activity previously reported for diazonamide A was confirmed with values in the low nanomolar range [Three new macrocyclic peptides, diazonamides C–E , a novel phosphate-containing macrolide, was isolated from a Papua New Guinea collection of the marine sponge Cinachyrella enigmatica [58 is comprised of an 18-membered phosphomacrolide that contains an embedded exomethylene-substituted tetrahydropyran ring and an acyclic portion that spans an oxazole moiety. Two additional analogues, 15-O-methylenigmazole A and 13-hydroxy-15-O-methylenigmazole A, were also isolated. The enigmazoles are the first phosphomacrolides from a marine source and 58 exhibited significant cytotoxicity in the NCI 60-cell line antitumor screen, with a mean GI50 of 1.7 μM.Enigmazole A (igmatica . Compoun42), 59), The relative stereochemistry of the metabolite (+)-neopeltolide  triflate promoted the macrocyclization of 62 in a Prins type transformation. The synthetic neopeltolide inhibited proliferation of the murine leukemia line P388 with an IC50 of 0.6 nM and the human breast adenocarcinoma line MCF-7 with an IC50 value of 2.2 nM. Validating the revised structure as the correct one; diastereomer which corresponded to the originally proposed structure, was approximately 100-fold less active in both cell lines [Scheidt and co-workers performed a macrocyclization of the linear precursor ll lines .59 have been reported in 2008 [Five total syntheses of  in 2008 –58 and t in 2008 –64.58), 64), The first total synthesis of the cytotoxic marine macrolide enigmazole A (65), 65 was reported, the first to yield a product that corresponds with the natural product. The synthesis involved the macrolactamization of the intermediate 66, followed by elaboration of the central oxazole ring in 65 as a late step in the overall synthesis [Stereochemistry of (−)-ulapualide A (ynthesis .67), An enantioselective, convergent, total synthesis of the antiviral marine natural product (−)-hennoxazole A (68) and B (69), Herpetosiphon species [Siphonazoles A  and acute T-cell leukemia TIB-152 , with 68 being significantly more active than 69 [Other synthesis of siphonazoles was performed by Ciufoloni and co-workers involving the iterative use of a conjunctive oxazole building block that acts as a carrier of synthon  than 69 .Jaspis sponge that display potent antifungal and anthelmintic activity, and have a unique structure containing two oxazole rings flanking a single carbon atom. Three new total synthesis of bengazoles [The bengazoles are a family of metabolites isolated from a ngazoles –76 and angazoles –79 were 71), Haraldiophylum sp. was synthesized in four steps. The key step, construction of the central 2,5-disubstituted oxazole ring, was based on the aza-Wittig reaction of the iminophosphorane derived from the α-azidoacetyl indole and (S)-N-phthaloylphenylalanyl chloride [The marine alkaloid almazole C (chloride .72), 73. Synthesis of 73 was performed by coupling the oxotryptophan methyl ester with N,N-dimethyl-L-phenylalanine and subsequent treatment with POCl3 [The structure of Almazole D (th POCl3 .50 < 1.58 × 10−9 M against the NCI panel of 60 tumor cell lines. Phorboxazole B (74), R)-2,3-dibenzyloxypropanal and the Brassard diene EtO(Me3SiO)C:CHC(OMe):CH2 in presence of Eu(fod)3 provided dihydropyranone I, enabling the synthesis of the C18–C46 fragment of phorboxazole B. Coupling of the C18–C46 fragment with a suitable C3–C17 partner, followed by the late-stage formation of the C16–C18 oxazole unit and stereoselective macrocyclization and deprotection, rendered synthetic phorboxazole B [The complex structure of phorboxazoles and their unique biological activity is a driving force for continued interest from the synthetic community. The phorboxazoles are among the most potent discovered cytostatic agents, exhibiting a mean GIxazole B .Highly convergent syntheses of eight phorboxazole congeners and their evaluation against a diverse panel of human solid tumor cancer cell lines have been achieved. Specifically, the C(45–46) alkyne, alkene, and alkane phorboxazole A analogs were constructed and found to display single digit nanomolar cell growth inhibitory activities in a series of human cancer cell lines. The structurally simplified C(11–15)-acetal congener also proved potent, albeit reduced, when evaluated against the same cell line panel. Importantly, (+)-C(46)-chlorophorboxazole A displayed picomolar (pM) inhibitory activity in several cell lines .75), cis-tetrahydropyrans inscribed with the phorboxazole architecture, a convenient method to prepare E and Z-vinyl bromides from TMS-protected alkynes utilizing radical isomerization of Z-vinylsilanes, and a convergent late-stage Stille union to couple a fully elaborated C(1–28) macrocyclic iodide with a C(29–46) oxazole stannane side chain, to establish the complete phorboxazole skeleton. The synthesis, achieved with a longest linear sequence of 24 steps, resulted in 4.6% overall yield [A highly convergent second-generation synthesis of (+)-phorboxazole A  coordination chemistry of westiellamide, as well as of the three synthetic analogues (76) with an [N-heterocycle-N-peptide-N-heterocycle binding site is highly preorganized for copper(II) coordination. The macrocyclic peptides have been found to form stable mono- and dinuclear copper(II) complexes.The synthesis of westiellamide analogues of type  with an azacrown- with an . The aut


Serum-free supernatants from in vitro maintained gastrointestinal cancer and melanoma cell lines inhibit the generation of lymphokine (IL-2) activated killer (LAK) cells in a time and dose-related manner. Concentrations as low as 5% can inhibit the generation of LAK cytotoxicity but inhibition of proliferation is not observed until higher concentrations are included in the culture system. Inhibition is not observed with supernatants from a breast cancer cell line nor with supernatants from normal cells. There was complete concordance between the capacity of the tumour cells themselves to inhibit LAK generation and the presence of inhibitory activity in the corresponding supernatant. The inhibitory factor(s) is stable after heating to 44 and 56 degrees C. Production of the inhibitory factor(s) is sensitive to metabolic inhibitors and has a molecular weight greater than 25 kD. The inhibition of LAK cell stimulation by tumour cells may partially explain the failure of adoptively transferred LAK cells and IL-2 therapy to cause tumour regression in man.


Thirteen human peripheral lung tumours have been studied in both light and electron microscopy. They were classified as epidermoid carcinoma, mucus-secreting cell adenocarcinoma, and alveolar cell adenocarcinoma, the latter made up of granular pneumocytes. Alveolar cell cancer, as defined by ultrastructural features, could assume different gross histological patterns in light microscopy, and therefore electron microscopy is required for its identification.Since neither squamous nor mucous metaplasia was observed in any alveolar cell tumour, it is tentatively suggested that all peripheral lung tumours which lack these features may be derived from granular pneumocytes, irrespective of whether they appear to be adenocarcinomata or large cell carcinomata when examined by light microscopy.


Irinotecan (CPT-11) is a semisynthetic camptothecin derivative with a broad spectrum of anti-tumour activity. Carboxylesterase (CE) catalyses the conversion of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin), the active form of CPT-11. The antiproliferative effects of CPT-11 and SN-38, CE-activity and topoisomerase I protein expression were investigated in five human small-cell lung cancer (SCLC) cell lines and four human non-small-cell lung cancer (NSCLC) cell lines. Antiproliferative activity, expressed as IC50 values, was determined using the MTT assay. CPT-11 was significantly more active in SCLC than in NSCLC cell lines (P = 0.0036), whereas no significant difference between histological types was observed with SN-38. A significant correlation  was observed between CE activity and chemosensitivity to CPT-11 but not to SN-38, and significantly higher CE activity was observed in SCLC compared with NSCLC cell lines (P = 0.025). Western blotting experiments showed topoisomerase I protein expressions within a factor of 2, and a granular nuclear staining was detectable in all cell lines by immunocytochemistry of cytospins. No correlation was observed between protein expression and sensitivity to CPT-11 or SN-38. Cellular and medium concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography (HPLC) in one SCLC cell line with high CE activity and high sensitivity to CPT-11, and one NSCLC cell line with low sensitivity to CPT-11 and CE activity. Intracellular concentrations of CPT-11 and SN-38 were higher in the SCLC cell line, and this was associated with an increase in cellular uptake of CPT-11 compared with the medium, and an increased intracellular formation of SN-38. In conclusion, CE activity appears to be associated with higher sensitivity to CPT-11 in human lung cancer cell lines and may partly explain the difference in the in vitro sensitivity to CPT-11 between SCLC and NSCLC cells. The assessment of CE activity in clinical material of lung cancer patients undergoing treatment with CPT-11 may be warranted. However, other mechanisms may influence sensitivity to CPT-11, possibly including drug transport.


Mast cell growth was inhibited by Mu-rIFNs when Sp C were exposed throughout the entire culture period to Mu-rIFNs. The most potent inhibitor of mast cell growth was Mu-rIFN-γ, followed by Mu-rIFN-β; Mu-rlFN-α had little effect. When added to IC-2 cells, clonal mast cell progenitor, both Mu-rlFN-β and −γ), significantly inhibited proliferative response of the target cells. The suppressive effect of Mu-rIFNs on IC-2 cells was selectively abolished by monoclonal antibodies against Mu-rIFN-β and −γ.Preparations of murine recombinant interferon (Mu-rIFN)-alpha, -beta and -gamma were assessed for their influence on


Epidemiological and experimental studies suggest that dehydroepiandrosterone (DHEA) exerts a protective effect against breast cancer. It has been proposed that the non-competitive inhibition of glucose-6-phosphate dehydrogenase (G6PD) contributes to DHEA antitumor action. We evaluated the effects of DHEA on G6PD activity and on the in vitro proliferation of two human breast cancer cell lines, MCF-7 (steroid receptor positive) and MDA-MB-231 (steroid receptor negative), in a serum-free assay. DHEA inhibition of G6PD was only found to occur at concentrations above 10 microM; at these high concentrations, the growth curve was parallel to the enzyme inhibition curve in both cell lines. In contrast, at concentrations in the in vivo breast tissue concentration range, neither cell growth nor enzyme activity was inhibited. The results failed to confirm DHEA's putative anti-tumor action on breast cancer through G6PD inhibition, as the enzyme blockade only becomes apparent at pharmacological concentrations of the steroid.


PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the 50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response.Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade.Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin DThese studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991. Breast cancer is a worldwide health concern with approximately 1,000,000 million new cases each year -4. This 0 (quiescence) followed by G1 (pre-DNA synthesis), S (DNA synthesis), G2 (pre-division), and M (cell division). The careful regulation of this system is of fundamental importance, and dysregulation can result in several disease processes including cancer. The progression from G1 to S is a key checkpoint in protecting the cell from abnormal replication. Key to passage through this restriction point is the interaction between the cyclin-dependent kinases (CDKs) and cyclin proteins. CDKs are a subgroup of serine/threonine kinases that play a key role in regulating cell cycle progression by associating with cyclins. Hyperphosphorylation of the retinoblastoma (Rb) gene product pRb is mediated in early G1 by CDK4 and CDK6 interacting with cyclin D1. This results in pRB inactivation and release of transcription factors that allow progression to the S phase. Negative regulators of CDK4/6-cyclin activity include the INK4 family  .Microarray slides were read using an Agilent Scanner, and Agilent Feature Extraction software version 7.5 was used to calculate gene expression values. The feature extracted files were imported into the Rosetta ResolverCells were seeded in duplicate at 5,000 to 10,000 cells per well in 24-well plates. The day after plating, PD 0332991 was added at 1 μM and twofold dilutions over six concentrations were performed to generate a dose-response curve. Control wells without drug were also seeded. Cells were counted on day 1 when the drug was added as well as after 6 days when the experiment ended. After trypsinization, cells were placed in Isotone solution and counted immediately using a Coulter Z2 particle counter . Suspension lines were counted using a Coulter Vi-Cell counter (Beckman Coulter Inc.).Growth inhibition was calculated as a function of the number of generations inhibited in the presence of PD 0332991 versus the number of generations over the same time course in the absence of PD 0332991. In addition, lethality was defined as any decrease in cell number in treated wells versus the baseline number of cells pre-treatment at day 1 of exposure.For tamoxifen studies with the MCF7 tamoxifen-resistant cell line, proliferation studies were performed as above except cells were plated without FBS and were supplemented with 0.5 nM β-estradiol (Sigma). Proliferation assays were then performed as above.Similar to above, the ER-positive cell lines MCF-7, T47-D, and EFM-19 were plated and treated with PD 0332991 alone, with 4-hydroxytamoxifen (Sigma) alone, or with the combination, in duplicate, over six twofold dilutions at a fixed molar ratio. For combination studies with trastuzumab, BT-474, EFM-192A, and MDA-361 lines were plated as above and treated with PD 0332991 alone, with trastuzumab  alone, or with the combination, in duplicate, over six twofold dilutions at a fixed molar ratio.r value) was calculated. Multiple drug effect analysis was performed using computer software as previously described [For each assay, the log of the fraction growth inhibition was plotted against the log of drug concentration, and the linear regression curve fit correlation coefficient (escribed .t test) to determine whether the mean CI values at multiple concentrations were significantly different from CI = 1. In this analysis, synergy is defined as CI values significantly lower than 1.0, antagonism as CI values significantly higher than 1.0, and additivity as CI values equal to 1.0. All CI values were calculated using the conservative assumption of mutually nonexclusive drug interactions. All experiments were carried out at least twice. Combination studies were performed as above with the MCF7 tamoxifen-resistant clones with addition of estrogen back to the media at the time of the experiment (as described in cell culture above).Combination index (CI) values were derived from parameters of the median effects plots, and statistical tests were applied  . Protein content was resolved by SDS-PAGE electrophoresis, and was transferred to nitrocellulose membranes (Invitrogen). Total pRb expression was detected using a rabbit polyclonal antibody to pRb . Rb phosphorylation was detected using rabbit polyclonal antibody to phospho-serine 780 . Blots were washed and incubated with a goat-anti-rabbit IgG horseradish peroxidase conjugate , developed using ECL Plus chemifluorescent reagent , and imaged using chemifluorescence.Cells in log-phase growth were treated with 100 nM PD 0332991 and were harvested at various timepoints by washing in PBS and lysis at 4°C in RIPA lysis buffer. Insoluble material was cleared by centrifugation at 10,000 × Effects of PD 0332991 on the cell cycle were assessed using Nim-DAPI staining . Cells were plated evenly in control and experimental wells, were allowed to grow to log phase and were then treated with 100 nM PD 0332991 for the defined times. To perform cell cycle analysis, cells were washed with PBS; then trypsin was applied to release cells, which were then centrifuged at 10,000 × g for 5 minutes. Supernatant was aspirated and cells were then resuspended in 100 μl Nim-DAPI (NPE Systems) and gently vortexed.Cells were analyzed with UV using a Cell Lab Quanta SC flow cytometer (Beckman-Coulter Inc.). Apoptosis assays were performed using an Annexin V-FITC apoptosis detection kit  and flow cytometry. Cells were plated and treated as for cell cycle studies and were exposed to 100 nM PD 0332991 for 5 days. After incubation, cells were processed as directed in the kit and were analyzed using a FITC signal detector and propidium iodide detector using a Cell Lab Quanta SC flow cytometer.Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 . The Pearson chi-square test was used to assess the relationship between response and subtype.50 < 150 nM) and the most resistant cell lines . All ANOVAs were performed using the Benjamini-Hochberg False Discovery Rate (FDR) multiple test correction and a statistical cutoff value for sequences of a twofold change in at least three experiments. The criteria used to determine differentially expressed genes were P < 0.05 with a difference between average expression for each group of at least |0.2|. Sequence sets were compared using the Venn Diagram tool in the Resolver system. The two-dimensional cluster analysis was performed using an agglomerative hierarchical clustering algorithm based on the cosine correlation similarity metric.Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA) and hierarchical cluster analysis of the breast cell line expression profiles were used to compare the most sensitive cell lines , and GATA3. The calculated IC, and GAT2 < 0.05). The subtypes most sensitive to growth inhibition by PD 0332991 were ER-positive. In addition, 10/16 HER2-amplified cell lines were sensitive. PD 0332991 inhibited growth in a cytostatic manner in these cells with no lethality observed (data not shown).There was a statistically significant correlation between molecular subtype and sensitivity to PD 0332991  was identified, where 253 genes were upregulated in sensitive cell lines and 197 genes were upregulated in resistant lines. Hierarchical clustering of the 43 cell lines across these 450 differentially expressed genes was performed  were differentially expressed - with higher levels of RB1 and cyclin D1, and lower levels of p16, in the sensitive group. Of note, these three genes are cyclin related and are not in the breast cell type gene set.A significant overlap was found between the gene set associated with response to PD 0332991 and the gene set that defines breast cancer cell subtypes. Of the genes more highly expressed in the sensitive cell lines, 193/253 (76%) are luminal markers and 0/253 are nonluminal markers. In the resistant gene set, 117/197 (59%) are nonluminal markers and 0/197 are luminal markers. RB1, cyclin DFull results of the ANOVA with the exact genes and Venn diagrams demonstrating the overlap between the cell classification markers and response markers are available online  compared with those with higher IC50 values   were associated with sensitivity to the effects of PD 0332991 on the cell cycle and growth inhibition. Western blot analysis confirmed that Rb phosphorylation is decreased in sensitive cell lines after PD 0332991 treatment, while resistant lines that had detectable pRb were relatively resistant to the effects of PD 0332991 on Rb phosphorylation. In this case, the presence of pRb alone is not predictive of response to PD 0332991. pRb presence in the background of luminal ER-positive breast cancer, however, is predictive of response to the compound. pRb is present in some nonluminal breast cancer cell lines but these lines are resistant to both the antiproliferative effects of PD 0332991 and its ability to block Rb hyperphosphorylation.Using baseline Agilent gene expression profiles, we first demonstrated that luminal ER-positive and HER2-amplified breast cancer cell lines were more sensitive to CDK4/6 inhibition of proliferation and cell cycle arrest. ANOVA analysis of these data identified a set of genes that were associated with response to PD 0332991. While the majority of these genes were associated with the luminal subtype, increased RB1 and cyclin DFurther studies will be required to determine why PD 0332991 cannot block hyperphosphorylation in cell lines that do contain pRb. One can speculate that potentially CDK4/6 is mutated in these cell lines and does not allow PD 0332991 binding and kinase inhibition, as is the case in resistance to BCR-ABL inhibitors in chronic myelogenous leukemia . Alternain vitro observations. The lack of activity of a CDK4/6 inhibitor in cell lines and tumors that lack pRb can be explained by the fact that cyclin D1 does not offer G1 control in the absence of pRb [Resistance to PD 0332991 in many of the nonluminal breast cancer cell lines may be explained by the absence of pRb. Recent publications highlighted the lack of pRb in basal-like breast cancer tissue  and obsee of pRb .1 in breast cancer support the current observations of the activity of a CDK4/6 inhibitor in luminal ER-positive breast cancer, its synergism with tamoxifen in cell lines that are sensitive to hormone manipulation, as well as the reversal of resistance of those that have acquired a resistant phenotype in the face of anti-estrogen therapy. Estrogen effects on cell cycle progression are tightly linked to expression of cyclin D1 [1 amplification and/or overexpression has been more commonly associated with an ER-positive breast cancer subtype [1 can directly activate ER in a hormonally independent manner that is also independent of CDK and pRb function [1 splice variant - named cyclin D1b - that occurs in breast cancer tissue and cell lines, and whose expression can overcome cell cycle arrest induced by anti-estrogens via a CDK4 interaction [Published studies evaluating the role of cyclin Dyclin D1 . Cyclin  subtype  and, as  subtype ,20. Somefunction ,22. The function . In the eraction .1 function in HER2-mediated transformation has been described using transgenic mouse models [1 to activate CDK4 is critical for driving tumorigenesis in these models. Moreover, CDK4-associated kinase activity is required to maintain breast tumorigenesis in this system [1 levels. The authors hypothesized that this 'subset may benefit from inhibiting CDK4 kinase' [1 alone in the HER2-amplified cell lines. In addition, synergistic efficacy was observed between trastuzumab and PD 0332991 that may also be independent of cyclin D1 measurement. Earlier work evaluating the nonspecific cyclin-CDK inhibitor flavopiridol in combination with trastuzumab also demonstrated similar activity in HER2-amplified cell lines [In addition, a pivotal role for cyclin De models . More res system ,45 and a kinase' . Our datll lines . Finallyll lines .de novo resistance to trastuzumab remains a management challenge in clinical oncology. While limited data exist about the role of CDK4-cyclin D1 interactions and trastuzumab resistance, these data suggest a role for dual targeting of the HER2 pathway and CDK4/6. Molecular profiling of the JIMT-1 human breast cancer cell line derived from a woman with progressive HER2-amplified disease while receiving trastuzumab did identify a small amplicon on 12q14.1, which contains the CDK4 gene [Acquired and DK4 gene .The goal of this preclinical study was to guide patient selection as this novel agent moves into the clinic. Given the data from the current study as well as published work, there is a strong rationale for clinical development of PD 0332991 focusing on ER-positive luminal breast cancer as well as HER2-amplified disease and combining CDK4/6 inhibition with anti-estrogen or anti-HER2 therapy, respectively.50: concentration that inhibits growth by 50% of control; PBS: phosphate-buffered saline; PSF: 1% penicillin G-streptomycin-fungizone solution; Rb: retinoblastoma.ANOVA: analysis of variance; CDK: cyclin D kinase; CI: combination index; DMEM: Dulbecco's modified Eagle's medium; ER: estrogen receptor; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; ICRSF and DJS research funding from Pfizer, Inc. IC, CF, and GL are employees of Pfizer, Inc. The other authors declare that they have no competing interests.in vitro experiments and performed molecular biology. CG performed all microarrays and analyzed data. MA created the tamoxifen-resistant cell line and designed experiments. IC, CF, and GL collaborated in experimental design, data analysis, and manuscript writing. All authors read and approved the final manuscript.RSF and DJS designed and supervised the study, analyzed data, and drafted the manuscript. JD performed data analysis. DC, OK, DJC, and AD performed Excel file containing a table that lists the genes from the ANOVA of sensitive cell lines versus resistant cell lines.Click here for filePowerPoint file containing a figure that shows a Venn diagram demonstrating the overlap between resistant and nonluminal markers in breast cancer cell lines.Click here for filePowerPoint file containing a figure that shows a Venn diagram demonstrating the overlap between sensitive and luminal markers in breast cancer cell lines.Click here for filePowerPoint file containing a figure that shows a cluster of differentially expressed genes between the sensitive and resistant cell lines that excludes genes associated with cell subtype.Click here for filePowerPoint file containing a figure that shows a cycle analysis of PD 0332991 in combination with tamoxifen.Click here for filePowerPoint file containing a figure that shows a cycle analysis of PD 0332991 in combination with trastuzumab.Click here for file


These results suggest that vesnarinone plus glucocorticoid might be useful in lung cancer therapy. © 1999 Cancer Research CampaignVesnarinone, an oral cardiotonic, inhibited the growth of several human non-small cell lung carcinoma cell lines, and its anti-proliferative effects in vitro and in vivo were greatly enhanced by combination with glucocorticoids, but not other steroids. Simultaneous treatment with vesnarinone and dexamethasone is the most effective to evoke the synergistic effect in the growth inhibition of lung carcinoma EBC-1 cells. Dexamethasone and other glucocorticoids induced morphological changes in EBC-1 cells and these agents together with vesnarinone induced alkaline phosphatase activity, which is a typical marker of type II pneumocyte maturation. This treatment arrested the growth of the cells at the G


Replacement of enriched CMRL 1066 medium by cell-free ascites from tumour patients in the human tumour clonogenic assay described by Hamburger and Salmon (1977) increased plating efficiency for ovarian cancer cells by a median of 8-fold (range 0.4-1012 fold). In 40 experiments, two cases had a lower plating efficiency when cultured in cell-free ascites, 10 grew neither in standard medium nor in cell-free ascites and in two cases, growth was only observed in cell-free ascites. With standard medium, we observed 53% growth (greater than 5 colonies/dish) and 41% evaluable for chemosensitivity testing (greater than 30 colonies/dish). With cell-free ascites as culture medium, these figures were 71% and 63%, respectively. While under standard conditions the highest plating efficiency obtained was 0.25%, in 21% of the experiments done with cell-free ascites a plating efficiency higher than 1% was reached. We conclude that cell-free ascites is able to stimulate proliferation of ovarian cancer cells in agar and that the use of it extends the applicability of the clonogenic assay.


Cisplatin is one of the most active cytotoxic agents in the treatment of testicular cancer, but its clinical use is associated with side-effects such as ototoxicity, neurotoxicity and nephrotoxicity. Long-term kidney damage from cisplatin particularly affects the proximal tubular apparatus and can be detected by increased urinary excretion of brush-border enzymes, such as L-alanine-aminopeptidase (AAP), and magnesium. In the current study, the flavonoid silibinin was used as a nephroprotectant for cisplatin-induced nephropathy in a rat animal model. Infusion of silibinin before cisplatin results in a significant decrease in glomerular (indicated by creatinine clearance and serum urea level) and tubular kidney toxicity (excretion of brush-border enzymes and magnesium). Silibinin given alone had no effect on renal function. In order to exclude an inhibition of the anti-tumour activity of cisplatin and 4-hydroperoxy-ifosfamide by co-administration of silibinin, in vitro studies were performed in three established human testicular cancer cell lines. Dose-response curves for cisplatin (3-30 000 nmol) combined with non-toxic silibinin doses (7.25 x 10(-6) or 7.25 x 10(-5) mol l-1) did not deviate significantly from those of cisplatin alone as measured by relative cell survival during a 5 day assay using the sulphorhodamine-B staining technique. Also silibinin did not influence the cytotoxic activity of 4-hydroperoxy-ifosfamide (30-10 000 nmol) in vitro. In summary, these in vitro data rule out a significant inhibition of the anti-tumour activity of the major nephrotoxic components, cisplatin and 4-hydroperoxy-ifosfamide, by co-administration of silibinin in a human germ cell tumour cell line model. Together with these demonstrated cytoprotection effects in the rat animal model, these data form the basis for a randomised clinical trial of silibinin for the protection of cisplatin-associated nephrotoxicity in patients with testicular cancer.


Previous studies have shown that multidrug resistance (MDR) in the doxorubicin-selected lung tumour cell lines COR-L23/R, GLC4/ADR and MOR/R is associated with overexpression of the MRP gene. In this study we report that resistance to daunorubicin, vincristine and rhodamine 123 can be partially reversed in these cell lines by exposing the cells to buthionine sulphoximine (BSO), an inhibitor of glutathione (GSH) synthesis. This effect of BSO on drug resistance was associated with an increased intracellular accumulation of daunorubicin and rhodamine 123, owing to inhibition of the enhanced drug efflux. In contrast, the accumulation of daunorubicin was not increased by BSO treatment in a P-glycoprotein (P-gp)-mediated MDR cell line. BSO treatment  of the cell lines resulted in 60-80% depletion of cellular GSH levels. The effects of BSO on daunorubicin accumulation in the COR-L23/R and GLC4/ADR cells were associated with cellular GSH depletion. In addition, increase of cellular GSH levels in BSO-treated COR-L23/R and GLC4/ADR cells as a result of incubation with 5 mM GSH ethyl ester restored the accumulation deficit of daunorubicin. However, the transport of daunorubicin did not increase the GSH release in any of the cell lines. These results demonstrate that drug transport in MRP- but not in P-gp-overexpressing MDR tumour cell lines can be regulated by intracellular GSH levels.


THBS-1 -transfected MDA-MB-435 breast cancer cells that overexpress thrombospondin-1. The role of urokinase plasminogen receptor in thrombospondin-1-mediated adhesion and invasion was studied by antisense inhibition, enzymatic cleavage and antibody neutralization. Tumour cell adhesion to collagen and laminin was evaluated. Tumour cell invasion was studied in a modified Boyden chamber collagen invasion assay. Tumour cell thrombospondin-1 induced a 2–7 fold increase in urokinase plasminogen activator receptor and cell-associated urokinase plasminogen activator expression and a 50–65% increase in cell-associated urokinase plasminogen activator and plasmin activities. Furthermore, tumour cell thrombospondin-1 promoted tumour cell invasion and decreased tumour cell adhesion through up-regulation of urokinase plasminogen activator receptor-controlled urokinase plasminogen activator and plasmin activities. We conclude that tumour cell-produced thrombospondin-1 may play a critical role in the regulation of tumour cell adhesion and tumour cell invasion. © 2000 Cancer Research CampaignWe have previously shown that platelet-produced thrombospondin-1 up-regulates the urokinase plasminogen activator and its receptor and promotes tumour cell invasion. Although tumour cells produce thrombospondin-1 in vivo, they produce only minimal amounts of thrombospondin-1 in vitro. To determine the effect of tumour cell-produced thrombospondin-1 in the regulation of the plasminogen/plasmin system and tumour cell invasion, we studied


Whilepreclinical activity has been observed in many cancer types,clinical activity has been observed only in pilot and phase IItrials in liposarcoma and prostate cancer. Most studies addressagonist compounds, with substantially fewer reports on anticancereffects of PPARγ antagonists. In cancer model systems, someeffects of PPARγ agonists were not inhibited by PPARγ antagonists,suggesting noncanonical or PPARγ-independent mechanisms. Inaddition, PPARγ antagonists, such as T0070907 and GW9662, haveexhibited antiproliferative effects on a broad range ofhematopoietic and epithelial cell lines, usually with greaterpotency than agonists. Also, additive antiproliferative effectsof combinations of agonist plus antagonist drugs were observed.Finally, there are preclinical in vivo data showing thatantagonist compounds can be administered safely, with favorablemetabolic effects as well as antitumor effects. Since PPARγantagonists represent a new drug class that holds promise as abroadly applicable therapeutic approach for cancer treatment, itis the subject of this review.PPAR Since it has apredominantly nuclear location, regardless of whether cognate ligands arepresent, it is classified as a type II NR. It functions as a transcription factor by heterodimerizing with theretinoid X receptor (RXR), after which this complex binds to specific DNAsequence elements called peroxisome proliferator response elements(PPREs) [γ must be bound by ligand. RXR can be affectedby binding its own cognate ligands, usually resulting in incremental increasesin transcriptional activity. After the PPARγ/RXR heterodimer binds to PPREs in promoterregions of target genes, coactivator proteins, such as p300 (CBP),SRC-1, and Drip205 (or TRAP220) family members, are recruited to this complexto modulate gene transcription [γ ligands appear to be able to recruit different coactivators, which may explain differences in the biological activitybetween ligands [PPAR(PPREs) . In ordecription . Differeγ is the induction of differentiation ofadipocytes, the cell type that expresses the highest levels of PPARγ amongst normal tissues. Lower levels of PPARγ are, however, found in other normal tissuesand cell types such as skeletal muscle, liver, breast, prostate,colon, type 2 alveolar pneumocytes, some endothelial cells as well as monocytes,and B-lymphocytes. There are three γPPAR mRNA isoforms  and two major protein species (γ1 and γ2). ThemRNA isoforms are generated by alternate promoter usage, resulting in anadditional 28 amino acids at the N-terminus of PPARγ2 compared with PPARγ1. Most tissues express PPARγ1, whereas the PPARγ2 isoform is expressed mostly by adipocytes.The longer N-terminal domain of PPARγ2 may affect function, since this isoform wasshown to confer a higher level of ligand-independent transcriptional activity,which was further increased by physiologic concentrations of insulin [γ expression by fat and its role in adipogenesisled to the recognition that agonistic PPARγ ligands have antidiabetic effects. Thechemical class of PPARγ agonists known as thiazolidinediones (TZDs)demonstrated high-affinity binding to PPARγ [The cardinal biologic activity of PPAR insulin . Highleto PPARγ  as well γ and its expression in normal tissues [γ expression was observed in an array of primarycancers and derivative cell lines. Its expression was reported initially inliposarcoma [γ and to have antiproliferative effects incancer models, such as the naturally occurring eicosanoid, 15-deoxy-Δ12,14-prostaglandin J2(15-d-PGJ2), the N-aryl tyrosine derivative, GW1929 [γ agonist activity, such as TZDs, have PPARγ-dependent antiproliferative effects, they havealso been shown to have antiproliferative effects in cell types that aregenetically PPARγ-null [γ ligands has resulted from variability in theclassification of some compounds  [Not long after reports of the cloning of PPAR tissues , 9,PPARosarcoma , and sooosarcoma –14. In aosarcoma , 16. Com, GW1929 , and the, GW1929 . Whilecivities) , 21.μM concentrations of BADGE, whichwould tend to increase off-target effects. Subsequently, Seargent et al. [γ antagonist, GW9662, had direct antiproliferativeeffects on three breast cancer cell lines of differing phenotypes . This antagonist compound was somewhat more potent in its effectsthan an agonist (rosiglitazone). In this report, the role of PPARγ in mediating growth inhibition was addressed,but not fully elucidated. All three cell lines expressed it and the predicted,canonical PPARγ-related transactivation effects weredemonstrated, with the agonist inducing transactivation and the antagonist suppressingit, thus excluding PPARγ-mediated transactivation as the mechanism ofthis effect. There are data, however, thatsuggest that antagonist-type compounds may also act via other PPARγ-dependent pathways. Lea et al. reported similar results usinga range of agonist and antagonist compounds on both murine and human cell lines[γ antagonist, T0070907, onhepatocellular carcinoma cell lines was attenuated by knockdown of PPARγ by siRNA [γ-mediated transrepression mechanism, which hasbeen demonstrated with respect to anti-inflammatory effects of PPARγ ligands mediated by the NF-κB signaling pathway. Pascual et al. showed similareffects of a pure agonist (rosiglitazone) and a mixed agonist/antagonist (GW0072)on the repression of a NF-κB-regulated gene, iNOS, suggesting that pure antagonists may also be capable ofmediating this effect [The initial report of Fehlberg et al.  showed at et al.  showed tll lines. Schaefeby siRNA . These s effect .γ ligands (both agonist and antagonist) exert PPARγ-independent effects suggesting other cellulartargets of these compounds. This was demonstrated clearly by Palakurthiet al., who demonstrated invitro and in vivo growthinhibition of two agonist compounds, troglitazone and ciglitazone, inexperiments utilizing PPARγ−/− and PPARγ+/+ embryonic stem cell lines (ES),both of which exhibited very similar sensitivity to these compounds [γ. Theeffect of antagonist compounds on this pathway has not been reported. As noted, BADGE had similar proapoptoticeffects in a colon cancer line expressing PPARγ and a T-lymphoma line that showed nodetectable expression of it (by immunoblotting and RT-PCR) of this target [There are also data that PPARompounds . Thisefs target .But, giγ antagonist compounds have alsobeen shown to affect cell shape, adhesion, and invasiveness of cancer celllines. Masuda et al. evaluatedthe effects of the PPARγ antagonists, BADGE, GW9662 andT0070907, on four squamous carcinoma cell lines derived from tumors of the oralcavity. Antiproliferative effects wereshown for the three antagonists, but not for the agonist, pioglitazone [γ-dependent effect. Since adhesionand detachment are related to cytoskeletal structure and function, this wasassessed by fluorescent staining of F-actin. Using confocal microscopy, T0070907 was shown to cause dose-dependentdisruption of F-actin, associated with rounding of the cells. Additionalexperiments showed inhibition of FAK and MEK-ERK signaling pathways, as well asdecreased expression of integrin α5 and CD151, both of which are adhesionproteins that have been implicated in cancer cell invasion and metastasis. Schaeferet al. showed similar effectsof PPARγ antagonists on hepatocellularcarcinoma cell lines including inhibition of adhesion, induction of anoikis, andinhibition of phosphorylation and activation of FAK [γ inhibition, and could be mediatedby the antagonist or knockdown of PPARγ via specific, cognate siRNA.T0070907 was also shown to have substantially greater growth inhibitory effectson the HepG2 line compared with the agonist drugs, troglitazone, androsiglitazone. Takahashi et al. demonstrated anti-invasive andgrowth inhibitory effects of the antagonists, GW9662 andT0070907, onesophageal cancer cell lines. The anti-invasive effects were observed at levelssubstantially lower than those required for growth inhibition [γ antagonist compounds have showeffects on cell growth, adhesion, and invasion in multiple epithelial cancermodels.PPARlitazone . Effectsn of FAK . These ehibition . In summγ-dependent, but the potential role of othertargets is suggested by the similar effects of BADGE on a PPARγ+ colon cancer line and a PPARγ-negative T-lymphoma line. Also, thesubstantially different concentrations of PPARγ antagonists needed to induce anti-invasiveeffects versus growth inhibition in esophageal cancer lines suggest differentmechanisms with differing degrees of PPARγ dependence or lack of involvement of the PPARγ-signaling pathway for some effects. A PPARγ-independent effect of antagonists oncolorectal cancer cell lines and in an invivo tumor xenograft derived from one of the lines was shown in a morerecent report by Schaefer et al.[γ, PPARδ,and proteasome function. This downregulation of tubulins α and β may explainthe antimigratory, anti-invasive, and antimetastatic effects that wereobserved. Thus, in summary, PPARγ antagonist compounds with varying chemicalstructures (though GW9662 and T0070907 are similar) have several significantanticancer effects in vitro andin vivo in epithelial cancermodel systems including breast, colon, aerodigestive squamous cell, andhepatocellular.Some of these effectsare PPARr et al.. A decreγ antagonists on hematopoietic cell lines. Initial screening showed that severalmyeloma (MM) cell lines had the greatest sensitivity to the antiproliferativeeffects of the antagonists, GW9662 and T0070907. Thus multiple MM lines weretested, including one that is IL-6-dependent, for sensitivity to thesecompounds as well as to the agonist, pioglitazone. MM lines as well asnon-Hodgkin lymphoma (NHL) lines showed significantly greater sensitivity tothe growth inhibitory effects of the two antagonist drugs compared with theagonist [Studies were conductedin our lab to assess the effects of PPARagonist . As a gr50 values for the panel of 16 cell linestested in these studies are shown in 50 values of the antagonistcompounds and the agonist drug, pioglitazone, were observed . While the MM lines showed thegreatest sensitivity to the antagonists, similar degrees of sensitivity to theantagonists were also seen in the subset of breast cancer lines, which includedtwo lines that are estrogen receptor-negative. Though not quite as sensitive asa subset, significant differences between the antagonists and the agonist werealso observed in the renal cell lines, which are among the most chemoresistantepithelial lines. The differential sensitivities within and across cell linesdid not appear to be related to the levels of PPARγ expression. Also, neither the agonist nor theantagonist induced significant upregulation of PPARγ as has been reported in some studies with PPARγ ligands. Consistent with prior reports, combinationsof the agonist and with each of the antagonists did not result in attenuationof growth inhibitory effects. In fact, schedule-dependent increases in growthinhibition were observed, particularly when the antagonists were added to cells24 hours prior to the agonist. Aspects of the mechanisms of cytotoxicity of theantagonists and agonists were also compared. It was shown that both classes of PPARγ ligand-induced apoptotic effects, but thiseffect was found to be caspase-independent for the agonist, pioglitazone [The IClitazone .γ antagonists, since this is a cytokine thatplays a central role in the pathogenesis and progression of MM, as well asother cancer types. For these studies, 4 of the 5 MM lines that were utilizedwere IL-6-independent in order to follow up on a previousreport of Wang et al. thatanalyzed the responses of three MM lines to the PPARγ agonists, 15-d-PGJ2 andtroglitazone. This report showed that growthinhibition and certain downstream signaling events were PPARγ-dependent, and alsothat two IL-6-dependent MM lines expressed PPARγ while an IL-6-independent line did not [γ, while the autocrine IL-6-dependent line,U266B1, did not express PPARγ by immunoblotting. Also, three of the four of IL-6-independent MM lines were more sensitive to the growthinhibitory effects of both of the two PPARγ antagonist compounds compared with theIL-6-dependent line, U266B1 . MTT assays were performed in thepresence and absence of exogenous IL-6 (5 ng/mL). For both of these MM lines,addition of IL-6 did not induce resistance, but instead appeared to increasethe sensitivity of these lines to T0070907, with a similar trend observed withGW9662 [In MM cell lines, whichare more often IL-6 dependent compared with other B cell lines, the strict dependenceon exogenous IL-6 is indicative of ongoing requirement for this signalingpathway, which in pathophysiologic states, such as MM, usually depends on productionof this cytokine by stromal cells. InMM, clinically more aggressive or treatment-resistant disease is associatedwith production of IL-6 by the myeloma cell themselves as opposed to the bonemarrow stroma . MM linehGW9662 .γ antagonistcompounds, GW9662 and T0070907, differ in their antiproliferative dose-responseeffects compared with the agonist as well as other agents. Not only are the corresponding IC50 values for theantagonists significantly lower than the agonist, pioglitazone, but a greaterdegree of growth inhibition (85–97% versus 50–80%) was observedwith the former compounds. Also, of note was that the maximal effects of theseagents were seen at concentrations that were only 2- to 3-fold greater than theIC50 across the entire panel of cell lines tested that included celllines with relative and very high levels of chemoresistance . The dose-response curves were much steeper with the antagonistcompounds compared to the agonist, pioglitazone, and also much steeper thanwhat is observed with most other agents, including standard chemotherapy drugsand other agents . This is a promising initial preclinicallead given that hLL1 is now being evaluated in several phase I/II clinicaltrials in B-cell cancers such as NHL and MM, and appears to be safe and welltolerated.Since MM lines as a group were the most sensitive ofthe cell lines we tested, interaction with other novel agents for therapy of MMwere evaluated. One such agent is anti-CD74 monoclonal antibody (mAb). CD74 wasshown to be strongly expressed by the malignant plasma cells in the vastmajority of clinical MM specimens as well as the majority of MM lines . Itwas γ ligands are mediated by various mechanisms.Some studies show or suggest canonical PPARγ-mediated effects ,as exemplified by early in vitro studies with agonist compounds thatshowed fat accumulation, a major PPARγ-mediated effect, in both breast cancer andliposarcoma cell lines [γ agonist compounds on MM lines was seen only inlines expressing PPARγ and that these effects were reversed bycotreatment with an antagonist compound [γ-independent effects were demonstrated for bothagonist and antagonist compounds in reports from Palakurthi etal. [γ-wild type and PPARγ-null (knockout) embryonic stem cell lines,both in vitro and in vivo [γ-independent growth inhibitory andantimetastatic effects of several antagonist compounds were shown in both in vitro and in vivo studies using threecolon carcinoma cell lines. These effects were associated with reductions intubulin levels and were also shown to be independent of PPARδ and proteasome function. The PPARγ-independent effect of agonist compounds wasshown to be associated with inhibitory effects on the protein translationpathway. The mechanism of PPARγ-independent effects of antagonist compounds ontubulin levels has not been elucidated.The studies reviewed above have shownthat the effects of PPARll lines , 12. Thill lines . The stucompound . In conti etal.  and Schai etal. . This wa in vivo . PPARγ-iγ-mediated transrepression may explain some ofthe effects of antagonist compounds. This was suggested by the attenuation ofthe effects of antagonist compounds by PPARγ knockdown by siRNA in hepatocellular carcinomacell lines [γ agonist and antagonist compounds result inadditive antiproliferative effects in various cancer cell lines [κB signaling pathway, which is central to inflammation and to the proliferation and survival of multiple cancer types including hepatocellular andcolon carcinomas as well as multiple myeloma. The potential role of this andother mechanisms remain to be determined.Themechanism of PPARll lines . Also, tll lines , 30 iscγ antagonists have in vitro and preclinical in vivo anticancer effects that are asbroad and potent as agonist compounds. These effects have been demonstrated ina wide range of epithelial cancer cell lines as well as hematopoietic cancercell lines. Exploration of theunderlying mechanisms of action for antagonist compounds has shown either involvementof PPARγ or a PPARγ-independent effect. One study suggested theinvolvement of the canonical transactivation mechanism in that antagonisteffects were antagonized by coincubation with an agonist compound, pioglitazone[γ affected responses to antagonist compounds,the effect was not consistent with the canonical transactivation mechanism, butmay be consistent with a transrepressive mechanism [γ, PPARδ, or the proteasome [The studies reviewed above have shown that PPARlitazone. In anotechanism . Anotheroteasome .γ agonists, there have been relatively few withantagonist compounds. Also agonists have been tested clinically. Some studies withantagonists have been conducted in noncancer models at low doses (≤1 mg/kg),which were not toxic and biologically active [γ antagonist, SR-202, has been synthesized and evaluatedin preclinical models (While there have been numerous preclinical in vivo studies in cancer models withPPARy active , 38. A cl models . It was


The effect of mitomycin C on two human ovarian cancer cell lines was measured during several exposure times and concentrations using the Human Tumour Colony-forming Assay (HTCA). Changes in exposure time and concentration resulted in considerable differences in tumour cell survival. It is concluded that several exposure times and concentrations are necessary for in vitro sensitivity testing. We suggest alternative criteria derived from pharmacokinetic data in patients instead of one-tenth of the peak plasma level which is the usual practice.


Authenticity was confirmed by genetic fingerprinting, which also demonstrated the potential stability of long-term cultures. In vitro glucocorticoid resistance correlated well with that measured ex vivo, but all lines were significantly more sensitive to vincristine than primary specimens. Sensitivity to methotrexate was inversely correlated to that of glucocorticoids and L-asparaginase, indicating possible reciprocity in resistance mechanisms. A cell line identified as highly methotrexate resistant (IC50 >8000-fold higher than other lines) was derived from a patient receiving escalating doses of the drug, indicating in vivo selection of resistance as a cause of relapse. Many of these lines are suitable as models to study naturally occurring resistance phenotypes in paediatric ALL.Cell lines are important models for drug resistance in acute lymphoblastic leukaemia (ALL), but are often criticised as being unrepresentative of primary disease. There are also doubts regarding the authenticity of many lines. We have characterised a panel of ALL cell lines for growth and drug resistance and compared data with that published for primary patient specimens. In contrast to the convention that cell lines are highly proliferative, those established in our laboratory grow at rates similar to estimates of leukaemic cells  This is true not only for relapsed ALL . Plates were re-incubated for 6 h before addition of 100 μl of acidified isopropyl alcohol solution to dissolve formazan crystals and measurement of absorbance at 590 nm. Testing was performed using two-fold step dilutions of the following drug ranges: cytosine arabinoside  0.3 pg ml−1–2.5 μg ml−1; dexamethasone  30 pg ml−1–250μg ml−1; methylprednisolone  30 pg ml−1–250 μg ml−1; 6-thioguanine  12 pg ml−1–100 μg ml−1; 6-mercaptopurine  60 pg ml−1–500 μg ml−1; daunorubicin  0.2 pg ml−1–2 μg ml−1; doxorubicin  1 pg ml−1–8μg ml−1; L-asparaginase  2.4 × 10−6–20 IU ml−1; vincristine  23 fg ml−1–195 ng ml−1; methotrexate  60 pg ml−1–500 μg ml−1. The IC50 (drug concentration that inhibits cell growth by 50%) was used as the measure of drug resistance. Data represent the average of 2–6 separate experiments and linearity was checked between viable cell number and optical density for each cell line. Where 50% cytotoxicity was not achieved by even the highest dose, the IC50 was recorded as double the highest concentration tested.Resistance was tested using the MTT assay (50 range for bone marrow specimens obtained from paediatric ALL patients at the time of diagnosis (PD) or relapse (PR). Data reported in these studies as LC50 values are, for simplicity, referred to here in terms of IC50. Eligible studies satisfied the following criteria: (i) drug sensitivity was tested in primary ALL specimens using the MTT assay; (ii) drug incubation was for 3–5 days with no exposure to other agents before testing; (iii) resistance was reported using IC50 or LC50 values; (iv) patients were under 19 years of age and were divided into diagnosis and relapse cohorts. In the two cases where patients were stratified by immunophenotype -transformed cell line PER-607 was not included in any of the analyses. Significant differences in IC50 values between drugs (in molarity) were determined using the nonparametric Wilcoxon-matched pairs signed ranks test. All other differences were assessed using the Mann–Whitney U-test. Spearman's correlations were used to assess relationships between drug profiles . Delta IC50 profiles for MPRED, DEX, ASP and MTX were calculated by subtracting median Log2 IC50 values  from the Log2 IC50 score for each cell line.All comparisons were performed on LogThe 17 cell lines developed in our laboratory are listed in 50 values for each cell line are listed in ex vivo . Previous work in primary specimens has indicated that T-ALL specimens are more resistant than precursor B-lineage to several front-line drugs, especially ASP, VCR and glucocorticoids , whereas 6TG was 6–24 times more cytotoxic than 6MP . These observations are consistent with previous reports of the in vitro cytotoxicity of these agents (P>0.05).Daunorubicin was approximately three times more potent than DOX in both lineages  and so the reported LC50 range for patients at relapse is likely to be an underestimate.To assess how the spectrum of drug resistance observed in the cell line panel related to levels of resistance found in patients, we compared our data with that published from  patient . Howeverin situ inhibition of thymidylate synthetase , demonstrating that this is a valid approach for the identification of potential cross-resistance between compounds, although it should be emphasised that these relationships were not directly tested in the present study. DEX showed significant correlation  to all drugs, except MTX and the thiopurines; MPRED mirrored this pattern, particularly in regard to ASP, DNR and MTX, the latter showing an inverse relationship as it did with DEX. The relationship between MPRED, DEX, ASP and MTX in the T-ALL cell lines is demonstrated graphically in 50 score used in this analysis essentially ranks the cell lines for their resistance in comparison to the population median . Cell lines resistant to MPRED, DEX and ASP were generally the most sensitive to MTX and vice versa , indicating cross-resistance.Spearman's correlations were used to identify drugs with similar profiles across the T-ALL cell line panel, which represents a larger and more homogenous group than the B-lineage cell lines. ICf action , boxed wex vivo is ∼65–90 h, whereas leukaemic pre-B cells are thought to be more heterogeneous, ranging from 25 to 240 h  and thus represent an ideal model system for the investigation of resistance mechanisms that may occur naturally in vivo; to this end, experiments are underway to examine the gene-expression profile of the panel by high-density oligonucleotide microarray. However, the consistent hypersensitivity of all cell lines to VCR may be an indication that in vitro studies of resistance for this drug should be interpreted with caution. The observation cannot be explained as an artefact of high proliferation rates in culture as many of these lines are extremely slow growing (in vitro microenvironment may mitigate changes to cell architecture that render the cell more sensitive to the microtubule-blocking effects of VCR.In this study, we have not directly tested the drug resistance profiles of the patient specimens from which our cell lines were derived. However, comparison with previously published data indicates that for each of the drugs tested here, except VCR, there are several cell lines within the panel that accurately reflect the sensitivity of leukaemic blasts tested  growing , but may50 values for all of the drugs, except ASP for which it appears to have retained sensitivity (in vivo. The patient subsequently relapsed for a fourth time after having received high-dose ARA-C; a description of an additional cell line isolated from this later time point and demonstrating resistance to ARA-C has previously been published (PER-145 was found to be highly resistant to MTX and had relatively high ICsitivity . This cesitivity  and was 50 correlation between DNR, DOX, VCR and ARA-C, consistent with previous studies indicating mechanisms of cross-resistance for these drugs (In T-ALL cell lines, there was significant IC


Extracts containing tissue-specific DNA-inhibitory activity were prepared from normal FL (Swiss) and BALBc3T3 cells, from these cells transformed with sarcoma virus and from revertants cloned from the transformed cell lines. By testing all extracts on all cell lines we found that (1) production of and susceptibility to the inhibitors were decreased in transformed BALB/c cells (2) specificity varied with expression of the transforming genome, as an extract from a given cell line inhibited the growth of its cell of origin, e.g. revertant, more than normal or transformed cells, and (3) there was also a DNA-synthesis stimulator.


We examined the effect of selective thromboxane A2 (TXA2) receptor antagonists, calcium 5(Z)-1R, 2S, 3S, 4S-7-[3-phenylsulphonylaminobicyclo [2.2.1] hept-2-yl]-5-heptonoate hydrate (S-1452) and +/- -7--7-phenylhaptanoic acid (AA-2414), on sensitivity to cis-diamminedichloroplatinum (II) (CDDP) in non-small-cell lung cancer cell lines. IC50 values to CDDP using MTT assay were decreased 2.1- and 4.6-fold respectively by treatment with 250 or 500 microM S-1452, for a 2 h simultaneous drug exposure, and those of PC-9/CDDP, a CDDP-resistant cell line, were decreased 3.1- and 6.1-fold. Sensitivity to carboplatin was also enhanced by the treatment with S-1452. IC50 values to CDDP and carboplatin were decreased by treatment with AA-2414 in a dose-dependent manner. Isobologram analysis showed that the combination of CDDP with S-1452 or AA-2414 produced supra-additive or additive effects in each cell line. Neither glutathione content nor glutathione S-transferase activity was changed in either cell line by treatment with 500 microM S-1452. Accumulation of platinum into PC-9 and PC-9/CDDP was increased by the treatment in a dose-dependent manner. Na+, K+-ATPase activity of PC-9 and PC-9/CDDP was enhanced by the treatment of S-1452 in a dose-dependent manner. These data show that the TXA2 receptor antagonists may enhance the sensitivity of non-small-cell lung cancer cell lines to platinum agents. Increase in Na+, K+-ATPase activity induced by S-1452 may be the mechanism of its sensitising effect through increase in platinum accumulation.


It was found that in coordinating solvents themore soluble derivative 2 is less active than the phenyl one (4), and compounds 1 and 3 are even inactive.The         in vitro antitumour activity of ionic diphenyltin(IV) complexes 4 and 5 against seventumoural cell lines of human origin is also reported. The preparation and characterization (3 is mentioned too.In this paper, the


We have measured post-irradiation recovery  after fractionated radiation in plateau-phase cultures of two human tumour cell lines derived from tumours of different radiocurabilities (melanoma and breast). Although the radiation survival-curve parameters of these cell lines are similar, the repair of potentially lethal X-ray damage after fractionated X-ray treatment conferred significant radioresistance on the human melanoma cells but not the human breast carcinoma cells. We suggest that the repair of potentially lethal damage may correlate with clinical radiocurability.


Following 1 h incubation with211At-rituximab, in concentrations which gave an initial activity of 50 kBq ml–1, a high tumour cell to normal bone marrow cell toxicity ratio was obtained; 4.1 to 1.0 log cell kill. Biodistribution studies of211At-rituximab in Balb/c mice showed similar stability as that of the iodinated analogue. The data indicate that testing of211At-rituximab in human patients is warranted. © 2000 Cancer Research CampaignThe ability of an α-emitter conjugated to a chimaeric anti-CD20 monoclonal antibody to kill selectively human B-lymphoma cells in vitro is reported. Two B-lymphoma cell lines RAEL and K422, and normal haematopoietic progenitor cells from human bone marrow aspirates were incubated with


Polyamines regulate important cellular functions and polyamine dysregulation frequently occurs in cancer. The objective of this study was to use a systems approach to study the relative effects of PG-11047, a polyamine analogue, across breast cancer cells derived from different patients and to identify genetic markers associated with differential cytotoxicity.50 (dose required for 50% relative growth inhibition) were correlated with the omic profiles of the cell lines to identify markers that predict response and cellular functions associated with drug sensitivity.A panel of 48 breast cell lines that mirror many transcriptional and genomic features present in primary human breast tumours were used to study the antiproliferative activity of PG-11047. Sensitive cell lines were further examined for cell cycle distribution and apoptotic response. Cell line responses, quantified by the GI50 values with the omic profiles of the cell lines identified genomic, transcriptional and proteomic variables associated with response.The concentrations of PG-11047 needed to inhibit growth of members of the panel of breast cell lines varied over a wide range, with basal-like cell lines being inhibited at lower concentrations than the luminal cell lines. Sensitive cell lines showed a significant decrease in S phase fraction at doses that produced little apoptosis. Correlation of the GIA 13-gene transcriptional marker set was developed as a predictor of response to PG-11047 that warrants clinical evaluation. Analyses of the pathways, networks and genes associated with response to PG-11047 suggest that response may be influenced by interferon signalling and differential inhibition of aspects of motility and epithelial to mesenchymal transition.http://www.biomedcentral.com/1741-7015/7/78See the related commentary by Benes and Settleman:  Low molecular weight polycationic polyamines are found in cells in millimolar concentrations and are essential for mammalian cell proliferation, survival and function . PolyamiSeveral approaches to modulating the polyamine pathway have been utilized to date . TargetiN1, N12-bisethylspermine (BESpm) [in vitro [in vitro screens in order to provide an insight into the subpopulations of specific cancer types that might be amenable to treatment with PG-11047, while at the same time identifying putative molecular markers for the prediction of sensitivity or resistance to treatment.The compound utilized in this study, PG-11047, is a second generation polyamine analogue of in vitro . It has et al., personal communication). The basal A and basal B subtypes will be referred to as basal and claudin-low, respectively, in the following text. Our studies show that basal subtype breast cancer cell lines are most sensitive to treatment with PG-11047 based on the dose required to inhibit 50% relative growth (GI50). Correlations between cell line responses quantified by the GI50 and the transcriptional and genomic characteristics of these cell lines identified a set of 250 genes that are associated with response. A signature comprised of 13 of these genes is proposed as a predictor of response to PG-11047 treatment in breast tumours.Breast cancers develop through multiple genetic and epigenetic changes resulting in a wide range of cancer phenotypes. The resulting breast cancers have been classified, based on their gene expression profiles, into five distinct subtypes that are associated with diverse tumour characteristics and clinical outcome ,10 and iet al. [et al., personal communication). The recurrent genome copy number abnormalities in the collection of cell lines was similar to those in primary tumours, while hierarchical analysis of the transcriptional features of the cell lines defined three clusters designated luminal, basal and claudin-low [Breast cancer cell lines were obtained from the American Type Culture Collection and from collections developed in the laboratories of Drs Steve Ethier  and Adi Gazdar . Forty of the cell lines were characterized in great detail by Neve et al. , eight wudin-low .N1, N12bis(ethyl)-6,7-dehydrospermine tetrahydrochloride, previously known as SL-11047 and CGC-11047) was obtained from Progen Pharmaceuticals  and a stock solution of 100 mM was prepared in sterile water. For the dose response study, a set of nine doses  in 1:5 serial dilution were added in triplicate wells. Adenosine triphosphate content was measured as an estimate of relative cell number using the CellTiter-Glo (CTG) Luminescent Cell Viability Assay , with slight modification form manufacturer's protocol, at day 0 (time when drug was added) and day 3 of drug exposure. Briefly, the CTG reagent was diluted with phosphate-buffered saline  and the culture media was removed from the 96-well plate prior to adding 50 μl per well of the diluted CTG reagent. Luminescence from the assay was recorded using BIO-TEK FLx800.Cells were plated at proper density  in 96-well plates, so that they remained in logarithmic growth at the time of assay. The cells were allowed to attach overnight before being exposed to the polyamine analogue PG-11047 for 72 h. PG-11047 /(C- T0)) ×100, where T0 is the CTG luminescence at day 0, C is the untreated control CTG luminescence at day 3 and T is the CTG luminescence at the test concentration. The dose response curve was fitted with GraphPad Prism4 software . The GI50 and TGI  values were determined as the drug concentrations that caused 50% and 0% relative growth at 72 h drug exposure, respectively.Metrics describing the drug effects were calculated according to the methods described by the NCI/NIH DTP Human Tumor Cell Line Screen  and by Met al. . The % gThe fractions of cells in the G1-S and G2 M phases of the cell cycle were estimated from measurements of BrdUrd-DNA distributions . For thiThe extent of PG-11047-induced apoptosis was determined using the Caspase-Glo 3/7 assay kit from Promega . A positive control of 10 nM docetaxel treatment was included in all experiments. After incubation of cells with PG-11047 at the specified concentrations for 48 h or 72 h, 50 μl Caspase-Glo reagent was added to wells. After an incubation time of 1 h, luminescence was measured using a BIO-TEK FLx800 luminometer. Parallel experiments were set up to determine the relative number of viable cells using the CTG assay, as described above, in order to normalize the caspase activity by relative cell density.50 levels for all cell lines were correlated with the molecular features measured for each cell lines (microarray profiles for RNA expression and genome copy number and Western blot based protein profiles) as reported by Neve et al. [P-value corresponding to the F-statistic for the fit [P-values were corrected for multiple hypotheses testing using the false discovery rate method [et al, in preparation). The final model was determined via leave-one-out cross-validation. Ingenuity Pathway Analysis  knowledgebase molecular interactome was applied to the 250 predictor genes to identify the networks of genes, generated algorithmically based on their connectivity. Network genes were further analysed for significant pathways associations.The PG-11047 GIe et al. . We usede et al. . Briefly the fit . P-valuee method . This pre method . The come method  . The TGI (dose required to inhibit 100 percent of the cell growth from T0) showed similar subtype specificity  and protein level (Western data) identified additional markers significantly associated with response. These are listed in Additional Files Correlative analysis of GIWe examined the effect of PG-11047 on cell cycle distribution and apoptosis in order to understand the mechanism by which PG-11047-induced growth inhibition occurred. Six sensitive and three resistant cell lines were plated and treated as stated previously. These were labelled with BrdUrd at 48 and 72 h after PG-11047 treatment. The sensitive cell lines showed a greater reduction in fraction of cells incorporating BrdUrd than did the resistant cells. The cell cycle distributions of these cell lines treated with 0.3, 10 and 300 μM of PG-11047 for 72 h are shown in Figure 50 response profiles. Basal subtype breast cell lines mirror many molecular features of basal-like primary breast tumours [et al., personal communication). This suggests that, PG-11047 may be preferentially effective against this more aggressive breast cancer subtype. This is consistent with observations made in a study of the polyamine analogue, N1, N11-diethylnorspermine (DENSPM) [et al. [Polyamines are required for cellular viability and elevated levels are found in many tumour types, including breast cancer , making  tumours ,16 inclu(DENSPM) . An anal [et al.  also rep50 or TGI ranged over four orders of magnitude which meant that molecular features associated with response could be identified with confidence. The correlation of transcription profiles of the cell lines with their GI50 sensitivity identified 250 genes whose expression levels were associated with response to PG-11047. Network and pathway analyses of these genes are summarized in Figure The concentration of drug needed to inhibit growth measured as GIet al. [P = 0.08). The stratification improved if we restricted the analysis to high confidence predictions of the model (89/118 samples): P = 0.01. This analysis supports the utility of PG-11047 in the treatment of basal-subtype tumours.A further analysis of the 250 genes associated with response to PG-11047 identified 13 genes whose levels were strongly and independently associated with response  to PG-11047 and molecular features of breast cell linesGrowth inhibition response .e et al.  and SpelClick here for fileStatistically significant mRNA markers of response to PG-11047 (top 250 genes). Markers generated by correlation of growth inhibition (GI50) sensitivity with expression data of the cell lines reported by Neve et al[eve et al.Click here for fileStatistically significant genomic markers (BAC clones) of response to PG-11047. Markers generated by correlation of growth inhibition (GI50) sensitivity with array CGH data of the cell lines reported by Neve et al. [e et al. . Bold teClick here for fileStatistically significant protein markers of response to PG-11047. Markers generated by correlation of growth inhibition (GI50) sensitivity with Western profile data of the cell lines reported by Neve et al. [e et al. .Click here for file


However, the biological response of immune escape mechanisms might depend on malignant transformation of cancer cells. In this study we investigated different mechanisms of immune escape in 2 well-differentiated low-grade (RT4 and RT112) and 2 poorly differentiated high-grade (T24 and TCCSUP) bladder cancer cell lines. Fas, the receptor of Fas-ligand, is expressed and shedded by human transitional bladder carcinoma cell lines RT4, RT112, T24 and TCCSUP. Cytotoxicity and apoptosis assays demonstrate that in spite of the Fas expression, poorly differentiated T24 and TCCSUP cells are insensitive towards either recombinant Fas-ligand or agonistic apoptosis-inducing monoclonal antibody against Fas. In poorly differentiated T24 and TCCSUP cell lines we were able to detect marked Fas-ligand protein by flow cytometry and Western blot analysis. In grade 1 RT4 and RT112 cells only minor expression of Fas-ligand possibly because of proteinase action. Fas-ligand mRNA translation or post-translational processing seems to be regulated differentially in the cancer cell lines depending on malignant transformation. In co-culture experiments we show that poorly differentiated cells can induce apoptosis and cell death in Jurkat cells and activated peripheral blood mononuclear cells. This in vitro study suggests that bladder cancer cells can take advantage of different mechanisms of immune evasion and become more competent in avoiding immune surveillance during transformation to higher-grade malignant disease. © 2001 Cancer Research Campaign


Two P388 cell lines with acquired resistance to daunorubicin have been shown to exhibit cross-resistance to anthrapyrazolone (NSC 357885). The degree of cross-resistance observed in these cell lines (58 and 150 fold) is similar to those observed towards daunorubicin (34 and 142 fold). However the decreased drug accumulation observed for daunorubicin in the resistant cell lines (4-6 fold) is not observed for anthrapyrazolone. Similarly, verapamil can increase daunorubicin accumulation in resistant cells but has no effect on anthrapyrazolone accumulation. It is concluded that in contrast to daunorubicin, decreased anthrapyrazolone accumulation is not the resistance mechanism operative in daunorubicin resistant cell lines towards anthrapyrazolone.


In contrast, PNT2-C2 and PC-3 cells were found to be the least and the most invasive and motile epithelia respectively. Stromal cultures enhanced the invasion of both epithelial cells, but no differences were observed between the use of malignant and non-malignant tissues. All stromal cultures modestly stimulated PNT2-C2 motility but displayed a greater stimulation of PC-3 cell motility, while stromal cells derived from malignant tissue stimulated PNT2-C2 and PC-3 cell motility more than stromal cultures from non-malignant tissues. © 2000 Cancer Research CampaignTo study the effects of stromal epithelial cell interactions on prostate cancer metastasis, we have used primary human prostatic stromal cells derived from malignant and non-malignant tissues and established epithelial cell lines from normal (PNT1a and PNT2-C2) and tumour  origins. The effects of stromal cells on epithelial cell growth were studied in direct and indirect (using culture inserts) co-culture and by exposure to stromal cell-conditioned medium (assessed by MTT assay). The influence of stromal cells on epithelial cell invasion was measured using matrigel invasion chambers and on epithelial cell motility using time lapse microscopy. Results indicated that epithelial cell line growth was similarly unaffected or inhibited by stromal cells derived from malignant (


A new human embryonal rhabdomyosarcoma cell line, designated JR-1, is described that closely resembles the tumour from which it was derived. Comparative studies, by light and electron microscopy reveal morphological features such as myofibre formation, that are concordant with embryonal rhabdomyosarcoma. Immunohistological investigations using a panel of monoclonal antibodies indicate that the cell surface antigen profile of the JR-1 cell line is similar to other embryonal rhabdomyosarcomas. In addition the cell line expresses the cytoplasmic intermediate filament protein desmin, only found in cells of rhabdoid origin. Karyotyping JR-1 shows the cells to contain variable numbers of chromosomes (range 44-100). DNA flow cytometry indicates that cells have an DNA content which is approximately twice normal. The JR-1 cell line has a doubling time of 29 h in culture and, in common with several other human cell lines, produces xenografts in nude mice within 6 weeks of inoculation. With detailed studies on the original tumour and the JR-1 cell line, the latter should prove an excellent model system for investigating the biology of rhabdomyosarcoma.


Paclitaxel (Taxol), an anti-cancer drug derived from Taxus species, was tested for its anti-migrational, anti-invasive and anti-proliferative effect on two human glioma cell lines (GaMg and D-54Mg) grown as multicellular tumour spheroids. In addition, the direct effect of paclitaxel on glioma cells was studied using flow cytometry and scanning confocal microscopy. Both cell lines showed a dose-dependent growth and migratory response to paclitaxel. The GaMg cells were found to be 5-10 times more sensitive to paclitaxel than D-54Mg cells. Paclitaxel also proved to be remarkably effective in preventing invasion in a co-culture system in which tumour spheroids were confronted with fetal rat brain cell aggregates. Control experiments with Cremophor EL  in this study showed no effect on tumour cell migration, cell proliferation or cell invasion. Scanning confocal microscopy of both cell lines showed an extensive random organization of the microtubules in the cytoplasm. After paclitaxel exposure, the GaMg and the D-54Mg cells exhibited a fragmentation of the nuclear material, indicating a possible induction of apoptosis. In line with this, flow cytometric DNA histograms showed an accumulation of cells in the G2/M phase of the cell cycle after 24 h of paclitaxel exposure. After 48 h, a deterioration of the DNA histograms was observed indicating nuclear fragmentation.