BRAF mutations are present in approximately 50% of melanomas. Although different downstream target genes of the most common mutant V600E have been identified, the contribution of activating BRAF mutations to malignant transformation needs further clarification.Activating V600E mutations in comparison to cell lines without this mutation.Microarray gene analysis was performed for human melanoma cell lines harboring BRAFBAALC  was most prominently regulated, since it was up-regulated in mutated cell lines by a mean of 11.45. Real time PCR analyses with RNA from melanoma cell lines (n = 30) confirmed the BRAF-activation dependent up-regulation of BAALC.This analysis revealed a more than two fold down-regulation of 43 and an increase of 39 gene products. BAALC, which has been associated with cell dedifferentiation and migration, may function as a downstream effector of activating BRAF mutations during melanomagenesis. BRAF have been observed in approximately fifty percent of malignant melanomas [V600E mutation leads to a continuous stimulation of the MAP kinase cascade which results in a variety of cellular changes such as proliferation and dedifferentiation [BRAF mutations with respect to course and stage of melanoma is still not defined. On the one hand, comparable incidence of activating BRAF mutations in invasive cutaneous melanomas and benign melanocytic nevi indicate that BRAF mutations alone are insufficient to cause malignant transformation [BRAF mutations in radial growth phase melanomas, i.e. the early phase of melanoma progression, suggests a correlation with progression rather than initiation [BRAF mutations is associated with a significantly poorer prognosis [V600E in melanocytes rendered them tumorigenic in nude mice [V600E expression in human melanocytes induced cell senescence leading to cell cycle arrest. Hence, additional mutations have to occur to overcome this cell cycle arrest and make the cells tumorigenic [HRAS, MYC and the E2F family [BRAF gene when compared to wild type cell lines [V600E dependent expression of a newly identified potential effector gene, BAALC  was confirmed by real time PCR analyses in complimentary experiments in melanoma cell lines.Activating mutations of the protooncogene elanomas . The V60elanomas ,3. Thus,ntiation . Howeverormation ,3. In aditiation . On the ude mice , while aorigenic . VariousF family . Similarll lines -12. In tV600E mutations as well as 9 melanoma cell lines with activating RAS mutations and 5 human malignant melanoma cell lines without mutations in these genomic sections were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum  were examined by cDNA microarray analyses as described elsewhere  and contained the GF200 set of Research Genetics cDNAs. Fluorescence labeled cDNA was spotted in duplicates. A flip color experiment was included. Samples were hybridized to microarrays for 16 h at 55°C. Chips were scanned with a GMS 418 fluorescent scanner (MWG-Biotech), and the images were analyzed with IMAGENE 3.0 software.Three melanoma cell lines  with BRAFlsewhere . In brieT method. Total RNA was isolated from approximately 3 × 106 cells of human melanoma cell lines or from 25 six μm thick kryosections of melanoma tissue samples. Samples of total RNA were subjected to reverse transcription. Primers for BAALC were designed with Primer Express software  and read as following: sense 5'-AGC-CGC-CGC-CAG-AGC-CGA-CAG-3'; antisense 5'-GG-GAT-CCA-GTG-CCG-TGA-AGG-3'. For the evaluation of BAALC expression the thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min, then 43 cycles of three-step PCR including 94°C for 30 sec, 60°C for 30 sec and 72°C for 40 sec. GAPDH (Applied Biosystems) served as endogenous control. The relative expression level of BAALC, normalized to GAPDH and relative to the randomly selected human melanoma cell line FM79 (BRAF wt/wt) was calculated as T =  - . CT is defined as the cycle when the threshold level of fluorescence is reached.Relative expression of BAALC was determined by real time PCR analyses in Sybr green technology using the comparative ΔΔCIn microarray analyses, the threshold value for up or down regulation of gene expression was defined as a more than two fold change compared to the mean value. Data of real time PCR studies were expressed as box-and-whiskers plot. Differences between the values were evaluated by Mann-Whitney test. p < 0.05 was considered to be significant.RAS and BRAF result in a constant activation of the MAP kinase pathway and eventually contribute to proliferation and dedifferentiation of cancer cells [V600E mutated cell lines a more than two fold decrease of 43 and increase of 39 gene products was detected , since (i) it was highest up-regulated in all three cell lines harboring BRAF mutations by an average of 11.25 fold and (ii) it was not yet described by other groups analyzing the effect of BRAF mutation on gene expression in melanoma [Activating mutations of er cells ,14. Althed table . Due to melanoma -12,15. Tmelanoma ,17. ThesBAALC gene has been previously discussed to be involved in cell dedifferentiation and motility. In differentiated cells BAALC is almost exclusively expressed in the central nervous system and in other neuroectodermal derived tissues. Moreover, BAALC expression has been described for CD34 positive hematopoietic progenitor cells from the bone marrow [BAALC during differentiation of hematopoietic progenitor cells suggests that it might function in sustaining an undifferentiated state of these cells [BAALC expression in both normal and malignant astrocytes is increased upon differentiation, suggesting complementary functions in different cell types [BAALC may belong to this group of genes. Recent studies performed in patients with leukaemic malignancies indeed suggest a role of BAALC in tumorigenesis. For example, blast cells from 28% of patients with acute myeloid leukaemia and 65% with acute lymphatic leukaemia displayed an overexpression of BAALC. In addition, high BAALC expression was identified as an independent risk factor in acute myeloid leukaemia [The product of the e marrow . Notablyse cells ,20. Intell types . Neverthll types  and BAALeukaemia . Furthereukaemia .BAALC expression in all three BRAFV600E mutated melanoma cell lines suggests a possible function of BAALC associated with activated BRAF mutations in melanoma and mediating cell dedifferentiation and motility. As mentioned before, complementary experiments using real time PCR analyses to measure the relative mRNA expression of BAALC have to be performed to confirm this correlation. To this end, BAALC expression was determined in 16 cell lines mutated for BRAF, 5 wild type BRAF melanoma cell lines as well as 9 melanoma cell lines in which the MAP kinase pathway was activated by RAS mutations. These analyses validated the first observations; BRAFV600E cell lines showed significantly (p < 0.01) elevated mRNA levels of BAALC (Fig. BAALC was also significant when compared to RAS mutated cell lines (Fig. The distinct increase of ALC Fig.  when comnes Fig. . ImportaIn summary, BAALC may function as an additional mediator of activating BRAF mutations. Future studies will have to clarify its exact role in malignant transformation of melanocytic lesions.


Nitric oxide induced apoptosis in a dose-dependent manner which preceded frank cell death (failure to exclude Trypan blue). These data suggest that epithelial cell death may be NO dependent and via apoptosis, in states of gut inflammation.Chronic inflammation is associated with inducible nitric oxide synthase expression in infiltrating and resident cells  and an exaggerated release of nitric oxide. NO can induce apoptosis in macrophages and tumour cell lines. We investigated whether NO induced cell death in an epithelial (T84) cell fine via apoptosis. Culture T84 cells were exposed to a bolus of NO (40 or 80 μM) dissolved in Hank's balanced salt solution (HBSS) supplemented with 10% fetal calf serum (FCS). After incubation for 4 h at 37


In contrast to reports of extensive hypoxia in human gliomas in situ measured by pO2 histography, non-invasive methods of assessing glioma oxygenation, including nitroimidazole binding, have yielded surprisingly contradictory results. In order to investigate the relationship of necrosis, hypoxia, nitroreductase activity and cellular respiration in human gliomas, subcutaneous models using the human glioma cell lines M059K, M006 and M010b were developed in the murine SCID host. Intracranial growth of the M006 line was achieved in nude rats. The nitroreductive capacity of glioma cell lines was assessed and found to be similar to transplanted tumours previously reported in the literature. This suggests that if substantial numbers of viable hypoxic cells were present in situ in gliomas, then nitroimidazole-binding techniques should be capable of identifying them. Inter-tumour variability in the amount of necrosis was seen. M006 xenografts growing in subcutaneous and intracranial sites revealed extensive necrotic regions histologically, some of which were surrounded by cells labelled heavily for [3H]misonidazole, while other areas were lightly labelled. Similar binding patterns were seen for subcutaneous M059K tumours, while subcutaneous M010b tumours display necroses of which almost all were surrounded by heavily labelled cells. The oxygen consumption rates of tumour cell lines grown in vivo, in which venous pO2 concentrations are of the order of 2-5%, were two to sevenfold less than those of the same lines grown as monolayers in vitro under oxygen concentrations of 18%. We postulate that glioma cell lines behave as 'oxygen conformers', in that their rate of oxygen consumption appears to vary with the availability of oxygen. Together with the misonidazole-binding data, the results in this glioma tumour model are consistent with coordinate inhibition or down-regulation of respiration under moderate hypoxia.


High constitutive NF-κB DNA-binding activity was detected in all chemoresistant cell lines. The upregulated NF-κB activity was composed of NF-κB subunits p50 and p65. Four out of five resistant cell lines constitutively overexpressed NF-κB p50 and p65 mRNA and protein. One resistant cell line with the highest NF-κB DNA-binding activity showed normal p50 and p65 protein expression. No NF-κB gene amplification was detected in resistant cell lines. Transient exposure of wild-type RKOWT and H630WT cells to 5-FU induced NF-κB DNA-binding activity but had no effect on NF-κB protein expression in these cells. Our results indicate that high constitutive NF-κB activity caused by its gene overexpression is an intrinsic character of TS inhibitor-resistant cells. NF-κB can antagonise anticancer drug-induced apoptosis. High NF-κB expression and nuclear activity in TS inhibitor-resistant cancer cells may play an important role in the chemoresistance.In this study, the gene copy number, mRNA and protein expression levels and nuclear DNA-binding activity of nuclear factor kappa B (NF- Since this reaction is catalysed by thymidylate synthase  using folate cosubstrate 5, 10-methylene tetrahydrofolate and is the only source of de novo cellular thymidylate, it makes TS an attractive molecular target for cytotoxic drugs  is a transcription factor that is composed of five subunits , RelB and c-Rel). NF-κB can bind to DNA target sites (κB sites) and influence downstream gene expression (κB (IκB) is a natural inhibitor of NF-κB. In most normal cells, IκB binds to NF-κB and retains NF-κB in the cytoplasm as an inactive complex. Many chemical and biological stimuli can induce IκB phosphorylation, ubiquitinisation and subsequent degradation. NF-κB will be released from the NF-κB–IκB complex and translocated into the nucleus. The liberated NF-κB then binds to the promoter region of the relevant downstream genes and triggers a series of transcriptional events  , two 5-FU-resistant colorectal cancer cell lines (H6305-FU and R105-FU) and the relevant parent cell lines were chosen for this study . The characteristics and culturing conditions have been described previously  was incubated with 1 μg poly(dIdC)  in binding buffer (50 mM Tris (pH 7.6), 250 mM KCl, 25 mM DTT, 5 mM EDTA and 25% glycerol) for 10 min at room temperature (RT). Approximately 20 000 c.p.m. of 32P-labelled 22-mer double-stranded NF-κB DNA probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was added and incubated at RT for 20 min. Oct-1 (5′-TGTCGAATGCAAATCACTAGAA-3′) was used as loading control for EMSA. The EMSA conditions of Oct-1 were the same as those for NF-κB. For supershift assay and binding specificity determination, 5 μg of nuclear extract mixture from different resistant cell lines was incubated with 0.4 μg 2 μl−1 of NF-κB p65 or p50 antibody  or 20×wild-type or mutant (5′-AGTTGATATTACTTTTATAGGC-3′) unlabelled NF-κB probe for 30 min before EMSA analysis. The complexes were separated on a 6% polyacrylamide gel and exposed for autoradiography.The nuclear and cytoplasmic protein extraction and EMSA were carried out as previously described (μg lane−1) was electrophoresed through a 10% SDS–PAGE and transferred to a PVDF membrane . The blots were stained with rabbit polyclonal primary  and HRP-conjugated donkey anti-rabbit secondary  antibodies, respectively, for 1 h at RT. The loading quantity of protein was verified by staining the same membranes with monoclonal anti-α-tubulin or antivinculin antibody  and HRP-conjugated sheep anti-mouse secondary antibody . The signal was detected using an ECL Western blotting detection kit . The intensity of the bands was scanned and analysed using Molecular Analyst software . This programme was also used to analyse the band intensity of Northern and Southern blots.The cytoplasmic protein (40 6) were digested in denaturing buffer and protein was extracted twice with phenol (pH 4.3) : chloroform (1 : 0.3). Total RNA in the aqueous phase was precipitated with an equal volume of isopropanol and then washed with 70% ethanol. Total RNA (20 μg) from drug-sensitive and resistant cells was electrophoresed through a 1% agarose gel containing 1.9% formaldehyde. After transfer to a Hybond-N+ nylon membrane (Amersham), the blotted RNA was immobilised by exposing under 250 nm UV light for 3 min. Membranes were hybridised with [32P] dATP random labelled probes at 68°C overnight. The signals were detected by exposing the membranes to a FUJI Phosphor Plate overnight and scanned using a FUJI FILM-3000 Phosphor-Imager. Blots were stripped and rehybridised with different probes. NF-κB p50 and p65 subunits were subjected to Northern blotting analysis and GAPDH gene was used as an internal control. The probes were reverse transcribed and amplified from 1 μg RKOTDX total RNA using a one-step RT–PCR kit . The sequences of primers are as follows: NF-κB p50 (601 bp): forward 5′-CTGGTGATCGTGGAACAGCC-3′; reverse 5′-CAGAGCCTGCTGTCTTGTCC-3′; NF-κB p65 (659 bp): forward 5′-atgcgcttccgctacaagtg-3′; reverse 5′-acaatggccacttgtcggtg-3′; GAPDH (915 bp): forward 5′-AAGGTCGGAGTCAACGGATTTG-3′; reverse 5′-CTTGACAAAGTGGTCGTTGAGG-3′. The sequence of each amplified probe was confirmed by direct automated DNA sequencing.Total RNA was extracted using the previously described method (μg of DNA from each cell line was digested with MvaII (p50) or SacI (p65) for 3 h at 37°C. The digested DNA fragments were separated through a 0.6% agarose gel. After de-naturing in 0.1 N NaOH, the DNA was blotted onto a nylon mem-brane by overnight capillary transfer and fixed under 250 nm UV light for 3 min. The probes, hybridisation and image analysis con-ditions were the same as those for Northern blotting analysis. The same membrane was stripped and hybridised to different probes.The DNA was extracted using Nucleon DNA purification kit  following the supplier's instruction. A total of 20 κB nuclear activity was tested in five pairs of TS inhibitors (5-FU and TDX)-sensitive and -resistant cell lines. All resistant cell lines were cultured in drug-free medium for 10 days before EMSA analysis. There was substantial NF-κB activity in the chemosensitive parent cell lines, but the TS inhibitor-resistant cell lines demonstrated much higher nuclear NF-κB activity . Anti-VEGF antibody was used as a negative control for the supershift assay. To test if the high NF-κB nuclear activity in drug-resistant cells was caused by IκBα degradation, IκBα protein levels in drug-sensitive and -resistant cells were assessed by Western blotting analysis. It seems that IκBα degradation was not responsible for the high constitutive NF-κB nuclear activity in resistant cells because high and comparable IκBα protein levels were detected in both resistant and relevant parent cell lines . In comparison with relevant parent cell lines, drug-resistant cells demonstrated higher NF-κB DNA-binding activity. Thus, the high NF-κB activity in drug-resistant cells is a constitutive and intrinsic feature of the resistant cell lines. NF-κB is a transcription factor that antagonises apoptosis induced by some anticancer drugs (κB activity show a high expression of antiapoptotic gene products such as c-IAPs, IXAP, A1/Bfl-1 and IEX-IL (κBactivity in TS-inhibitor resistant cell lines may at least partially lead to their chemoresistance. κB activity could be induced by transient exposure of colorectal cancer cell lines to 5-FU. In combination with 5-FU, disulfiram, an NF-κB inhibitor, strongly enhances the cytotoxicity of 5-FU and totally reverses 5-FU resistance in two 5-FU-resistant colorectal cancer cell lines in vitro (κB may be a new measure to overcome TS inhibitor resistance.5-FU and TDX have been successfully used in human solid cancer chemotherapy. Both drugs inhibit TS, the enzyme converting 2′-deoxyuridine to thymidylate by reductive methylation. 5-FU can also induce metabolic disruption and cell death by incorporating into DNA and RNA. After chemotherapy, some cancer cells show acquired chemoresistance to TS inhibitors. The drug-resistant cells usually demonstrate TS protein and mRNA overexpression and TS gene amplification (ll lines . The haler drugs . Cancer κB p50 and p65 protein and mRNA was detected in four out of five drug-resistant cell lines. The overexpressed NF-κB protein levels in these cell lines were in parallel with those of enhanced NF-κB nuclear binding activity. Thus, the constitutive gene overexpression may be the main cause of the high NF-κB nuclear activity in these cell lines. Although NF-κB DNA-binding activity in drug-sensitive cancer cell lines could be induced by transient exposure to 5-FU, no alteration of NF-κB protein levels was observed in these cell lines (κB nuclear activity supported by gene overexpression is the intrinsic character of most TS inhibitor-resistant cancer cells. NF-κB deregulation in drug-resistant cell lines is mainly at the transcriptional, translational, nuclear transporting and DNA-binding levels because no gene amplification was detected by Southern blotting. We have tested mRNA expression patterns in the same panel of cell lines using Atlas nylon membrane cDNA array system before and could not identify the mRNA overexpression of NF-κB p65 that was spotted on the array membrane (High transcription factor activity in cancer cells is frequently induced by gene amplification and/or overexpression . In thisll lines . These rκB DNA-binding activity and protein expression patterns in H630TDX cells are intriguing. NF-κB nuclear DNA-binding activity in H630TDX cells was highly upregulated. The NF-κB proteins p50 and p65 in this cell line were similar to those in the parent cells. Apart from gene overexpression, high NF-κB nuclear activity can be caused by enhanced nuclear translocation and DNA-binding affinity (κB protein phosphorylation also plays an important role in the regulation of NF-κB DNA-binding and transcriptional activity (κB activity in TS inhibitor-resistant cell lines may be caused by different molecular mechanisms.Compared to other cell lines, the NF-affinity . It has activity . Our resκB degradation is thought to be a main reason for induction and maintenance of high nuclear NF-κB activity (κBα is the predominant member of the IκB family. IκBα degradation was involved in 5-FU-induced NF-κB activation in 5-FU-sensitive cell lines (κB activity in TS inhibitor-resistant cells was not conferred by degradation of IκBα. High levels of IκBα were detected in all drug-resistant cancer cell lines that demonstrated high nuclear NF-κB activity (κB activity in two chemoresistant lymphoma cell lines was caused by aberrant activation of IKK's and overexpression of defective IκBα (κBα in NF-κB signalling pathways in TS inhibitor-resistant cell lines remains to be elucidated.Iactivity . IκBα isactivity . It was


The effects of cell density on drug-induced cell kill kinetics were studied by means of clonogenic assay using 3 human leukaemia-lymphoma cell lines. Mitoxantrone, daunorubicin, doxorubicin, vincristine and bleomycin were progressively less efficacious when cell density increased (positive inoculum effects), whereas the effects of cis-platin and carboplatin were not influenced by cell density. Inoculum effects were related to the kind of chemotherapeutic agents tested, irrespective of the type of cell lines used. Preincubation of mitoxantrone or doxorubicin in the presence of cells in high density resulted in decreases in the cytocidal activity, whereas the effects of bleomycin, vincristine and cis-platin were unaffected. These results show that cell density affects the biological effect of certain chemotherapeutic agents. Inactivation of drugs by high densities of cells partially explains this phenomenon.


Penaeus is a potential reservoir of this virus.Taura syndrome virus (TSV) affects shrimp cultured for human consumption. Although TSV is related to the Cricket Paralysis virus, it belongs to the “picornavirus superfamily,” the most common cause of viral illnesses. Here we demonstrate that TSV also infects human cell lines, which may suggest that  Cripavirus genus, family Dicistroviridae of the “picornavirus superfamily” (The Taura syndrome virus (TSV) causes a disease affecting penaeid shrimp, the most important commercial family of crustaceans (Penaeus stylirostris) affected with TSV. The animals were collected from farms located in northwestern Mexico. Control cell lines were injected with filtered hepatopancreas extracts from healthy shrimp. Cultures were incubated at 37°C and periodically observed under a microscope until any sign of cytopathic effect was detected . Cells were then harvested and lysed. Fresh cell lines were inoculated with the lysate, incubated, and processed in a similar way. A third inoculation was again performed with the second lysate (Since Sabin strain LSc 2ab (Sabin 1), the poliovirus used for human vaccination, is usually replicated in monolayer culture cells of human rhabdomyosarcoma (RD), human larynx carcinoma (Hep-2C) , or Buffalo green monkey kidney (BGM) . These signs were clinically indistinguishable from those occurring in naturally infected animals and are considered as pathognomonic of the acute phase of infection by TSV (To confirm the presence of TSV in the cell culture media, a bioassay was performed by using media from the third passage. For this assay, healthy n by TSV . Shrimp –Penaeus could be considered as a reservoir of a virus that could become a potential pathogen to humans and other mammals (If one takes into consideration the capacity of viruses to modify receptor recognition and host cell tropism and the fact that cell receptors for many of the picornavirus superfamily members seem to be ubiquitous membrane molecules (e.g., decay-accelerating factor, different type of integrins, low-density lipoprotein receptor, sialic acid [


Analogues substituted in the 3′ position with I (DF 129), CH 3(DF 203), or CI (DF 229) possess an extended profile of antitumour activity with remarkable selective activity in cell lines derived from solid tumours associated with poor prognosis, e.g. breast, ovarian, renal and colon. Growth inhibition occurs via unknown, possibly novel mechanism(s) of action. Two cell lines have been derived from sensitive MCF-7wtbreast cancer cells  following long-term exposure to 10 nM or 10 μM CJM 126, MCF-710 μM 126 and MCF-710 μM 126respectively, which demonstrate acquired resistance to this agent (IC 50> 30 μM) and cross-resistance to DF 129, DF 203 and DF 229. Sensitivity to tamoxifen, benzo[a]pyrene (BP), mitomyin C, doxorubicin and actinomycin D is retained. Resistance may, in part, be conferred by the constitutively increased expression of bcl-2 and p53 proteins detected in MCF-710 nM 126and MCF-710 μM 126lysates. Significantly decreased depletion of CJM 126 (30 μM) from nutrient medium of MCF-710 nM 126cells was observed with predominantly cytoplasmic drug localization and negligible DNA strand breaks. N -acetyl transferase (NAT)1 and NAT2 proteins were expressed by all three MCF-7 sub-lines, but significantly higher expression of NAT2 was accompanied by enhanced acetylation efficacy in MCF-710 nM 126cells. In contrast, CJM 126 (30 μM) was rapidly depleted from nutrient medium of MCF-710 μM 126culture and accessed nuclei of these cells exerting damage to DNA. The major biotransformation product of CJM 126 in MCF-710 μM 126cells was 2-(4-aminophenyl)-6-hydroxybenzothiazole (6-OH 126). This metabolite possessed no antitumour activity. Accordingly, in this sub-line, low constitutive expression and activity of cytochrome P450 (CYP) 1A1 was detected. © 2000 Cancer Research Campaign2-(4-aminophenyl)benzothiazole (CJM 126) elicits potent growth inhibition in human-derived breast carcinoma cell lines, including oestrogen receptor-positive (ER+) MCF-7


Cyclooxygenase-2 (COX-2) expression has previously been identified in uveal melanoma although the biological role of COX-2 in this intraocular malignancy has not been elucidated. This study aimed to investigate the effect of a COX-2 inhibitor on the proliferation rate of human uveal melanoma cells, as well as its effect on the cytotoxic response of macrophages.Human uveal melanoma cell lines were transfected to constitutively express COX-2 and the proliferative rate of these cells using two different methods, with and without the addition of Amfenac, was measured. Nitric oxide production by macrophages was measured after exposure to melanoma-conditioned medium from both groups of cells as well as with and without Amfenac, the active metabolite of Nepafenac.Cells transfected to express COX-2 had a higher proliferation rate than those that did not. The addition of Amfenac significantly decreased the proliferation rate of all cell lines. Nitric oxide production by macrophages was inhibited by the addition of melanoma conditioned medium, the addition of Amfenac partially overcame this inhibition.Amfenac affected both COX-2 transfected and non-transfected uveal melanoma cells in terms of their proliferation rates as well as their suppressive effects on macrophage cytotoxic activity. Uveal melanoma is the most common primary intraocular tumor in adults. The ability of ophthalmologists to diagnose the primary tumor has increased from an average accuracy of 87.5 percent in 1980 . This inSeveral prognostic factors of uveal melanoma, such as cell type, have been utilized for decades. More recently identified prognostic factors include tumor associated macrophages (TAM) which have been shown to be a predictor of poor prognosis in uveal melanoma . The actCOX-2 is not only a prognostic factor but also a potential therapeutic target in uveal melanoma. There are three isoforms of the COX enzyme. COX-1 is expressed constitutively in normal tissues . COX-2 iThe aim of this study was to investigate the possible role of COX-2 expression and inhibition by a COX-2 inhibitor (Nepafenac) on the proliferation rates of human uveal melanoma cell lines. In addition, we wished to investigate the effect of soluble factors secreted by human uveal melanoma cell lines on the cytotoxic activity of macrophages. Amfenac, a COX-2 inhibitor formulated for topical administration to the eye ,20, was 2-enriched atmosphere [2 flasks  and observed twice weekly, at every media change, for normal growth by phase contrast microscopy. The cultures were grown to confluence and passaged by treatment with 0.05% trypsin in EDTA (Fisher) at 37°C and washed in 7 ml RPMI-1640 media before being centrifuged at 120 g for 10 minutes to form a pellet. Cells were then suspended in 1 ml of medium and counted using the Trypan Blue dye exclusion test for use in all subsequent assays.Four previously characterized human uveal melanoma cell lines  and one human transformed uveal melanocyte cell line (UW-1) were incubated at 37°C in a humidified 5% COmosphere . The celThe uveal melanoma cell lines 92.1, SP6.5, and MKT-BR were established by Dr. Jager , Dr. Pelletier  and Dr. Belkhou , respectively. Dr. Albert  established the OCM-1 and UW-1 cell lines ,23.® as per the manufacturer's recommendations (Invitrogen). Briefly, 1 × 105 cells were seeded in a 10 cm petri dish. Cells were then allowed to incubate overnight. The pcDNA3 plasmid (Invitrogen) encodes for resistance to Geneticin. This plasmid, with COX-2 cDNA inserted at the XBA I cleavage site, was used at a concentration of 2 ug per 10 ul of LipofectAMINE in 100 ul of serum-free OptiMem medium (Invitrogen). The cells were incubated for four hours with this solution. After removal of the solution, cells were grown in 5% FBS supplemented RPMI-1640 medium with 400 ug/ml of Geneticin . Cells were also transfected with the empty pcDNA3 plasmid and compared to the original non-transfected cell lines as controls.The four human uveal melanoma cell lines and one transformed melanocyte cell line were transfected to constitutively express COX-2 using lipofectAMINECOX-2 expression was verified by Western blot and immunohistochemistry performed on cytospins of the five cell lines.Western blot analysis was done as previously described . BrieflyThe cytospin slides were removed from -20°C and left at room temperature overnight before being fixed with 2% paraformaldehyde for 30 min. The paraformaldehyde-fixed samples were then tested for COX-2 expression by immunocytochemical analysis using a monoclonal mouse anti-COX-2 antibody  at a dilution of 1:50. Immunohistochemistry was performed using the Ventana BenchMark fully automated staining machine with a sample of cutaneous melanoma as a positive control.3 cells per well, with a minimum of six wells per cell line. A row of 8 wells containing only RPMI-1640 medium was used as a control. Cells were allowed to adhere overnight. The active metabolite of Nepafenac, amfenac, was added to each well at its IC50 of 150 nM [The Sulforhodamine-B based assay  was performed according to the National Cancer Institute protocol . Each off 150 nM . Cells w6 cells/ml. The cells were labelled with propidium iodide and a measurement of the cellular DNA was then performed using an Epics XL flow cytometer . The percent of cells in the S phase fraction (SPF) were then recorded for each of the three replicates and averaged together.For flow cytometry cell cycle analysis, cells were passaged and counted as described previously . Cell pe6 cells/ml in 5% FBS supplemented RPMI-1640 medium. Eighteen hours after seeding the melanoma cells, medium was removed from each well and centrifuged at 120 g for 10 minutes to remove cells. The supernatant was then used as melanoma conditioned medium (MCM). 28SC was then exposed to MCM from the transfected and non-transfected cell lines with and without amfenac at the recommended IC50 of 150 nM. 28SC incubated in fresh medium was used as control. Twenty-four hours after media transfer, 28SC were stimulated with 100 U/ml of Interferon gamma (IFNγ) and 100 ng/mL of lipopolysacharide (LPS). The experiment was done in triplicate. After 24 hours of stimulation supernatants were removed and assayed for nitrite levels using the Greiss reaction. One hundred μl of medium was reacted with an equal mixture of 1% sulphanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid. The mixture was incubated at room temperature for 10 minutes and then the absorbance was read at 550 nm using a BioTek EL 800 microplate reader (Fisher). Readings were compared to a standard curve to calculate nitrite levels.The four human uveal melanoma cell lines  and the transformed melanocyte cell line (UW-1) with and without COX-2 transfection, as well as one monocyte cell line (28SC), were seeded in 6 well plates at a concentration of 1 × 10The Student's t-test was used to compare proliferation results between transfected and non-transfected cell lines, as well as the same cell lines exposed to amfenac. It was also used to compare results of 28SC incubated with conditioned medium from COX-2 transfected versus non-COX-2 transfected melanoma cell lines as well as 28SC incubated with conditioned medium from these cell lines with and without amfenac. Flow cytometry results were compared using a one-way ANOVA test with LSD post hoc analysisNeither the five human uveal melanoma cell lines nor the macrophage cell line constitutively expressed detectable levels of COX-2 as measured by western blot analysis Figure  or immunWith the Sulforhodamine-B assay, an increase (p < 0.05) in proliferation was seen in four of the cell lines expressing COX-2 , while a decrease in proliferation was seen in one cell line (OCM-1) as compared to their non-transfected counterparts (p < 0.05). The addition of amfenac inhibited the proliferation of all cell lines (p < 0.05), with an inhibition that ranged between 20–22% for all cell lines including OCM-1. Figure The percentage of cells in the S phase fraction (SPF%) is shown in Table As compared to control medium, MCM from the four uveal melanoma cell lines significantly reduced macrophage production of NO (p < 0.001). The addition of conditioned medium from UW-1, the transformed melanocytic cell line, yielded no significant decrease in NO production by macrophages. Different levels of macrophage NO production were seen with the MCM from different uveal melanoma cell lines. As a result the different MCMs could be ranked according to their differential effects on macrophage NO production (most macrophage NO production UW-1 > OCM-1 > MKT-BR > 92.1 > SP6.5 least macrophage NO production).As compared to control medium, conditioned medium from the uveal melanoma cell lines transfected to express COX-2 also significantly decreased macrophage NO production (P < 0.001). This decrease in macrophage NO production was significantly different than that caused by medium from the non-transfected cell lines. The addition of conditioned medium from the COX-2 transfected UW-1 cell line yielded a significant decrease in NO production (P < 0.001) as compared to control medium. The addition of conditioned medium from the COX-2 transfected UW-1 cell line also yielded a significant decrease in NO production (P < 0.0006) as compared to MCM from the non-transfected UW-1 cell line. The different MCMs from COX-2 transfected cell lines could be ranked according to their differential effects on macrophage NO production (most macrophage NO production OCM-1 > UW-1 > MKT-BR > 92.1 > SP6.5 least macrophage NO production).The addition of amfenac to the MCM of the four uveal melanoma cell lines resulted in increase in macrophage NO production as compared to MCM without amfenac from the same cell lines. This increase was seen with MCM from both transfected and non-transfected uveal melanoma cell lines, with the exception of the non-transfected OCM-1 cell line. The addition of amfenac to the MCM of both transfected and non-transfected UW-1 cells caused an increase in macrophage NO production as compared to MCM without amfenac from the same cell lines. Figures in vitro studies. We also sought to demonstrate the effects of a COX-2 inhibitor on the proliferation and macrophage cytotoxic activity in response to soluble factors secreted by the tumor cells.COX-2 expression has been identified in a wide variety of human malignant tumors -12, inclin vitro studies of prostate [2 [COX-2 has been shown to play a role in proliferation prostate  and breaprostate . The prestate [2 . FurtherThe present study also showed that the addition of amfenac, the active metabolite of Nepafenac which is a COX-2 inhibitor ,20, signIn vitro conditioned medium studies have shown cutaneous melanoma cells to inhibit macrophage tumoricidal activity [This study is also the first to investigate the effect of uveal melanoma soluble factors on macrophage cytotoxic potential. In uveal melanoma, the presence of tumor associated macrophages (TAM) has previously been shown to be an independent prognostic factor of poor prognosis , suggestactivity ,33 and tactivity . A majoractivity . For ouractivity .The addition of melanoma conditioned medium to macrophages significantly inhibited the production of NO by four of the five non-transfected human uveal melanoma cell lines. It is interesting to note that the sole cell line that did not significantly inhibit NO production was UW-1. The addition of conditioned medium from the COX-2 transfected cell lines did not give significantly different results for the four uveal melanoma cell lines. In comparison, however, the UW-1 transfected cell line did give a significant decrease in NO production compared to its non-transfected counterpart. This cell line was established from normal melanocytes whereas the other four cell lines were established from the primary uveal melanoma tumors of patients ,23. We sThe inhibitory effect of conditioned medium from the five cell lines used on macrophage NO production was ranked for both the transfected and non-transfected cells. In both cases the inhibition was greatest with the SP6.5 and 92.1 cell lines, while OCM-1 and UW-1 gave the least inhibition, with MKT-BR ranking in the middle. This ranking is the same as the proliferative and metastatic ranking of the cell lines that has previously been described , indicatWe have previously demonstrated that a "cross-talk" exists between uveal melanoma tumor cells and macrophages, causing an up-regulation of several soluble factors such as Vascular Endothelial Growth Factor, Interleukin-6, Hepatocyte Growth Factor, and Melanoma Inhibitory Activity -37. ThesWhen the COX-2 inhibitor amfenac was added to the MCM, the suppression of macrophage function was partially overcome in all instances. The addition of amfenac not only affected macrophages exposed to medium from COX-2 transfected cells, but also macrophages exposed to medium from non-transfected cells. This result again supports the hypothesis that amfenac may have a COX-2-independent mechanism of action.COX-2 inhibitors show promise for use as an adjuvant therapy in many tumor types. However, there has been recent discussion regarding the safety of systemic COX-2 inhibitors, most notably rofecoxib . In our In conclusion, COX-2 expressing uveal melanoma cell lines demonstrated an increase in proliferation over their non-COX-2 expressing counterparts. The anti-COX-2 molecule amfenac inhibited the proliferation rate of both COX-2 expressing and non-expressing uveal melanoma cell lines. In addition, amfenac partially overcame the suppression of macrophage function by conditioned medium from both COX-2 transfected and non-transfected uveal melanoma cell lines. Further trials should be undertaken to study the effect of COX-2 inhibitors as potential adjuncts to standard therapy for uveal melanoma and to investigate the potential COX-2 independent function of amfenac.In vitro work was done by JM, AC, SC. Statistical analysis was done by VS. All authors read and approved the final manuscript prepared by JM.


A novel sterically hindered platinum complex, AMD473 [cis-aminedichloro(2-methylpyridine) platinum (II)], has been selected for phase I clinical trials due to commence in 1997. AMD473 was rationally designed to react preferentially with nucleic acids over sulphur ligands such as glutathione. This report documents the in vitro circumvention of acquired cisplatin resistance mechanisms in human ovarian carcinoma (HOC) cell lines by AMD473. In a panel of 11 HOC cell lines, AMD473 showed intermediate growth inhibition potency (mean IC50 of 8.1 microM) in comparison to cisplatin (mean IC50 of 2.6 microM) and carboplatin (mean IC50 of 20.3 microM). AMD473 showed only a 30.7-fold increase in IC50 value from the most sensitive to the most resistant HOC cell line, whereas for cisplatin it was 117.9-fold and for carboplatin 119.7-fold. AMD473 also showed significantly (P < 0.05) reduced cross-resistance to cisplatin in a panel of three cell lines with known acquired platinum drug resistance mechanisms . Cellular accumulation of AMD473 was not reduced in two HOC cell lines (A2780cisR and 41McisR), in which reduced cisplatin accumulation is a major mechanism of acquired cisplatin resistance. AMD473 naked-DNA binding was significantly less affected (P < 0.05) than that of cisplatin by the presence of 5 mM glutathione. Also, AMD473 almost completely circumvented acquired cisplatin resistance in a cell line (A2780cisR) with fivefold elevated intracellular glutathione levels compared with the parent A2780 cell line when measured by clonogenic assay (RF 4.5 for AMD473 vs RF 18 for cisplatin). AMD473 also showed a lower increase in IC50 than cisplatin in an A2780 cell line model with artificially elevated glutathione levels. AMD473 DNA binding was slower than that of cisplatin on both naked and cellular DNA. AMD473 also formed DNA interstrand cross-links (ICLs) at a slower rate than cisplatin (peak ICL formation was at 5 h for cisplatin vs > or = 14 h for AMD473) after equitoxic doses were exposed to HOC cells for 2 h. AMD473 ICLs in the CH1 HOC cell line were slowly formed and showed no visible signs of being repaired 24 h after removal of drug. This was paralleled by a slower, longer lasting induction of p53 protein by equitoxic doses of AMD473 in HOC cell lines with wild-type p53. This new class of sterically hindered platinum compound, selected for clinical trial in 1997, may therefore elicit improved clinical response in intrinsically and acquired cisplatin-resistant tumours in the clinic.


We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines . Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1–100 nM) or AP2 (10–100 μM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1–1 nM) or AP2 (1–300 μM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours. © 2001 Cancer Research Campaign


A new human rhabdomyosarcoma cell line (HX170c) has been established from a paratesticular embryonal tumour in a 5-year-old male. The cells grew as an adherent monolayer with a doubling time of 32 h and showed pleomorphic features. Intermediate filament analysis revealed the line to be mesenchymal in origin (reactivity to vimentin and desmin antibodies). The line was tumorigenic in nude mice, possessed elevated levels of creatine phosphokinase (mainly of the MM isoenzyme form) and had a near diploid mean chromosome number of 50. In vitro cell cloning determinations gave colony forming efficiencies of 0.01% in soft agar and 24% in a monolayer anchorage-dependent assay. Radiosensitivity determinations using a monolayer clonogenic assay with feeder layer support showed the cells to be among the more radiosensitive human tumour cell types (surviving fraction at 2 Gy of 0.26) that have been investigated. Furthermore, experiments utilising continuous low dose rate radiation at 3.2 cGy min-1, showed that, under these experimental conditions, the cells possessed only a very low capacity to recover from radiation-induced damage . As other human tumour cells of an embryonal cell origin (e.g. neuroblastoma and germ cell tumours of the testis) have also been shown to be radiosensitive it appears that sensitivity to radiation may be a common property of this group of tumours.


Mononuclear cells from normal rabbit blood were cytotoxic to a number of cell lines in vitro. The cytotoxic cells were contained in the monocyte-enriched fraction adherent to plastic. Supernatants of the monocyte-enriched fraction had the same cytotoxic specificity as the parent cells. The cytotoxic factor precipitated in 50% saturated ammonium sulphate solution and on gel filtration was heterogeneous with a mol.-wt. range of 30--50,000 u. On the basis of specificity and molecular characteristics, this cytotoxic factor closely resembles rabbit tumour-necrosis factor (TNF) suggesting that TNF or a closely related factor is a normal product of mononuclear phagocytes.


A cell-free plasmid reactivation assay was used to determine the fidelity of DNA double-strand break (DSB) repair in a panel of eight DSB repair-proficient human tumour cell lines. Nuclear protein extracts derived from radiosensitive tumour cells were less capable of correctly rejoining EcoRI-induced DSBs than were similar extracts from radioresistant tumour cells. Linear regression analysis suggests that there was a significant  correlation between the fidelity of DSB rejoining and the SF2 values of the cell lines studied. This cell-free assay is clearly sensitive to differences in the nuclear protein composition that reflect the clinically relevant radiosensitivity of these cell lines. The fact that our cell-free assay yielded similar results to previous studies that used intracellular plasmid reactivation assays suggests that those differences in DSB mis-rejoining frequencies in radiosensitive and radioresistant cell lines may be due to inherent differences in nuclear protein composition and are not directly attributable to differences in proliferation rates between cell lines. The underlying cause for this association between DSB mis-rejoining frequencies and radiosensitivity is presently unknown, however restriction endonuclease mapping and polymerase chain reaction (PCR) amplification analysis revealed that approximately 40% of the mis-rejoined DSBs arose as a result of the deletion of between 40 and 440 base pairs. These data raise the possibility that the radiosensitivity of DSB repair-proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways.


Nucleotides such as ATP can act as extracellular effector molecules by interaction with specific cellular receptors known as P2-purinoceptors. Recently, we cloned the human P2U purinoceptor from osteoclastoma and demonstrated its expression in skeletal tissues. In the current study we have investigated the expression of P2U purinoceptors in human breast tumour cell lines and examined functional effects of extracellular nucleotides on these cells. By reverse transcription-linked polymerase chain reaction (RT-PCR) the expression of mRNA for P2U purinoceptors was demonstrated in four human breast cancer cell lines, Hs578T, MCF-7, SK-Br3 and T47-D. In MCF-7 cells, extracellular ATP (1-100 microM) elevated intracellular free calcium concentration [Ca2+]i, indicating that these cells express functional P2-purinoceptors. UTP elevated [Ca2+]i in an identical manner to ATP, whereas 2-methylthioATP was completely ineffective, and ADP only partially effective. This pharmacological profile suggests that the P2U subtype may be the only P2-purinoceptor expressed by these cells. The functional significance of P2U purinoceptor expression by MCF-7 cells was investigated by analysing the effects of extracellular ATP on cell proliferation. The slowly hydrolysed analogue of ATP, ATPgammaS , induced proliferation of MCF-7 cells when added daily to serum-free cultures over a period of 3 days. ATPgammaS-induced proliferation was demonstrated by three separate methods, detection by scintillation counting of [3H]thymidine incorporation, immunocytochemical detection of 5-bromo-2-deoxyuridine incorporation and direct counting of cell numbers. These data suggest that ATP, possibly released at sites of tissue injury or inflammation, may be capable of growth factor action in promotion of tumour proliferation or progression.


Enhanced DNA repair has been observed in cisplatin-resistant ovarian cancer cell lines. This resistance can be modulated, on co-incubation with aphidicolin in established cell lines and animal tumour models, by inhibiting DNA polymerases. We describe a study of the in vitro modulation effect of aphidicolin on cisplatin and carboplatin using fresh cells harvested from biopsy samples or ascitic fluids from 25 patients with ovarian adenocarcinoma. The MTT assay was used to measure cell survival after drug exposure. Aphidicolin (up to 30 microM) showed no cytotoxicity when tested alone. Forty-seven comparisons were made between drug with and without aphidicolin, and 37 (79%) cases demonstrated a significant increase in sensitivity to the platinum agents on co-incubation. Overall, there was a median 10-fold (range 1.64- to 58.5-fold) increase in sensitivity. When patients were grouped according to in vitro sensitivity to platinum, aphidicolin had a significantly greater effect in the "resistant' group, causing a median 13.5-fold increase in sensitivity compared with 2.4-fold in the "sensitive' group. Furthermore, a positive correlation between the LC50 for platinum and the corresponding fold increase in sensitivity suggests that aphidicolin overcomes platinum resistance in fresh cells from primary tumours. These results encourage the further development of this interesting compound.


L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC) cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested.Three murine renal cell carcinoma (mRCC) cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC.P = 0.01) reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity.Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (The depletion of L-arginine by mRCC induced the decrease expression of CD3ζ a key element for T-cell function.The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3ζ. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell growth and aid in restoring immune function by increasing L-arginine availability for T-cell use. Understanding the interplay between arginase II and its interaction with the immune system may provide future therapeutic benefits to treat patients with RCC. L-arginine is a basic amino acid that plays a central role in multiple systems including the immune system -3. Two iSeveral studies have shown that decreased plasma L-arginine levels and nitric oxide (NO) metabolites induced by trauma are associated with an increase in arginase I expression in mononuclear immune cells ,13, suggIn this study we demonstrate for the first time that only arginase II is produced by murine renal cell carcinoma (mRCC) cell lines and that high enzyme levels, specifically depletes extra cellular L-arginine. This amino acid deprivation induces the downregulation of CD3ζ expression in co-cultured Jurkat T-cells. Arginase inhibitors significantly suppressed cell growth in cell lines presenting high arginase II activity.Complete tissue culture medium consisted of RPMI-1640 containing 1,140 μM L-arginine and supplemented with 10% fetal calf serum , 25 mM HEPES, 4 mM L-glutamine, and 100 units/mL penicillin/streptomycin, 1 mM non-essential amino acids, and 1 mM sodium pyruvate. All other reagents were purchased from Lonza Walkersville Inc., Walkersville, MD.For this study we used mRCC cell lines SIRCC-1.2 (CL-2) and SIRCC 1.19 (CL-19), both of which are sub-clones derived from a streptozotocin-induced kidney tumor  and Renc6cells/hr), as described elsewhere -thymidine  and tested for [3H] incorporation at 24, 48, and 72 hours using a TOPCOUNT Microplate Scintillation Counter . Cell viability was checked by trypan blue exclusion at each time point. Each condition was tested in triplicate.Cells  were then added to the upper chamber of a trans-well system  and co-cultured for 24, 48, and 72 hours. CD3ζ expression was determined by flow cytometry as described elsewhere -thymidine at the same rate during the first 48 hours. However at 72 hours, significant differences in cell proliferation among the lines were apparent. CL-2 proliferation was significantly lower (P = 0.003) compared to CL-19, which presented the highest arginase activity. Interestingly, the Renca cell line, which had intermediate arginase activity, had the highest proliferation rate compared to the CL-19 (P = 0.01) and CL-2 (P = 0.003) cell lines  and Renca (P = 0.07). Cell viability of the cells was > 95% at the different points and conditions of the experiments.We then tested the effect of the arginase inhibition by nor-NOHA (2 mM) on cell proliferation at 24, 48, and 72 hours. Growth of CL-19, which had the highest level of arginase II activity, was significantly inhibited (P = 0.002 and P = 0.001 respectively)  the depletion of L-arginine as well as the accumulation of L-ornithine (P < 0.0001) after 48 hours compared to CL-19 cultures without the inhibitor  in the co-cultured CL-19 compared to the Jurkat control . L-arginine deprivation should promote the death of CL-19 cells, as reported previously to occur in other cancer cells lines ,37, indiL-arginine is a non-essential amino acid that plays a central role in several biological systems including the immune response. Paradoxically, L-arginine deprivation can cause tumor cell death as well as T-cell dysfunction. The loss of CD3ζ is the only arginase-triggered mechanism described so far that has proven to have direct relevance to T-cell function ,41. It hMost tumor cells have a great demand for amino acids to support rapid proliferation and L-arginine is the first amino acid depleted faster than other nutrients by normal cell metabolism. Therefore, L-arginine could be a reasonable target of deprivation strategy for the type of tumors with a low recycling efficiency. Taken collectively, these findings, demonstrate that the availability of L-arginine and L-ornithine could be the limiting factor to control cell proliferation. We believe that treating RCC cells downstream from the L-arginine metabolic pathway by blocking polyamine production will have a major impact in suppressing tumor growth. This is supported by the use of DL-α-difluoromethylornithine, which completely blocks the proliferation of these cell lines independent of the presence of L-arginine, L-ornithine and arginase in addition to the promising anti-tumor effect in human tumors -45.Renal cell carcinoma is a malignancy with poor prognosis due to its strong resistance to conventional cancer treatments and frequent metastases. With the standard immunotherapeutic treatment of IL-2 and IFNα for RCC, only 10–20% of the patients respond . This laArginase II produced by renal cell carcinoma cells can modulate L-arginine levels to regulate both cell growth and T cell function. Blocking arginase may lead to a decrease in RCC cell growth and aid in restoring immune function by blocking the formation of polyamines, thus providing a novel therapeutic advance.The authors declare that they have no competing interests.DJT participated in the design of the study, developed HPLC for the detection of amino acid levels, performed RT-PCR, analyzed the collected data and wrote the manuscript; DJV participated in the study design and conducted western blots and arginase activity assays; YAC participate in the analysis of HPLC data and design and conducted functional assays; TM participated in tissue culture and preparation of cell lysates and RNA extractions; JRP participated in performing arginase activity assays, western blots, amino acid assays and data collection; DHA was involved in the analysis and interpretation of data and critically revised the manuscript; AHZ designed the study, performed flow cytometry assays, analyzed and interpreted the data and wrote the manuscript. All authors read and approved the manuscript.


A major problem in the chemotherapy of colorectal cancers is their resistance to most cytotoxic drugs which may be due to insufficient cellular transport. Drugs conjugated to monoclonal antibodies recognising tumour antigens may overcome these difficulties by providing access of active agents to the tumour cells. The anti-tumour monoclonal antibody shown to localise in patients with colorectal cancer, 791T/36, has been investigated as a potential targeting antibody. Eight cell lines were established from surgically resected material and were shown to bind 791T/36 antibody. They were screened for their sensitivity to methotrexate, 5-fluorouracil and daunomycin. Although 5-fluorouracil is the drug of choice for chemotherapy of colorectal cancer it was the most cytotoxic drug in only 2 of the 8 cell lines. Only the 4 cell lines which were resistant to methotrexate showed less cytotoxicity with methotrexate than 5-fluorouracil. The cell lines which were resistant to methotrexate were more sensitive to 791T/36-methotrexate conjugates. Daunomycin was the most cytotoxic drug in 4 of the 8 cell lines. However, a similar cytotoxicity was observed for free drug and 791T/36 daunomycin in the two lines tested. Selective monoclonal antibody drug conjugates may offer a solution to treatment of tumours which are resistant to classical chemotherapeutic agents. This is the first report to show that newly established cell lines that are resistant to classical chemotherapeutic agents are rendered sensitive when the drug enters the cell as a drug monoclonal antibody carrier.


The non-aminobisphosphonate clodronate was administered for the same time periods (10−6M to 10−2M). Cell proliferation, apoptosis and mRNA expression was assessed. Both agents inhibited cell proliferation in a time- and dose-dependent manner. ELISA analysis demonstrated an increase in DNA fragmentation although there was no significant dose-related difference between the doses studied. Bisphosphonate-treated cultures had a greater subpopulation of cells exhibiting morphological changes of apoptosis. Expression of mRNA for osteopontin and RANKL was down-regulated by both agents, while the expression of mRNA for alkaline phosphatase, pro-α1(I) collagen and OPG was not altered. Out in vitro work suggests the bisphosphonates not only have direct effects on osteosarcoma cell growth and apoptosis, but also, by altering the relative expression of osteoclast-regulating factors, they may inhibit the activity of osteoclasts and their recruitment. © 2001 Cancer Research Campaignhttp://www.bjcancer.comLocal growth of osteosarcoma involves destruction of host bone by proteolytic mechanisms and/or host osteoclast activation. Osteoclast formation and activity are regulated by osteoblast-derived factors such as the osteoclast differentiating factor, receptor activator of NF-κB ligand (RANKL) and the inhibitor osteoprotegerin (OPG). We have investigated the in vitro effects of bisphosphonates on a clonal rat osteosarcoma cell line. The aminobisphosphonate pamidronate was added to UMR 106-01 cell cultures (10


Cisplatin (CDDP) resistance mechanisms were studied in a model of three germ cell tumour and three colon carcinoma cell lines representing intrinsically CDDP-sensitive and -resistant tumours respectively. The CDDP sensitivity of the cell lines mimicked the clinical situation. The glutathione levels of the cell lines correlated with CDDP concentrations inhibiting cell survival by 50% (IC50); total cellular sulphydryl content (TSH) was unexpectedly inversely correlated with IC50. IC50 correlated neither with glutathione S-transferase (GST) nor with GST pi expression, topoisomerase I or II activity. Immediately after 4 h incubation with CDDP, platinum (Pt) accumulation and Pt bound to DNA were not correlated, but after another 24 h drug-free culture, Pt binding to DNA in germ cell tumour but not in colon carcinoma cell lines correlated with IC50. With the exception of in vitro sensitivity and TSH, none of the parameters studied discriminated between the two groups of cell lines. Correction of CDDP sensitivity parameters for phenotypical differences did not influence statistical correlations. Analysis of variance revealed a correlation between IC50 and the combination of glutathione, GST activity and Pt bound to DNA. But at other CDDP cytotoxicity levels sensitivity was also correlated with Pt accumulation, topoisomerase II activity and TSH in various combinations. This model of intrinsic CDDP resistance showed that multiple parameters ought to be studied to explain CDDP resistance, but did not elucidate the cause of the unique sensitivity of germ cell carcinoma, although the unexpected values of TSH deserve further attention.


Morphological and growth characteristics are described of a rapidly growing cell line with epithelioid and giant-cell characteristics derived from a chondrosarcoma in a male patient 65 years of age. This cell line is of considerable interest because in these cells cross-reacting antigens with known animal oncorna-viruses are present. Biochemically, the cells contain particles with a density of 1-16 with "cores" of density 1'23 associated with a reverse-transcriptase-like enzyme and with 70S RNA. Occasionally, virus-like particles were demonstrated by electron microscope in material derived from the culture medium.


A brain tumour-associated marker, urokinase (UK), was investigated using rabbit anti-UK polyclonal and murine anti-UK monoclonal antibodies, which were prepared by immunization with low molecular weight UK (LMW-UK) and high molecular weight urokinase (HMW-UK) synthetic peptide respectively. The polyclonal antibody cross-reacted with both LMW-UK and HMW-UK, whereas the murine MAbs were specific for HMW-UK. These immunological probes were used to study urokinase in glioma extracts, tissues, sera and cell lines that had been prepared from primary cultures of freshly dissected gliomas. Radioimmunoassays showed that glioma extracts had much higher level (5- to 44-fold) of UK than normal human brain extracts. This result was confirmed by immunoblotting of electrophoresis gels of glioma and human brain extracts. Immunohistochemical study using anti-UK MAb demonstrated much higher levels of UK in glioma tissue than normal brain tissue. Immunohistochemical study using anti-UK MAbs localized UK on the cell surface of glioma cells. Anti-UK MAbs inhibited the proliferation of AA cell lines and GB cell lines (50% to > 90%) and exerted minor effects (< or = 20%) on normal human liver, intestine and lymphocyte cell lines. Taken together, these results suggest that anti-UK MAbs may have therapeutic potential for human gliomas and cancer metastasis.


Two cell lines derived from vaginal intraepithelial neoplasias (VAINs) expressing human papillomavirus (HPV) 33  and 16  E6-E7 mRNA were studied in organotypic culture for their keratins and cell cycle regulatory proteins in relation to replicative aging. Early-passage UT-DEC-1 and UT-DEC-2 cells reproduced epithelial patterns consistent with VAIN. Cells from later passages resembled full-thickness intraepithelial neoplasia (UT-DEC-1) and microinvasive cancer (UT-DEC-2). The morphological changes were compatible with these cell lines' ability for anchorage-independent growth at later passages. Simple epithelial keratins were aberrantly expressed in both cell lines. K18  and K17 expression increased in UT-DEC-1 and UT-DEC-2 cells at late passages. No marked differences in expression of p53 (wild type in both cell lines), mdm-2 or PCNA were detected in parallel with progression. The expression of p21WAF1/cip1 localized mostly to the upper half of the epithelium at early passage and was more intense in the HPV 16-positive UT-DEC-2 cell line expressing K10. In Northern blot analyses, the transcription pattern of the HPV 33 E6-E7 of the UT-DEC-1 cell line changed during later passages, whereas that of the HPV 16 E6-E7 of the UT-DEC-2 cell line remained unaltered. The present characterization of the phenotype of these cell lines derived from natural squamous intraepithelial lesions shows an association between simple epithelial-type keratin expression and progressive changes in growth and morphology, but fails to demonstrate consistent changes in the expression of cell cycle regulatory proteins studied in parallel with progression.


Monoclonal antibody against an osteogenic-sarcoma cell line (791T) was prepared by production and cloning of a somatic-cell hybrid between the mouse myeloma P3-NS1 and spleen cells from 791T-immunized mice. Three clones of hybridoma producing antibody against 791T, as detected by 125I-labelled Protein A binding, were tested against a range of normal and tumour cell targets to determine the pattern of expression of the antigen detected. The 3 clones had identical activity. They reacted strongly against 791T cells and another osteogenic sarcoma, 788T, and more weakly against a further 2 from a total panel of 10 osteogenic-sarcoma lines. The antibody was negative for fibroblasts from the donor of 791T, and for other fibroblasts, human red blood cells, human peripheral mononuclear cells and sheep red blood cells. When tested against a panel of unrelated tumours, they reacted against individual cell lines derived from carcinomas of colon, lung, bladder and cervix. These cross-reactions were not observed with other colon or lung carcinomas, and it is suggested that the antibody was reacting with a tumour-associated antigen expressed randomly on different tumour types, rather than specifically on osteogenic sarcomas.


Di-n-butyltin, tri-n-butyltin and triphenyltin terebates were screened against several human tumour cell lines and found comparably or more active than carboplatin, cis-platin, 5-fluorouracil, methotrexate and doxorubicin, some reference compounds used clinically.


The role of protein kinase C (PKC) in in vitro invasiveness of four different human renal cell carcinoma (RCC) cell lines of the clear cell type was investigated. Different PKC-inhibitors markedly inhibited invasiveness of the highly invasive cell lines, suggesting an invasion-promoting role of PKC in human RCC. Analysis of PKC-isoenzyme expression by protein fractionation and immunoblotting revealed that all cell lines expressed PKC-α, -ɛ, -ζ, -μ and -ι as known from normal kidney tissue. Interestingly, PKC-δ, known to be expressed by normal kidney epithelial cells of the rat, was absent on protein and RNA levels in all RCC cell lines investigated and in normal human kidney epithelial cells. PKC-ɛ expression levels correlated positively with a high proliferation activity, but no obvious correlation between expression levels of distinct PKC-isoenzymes and in vitro invasiveness was observed. However, by immunofluorescence microscopy, membrane localisation of PKC-α and PKC-ɛ reflecting activation of the enzymes, was associated with a highly invasive potential. In conclusion, our results suggest a role for PKC in invasion of human RCCs and might argue in favour of a particular role of PKC-α and PKC-ɛ. Our results further suggest that organ-specific expression patterns of PKC-isoenzymes are not necessarily conserved during evolution. © 2000 Cancer Research Campaign


HER2 overexpression or increased expression of EGFR was significantly associated with in vitro sensitivity . Lapatinib exerts growth inhibition in a PTEN-independent manner. Sensitive cell lines also exhibited increased expression of EGFR ligands or HER3. In contrast, lapatinib-resistant cell lines exhibited high androgen receptor (AR) levels or epithelial-to-mesenchymal transition (post-EMT) features. In endometrial cancer cells, at a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for lapatinib plus carboplatin, paclitaxel, docetaxel, and doxorubicin. These observations provide a clear biologic rational to test lapatinib as a single agent or in combination with chemotherapy in endometrial cancer with HER2 overexpression. Expression of EGFR, its ligands, HER3, AR, and post-EMT markers warrant further evaluation to help define patients with HER2-nonoverexpressing endometrial cancer most likely to benefit from lapatinib.In this study, we explore the therapeutic potential of lapatinib a selective inhibitor of both the EGFR and HER2 tyrosine kinases for the treatment of endometrial cancer. The effect of lapatinib on tumour cell growth and receptor activation was studied in a panel of human endometrial cancer cell lines. Candidate molecular markers predicting sensitivity were assessed by baseline gene expression profiling, ELISA, and western blot analyses. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions between chemotherapeutic drugs and lapatinib. Concentration-dependent anti-proliferative effects of lapatinib were seen in all endometrial cancer cell lines tested, but varied significantly between individual cell lines (IC Endometrial cancer is the most common malignancy of the female reproductive tract. Despite recent advances in the molecular characterisation of endometrial cancer, current treatment strategies do not include target-based therapies  in an unbiased way using a large panel of 19 endometrial cancer lines that express variable levels of HER2 and EGFR. Subsequently, all cell lines were molecularly characterised using Agilent Microarrays. Although this technology allows us to examine thousands of genes simultaneously the identification of a more limited number of markers that predict response to lapatinib in endometrial cancer cells might be more useful for independent validation in clinical studies. Importantly, for this study markers were selected on the basis of their potential biologic relevance in HER2 and EGFR signalling. Therefore, we correlated the IC50 values with the relative expression levels of EGFR, HER2, HER3, and HER4 as well as the ligands of EGFR such as TGF-α, EGF, amphiregulin, HB-EGF, betacellulin, epiregulin, neuregulin 1, and neuregulin 2. Next, we correlated the relative expression of the steroid hormone receptors oestrogen receptor-α (ER-α) or -β (ER-β), progesterone receptor (PR), and androgen receptor (AR) with in vitro response to lapatinib, because recent studies suggest that EGFR inhibitors may be particularly active in a subset of breast tumours described as ‘triple negative’  (in vitro effects of lapatinib between both of these subtypes.Our primary aim was to explore the ression) . To furtFinally, we used multiple drug effect/combination index (CI) isobologram analysis to study the efficacy of chemotherapeutic drugs plus lapatinib combinations tested against lapatinib-sensitive HER2-amplified/overexpressing or EGFR-expressing endometrial cancer cells.In summary, the current studies were intended to provide a rational to test lapatinib as a single agent or in combination with chemotherapy in patients with high-risk primary or metastatic endometrial cancer and to identify candidate markers that may help define subsets of patients most likely to benefit from treatment with lapatinib.M glutamine, and PSF . HEC1A cells were cultured in McCoy's medium supplemented with 10% heat-inactivated fetal bovine serum and PSF (Irvine Scientific). SNG-II and SNG-M cells were cultured in Ham's F-12 supplemented with 10% heat-inactivated fetal bovine serum and PSF (Irvine Scientific). The remaining cell lines were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, and PSF (Irvine Scientific). Lapatinib was provided by GlaxoSmithKline  as a 10-mM concentrated stock solution in dimethyl sulphoxide.The effects of lapatinib on malignant cell growth were studied in a panel of 19 established human endometrial cancer cell lines. The established human endometrial carcinoma cell lines KLE, RL-95-2, AN3CA, HEC1A, and HEC1B were obtained from American Type Culture Collection . The established human endometrial cancer cell lines MFE280, MFE296, MFE319, EFE184, and EN were obtained from the German Tissue Repository DSMZ . Ishikawa cells were obtained from the European Collection of Cell Cultures . The established human endometrial carcinoma cell lines HEC155, SNG-II, and SNG-M were obtained from the Japanese Health Science Research Resources Bank . The cell lines SPAC1S and SPAC1L were provided by the laboratory of Dr Y Hirai from the Department of Gynecology, Cancer Institute Hospital . The cell line EN1 was provided by Dr V Möbus from Department of Gynecology at the University of Ulm (Germany). USPC1 and USPC2 cells were provided by Dr A Santin from the Department of Obstetrics and Gynecology, Division of Gynecologic Oncology at the University of Arkansas . USPC2, RL-95-2, SPAC1L, SPAC1S, and HEC1B cells were cultured in RPMI medium 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mHER2 and EGFR protein content in all cell lines was measured by ELISA as described previously (5 and grown in cell line-specific media without or with increasing concentrations of lapatinib (ranging between 0.031 and 10 μM). Cells were harvested by trypsinisation on day 7 and counted using a particle counter . Growth inhibition was calculated as a percentage of the untreated controls. Experiments were performed three times in duplicate for each cell line. The log of the fractional growth inhibition was then plotted against the log of the drug concentration, and the IC50 values were interpolated from the resulting linear regression curve fit .Cells were plated into 24-well plates at a density of 2 to 5 × 10g for 10 min. Protein was quantitated using BCA , resolved by SDS-PAGE, and transferred to nitrocellulose membranes . HER2, EGFR, and PTEN expression were detected, respectively, by monoclonal anti-HER2 , anti-EGFR antibodies , and anti-PTEN antibodies . For the evaluation of concentration-dependent activity of lapatinib on HER2, EGFR, AKT, and ERK Phosphorylation, USPC1, USPC2, RL-95-2, HEC155, and SNG-II cells in log-phase growth were treated with increasing doses of lapatinib (0.5–5 μM) for 1 h prior to cell lysis. Tyrosine phosphorylation of HER2 and EGFR was analysed as follows. Immunoprecipitations were performed by allowing 250 μg protein lysate to incubate with 3 μg monoclonal anti-HER2 (Ab-3) or anti-EGFR antibody , and Protein A/G-agarose (Santa Cruz Biotechnology) at 4°C overnight with gentle agitation. The immunoprecipitates were washed three times in lysis buffer and then denatured in Lemmli's buffer prior to SDS-PAGE. Immunoblotting was performed using a monoclonal anti-phosphotyrosine antibody . Detection was performed using ECL Plus chemifluorescent reagent  and imaging of the resulting western blots was performed using the chemifluorescence method by Typhoon 9400 (Amersham Biosciences). Phospho-AKT and phospho-ERK were detected by polyclonal anti-pAKT (Ser473) and anti-pERK (Thr202/Thr204) antibodies .Cells in log-phase growth were washed in PBS and lysed at 4°C in lysis buffer. Insoluble material was cleared by centrifugation at 10 000 in situ hybridisation (FISH). In 12 established endometrial cancer cell lines . The cell lines were treated with Colcemid (0.05 g ml−1) for 2–4 h to obtain metaphase preparations. All samples were fixed in methanol/acetic acid (3 : 1). Specimen preparation, hybridisation, and microscopy were performed as previously described , experimental media containing either control media, lapatinib, chemotherapeutic agent, or the combination (lapatinib plus chemotherapeutic agent) were added to appropriate wells in duplicate, and serial two-fold dilutions were performed to span clinically relevant concentration ranges for the dose–effect analysis for lapatinib and drug combinations. Following incubation for 72–120 h, plates were washed with PBS and stained with 0.5% N-hexamethylpararosaniline  in methanol. Sorenson's buffer  (0.1 ml) was added to each well, and the plates were analysed in an ELISA plate reader at 540 nm wavelength. For each assay, the log of the fractional growth inhibition was plotted against the log of the drug concentration and the linear regression curve fit correlation coefficient  was calculated. Multiple drug effect analysis was performed using computer software (Biosoft) as described in detail . Microarray slides were read using an Agilent Scanner, and the Agilent Feature Extraction software version 7.5 was used to calculate gene expression values. Extracted data were imported into Rosetta Resolver 5.1 to create expression profiles for each individual endometrial cell line experiment. Cluster analysis was performed in Resolver and cell line profiles were exported to Excel  for additional analysis.Agilent microarray analyses were developed for each cell line. Briefly, cells were grown to log phase, and then RNA was extracted using the RNeasy Kit . The purified RNA was eluted in 30–60 50 values were analysed using Spearman's ρ correlation, and differences in the IC50 values between subgroups compared using the Mann–Whitney U-test. All statistical tests were two sided. The threshold for upregulation was set to log (ratio) >0.13 with a P-value <0.01, and downregulation was defined as log (ratio) ⩽0.13 with a P-value <0.01. The P-values were determined according to the Agilent error model when the feature-extracted data were imported into Resolver. This study was reviewed and approved by the Mayo Clinic Foundation Institutional Review Board.Associations between the expression levels of biomarkers and the ICin vitro studies was quantitated using ELISA  was identified using a wide range of lapatinib concentrations (0.031–10 μM). Lapatinib inhibited the proliferation of all endometrial cancer cell lines investigated in a concentration-dependent fashion; however, the IC50 values varied significantly between individual cell lines with up to a 200-fold difference in the IC50 values and ranged between 0.052 μM in HER2-overexpressing USPC2 endometrial cancer cells and 10.9 μM in MFE296 cells that express low levels of HER2 and EGFR , and EGFR expression was significantly inversely correlated with IC50 values . We next investigated the sensitivity of the endometrial cancer cell lines to lapatinib by using a binary classification, and thus two classes of cell lines were defined: sensitive and resistant. Using the median IC50 value as a cutoff cell lines with an IC50 below 3 μmol were classified as sensitive and those with higher IC50 values as resistant. Using this binary classification, 4/5 uterine serous papillary cell lines and all 6 cell lines with either high HER2 (USPC1 and USPC2) or EGFR  expression were classified as sensitive. In contrast, none of the resistant cell lines demonstrated HER2 overexpression or high levels of EGFR expression. Among those cell lines with low levels of HER2 and EGFR expression, only EFE184 demonstrated responsiveness to lapatinib at nanomolar concentrations (IC50 0.649 μM).After quantitation of EGFR and HER2 receptor levels in the endometrial cancer cell lines, growth assays were performed . The effand EGFR . Both HEin vitro resistance towards EGFR TKIs .As a measure of functional activity of the receptor tyrosine kinases, the phosphorylation state of HER2 or EGFR was assessed using immunoblotting techniques. Exposure of USPC1, USPC2, RL-95-2, HEC155, and SNG-II human endometrial cancer cells to lapatinib resulted in a dose-dependent reduction of phosphorylation of both the EGFR and HER2 receptor tyrosine kinases and their downstream signalling intermediates AKT and ERK . Of noten levels . Low expin vitro sensitivity to lapatinib , and HER3, the preferred oligomerisation partner of HER2 , suggesting that lapatinib-sensitive cell lines have higher levels of amphiregulin or HER3 expression .A key aspect of therapies with targeted agents is accurate selection of patients most likely to benefit from therapy. Our preclinical data demonstrate that increased expression of either HER2 or EGFR was significantly associated with apatinib . Moreovepression . Other Ent level . We foun50 5.84 vs 2.78, P=0.079). Conversely, we were able to demonstrate inverse correlations between IC50 values and the relative expression of CDH1 , CDH3 , and the KRT8 , 18 , or 19 , suggesting that the epithelial phenotype may be associated with lapatinib sensitivity.Induction of regulators of an EMT, such as Twist, Slug, and Snail, represses epithelial cadherin transcription in cancer cells, causing downregulation of this adhesive epithelial marker . SeveralμM for USPC1 and USPC2 cells, and 0.31 and 10.0 μM for RL-95-2 and HEC155 cells and were below the reported peak plasma concentrations achievable in humans (P=0.358) to 1.04 (P=0.564)) (P<0.001) to 0.91 (P=0.068)) and doxorubicin (0.62 (P<0.001) to 0.89 (P=0.341)) (P=0.001) to 0.78 (P<0.001)) in all four cell lines ) . Synergi=0.341)) . Synergill lines .Evidence for a role of HER2 and EGFR in the pathogenesis of various cancers has led to the rational design and development of agents that selectively target HER2 and EGFR. In unselected patients with endometrial cancer, HER2 gene amplification/overexpression represents a rare event. However, HER2 amplification/overexpression is seen more commonly in well-defined subtypes of endometrial cancer such as uterine serous papillary cancer, clear cell cancer, or grade 3 endometrioid cancer . These are the patients most likely to also benefit from adjuvant-targeted therapies. Our laboratory data provide a clear biologic rational to test lapatinib as a single agent or in combination with chemotherapy in HER2-overexpressing and/or possibly EGFR-expressing endometrial cancer. Patients most appropriate to screen for HER2 amplification are patients with nonendometrioid histologies (type II). Further evaluation of not only EGFR ligands and HER3 levels but also AR levels and markers of EMT may help to define patients with EGFR-expressing endometrial cancer most likely to respond to lapatinib.


Ten cell lines of human squamous carcinomas of the tongue and larynx have been established from surgical specimens removed from 36 unselected patients, in order to provide systems for investigating the invasive and tissue-destructive capacity of squamous carcinomas of the head and neck. The morphology, ultrastructure and growth characteristics of the 10 lines are described. Detailed cytogenetic analysis of the first 4 lines indicates that each is karyotypically unique, with no evidence of cross-contamination. Nine of the 10 cell lines secrete immunoreactive beta human chorionic gonadotrophin (beta-hCG) in the culture medium. No correlation was demonstrated between the ability of the cell lines to secrete plasminogen activator and their capacity to grow in soft agar or as xenografts in immune-deficient mice.


We tested the ability of hormones and growth factors to enhance the colony formation in soft agarose of breast carcinoma using two human breast carcinoma cell lines, MCF-7 and MDA-MB231, MCF-7 could clone in a basal medium supplemented only by insulin, transferrin, prostaglandin F2 alpha, and fibronectin. Combining oestradiol, dexamethasone, insulin, transferrin, and triiodothyronine with a basal medium supplemented with 5% (v/v) foetal bovine serum (FBS) increased colony forming efficiency (CFE) two-to three-fold over the best obtained in serum supplemented medium without hormones. While optimal CFE was seen in the hormonally supplemented medium plus 5% FBS, clonal anchorage independent growth could also be obtained without serum for both cell lines by substituting 0.5-1% (v/v) bovine serum albumin (BSA) for FBS. Although CFE was enhanced with the addition of hormones, they did not substantially alter the in vitro chemosensitivity patterns of the cell lines to 8 cytotoxic drugs. Hormonally-supplemented medium with 5% FBS increased the CFE of a small number of fresh specimens of human breast cancer compared with medium supplemented with serum alone. The systematic study of requirements for the in vitro growth of human breast cancer may improve drug sensitivity testing by increasing our ability to grow this neoplasm in culture.


The principles and design of a Hadamard transform UV absorbance detector for liquid chromatography are outlined, and some spectra of aromatic compounds passing through its flow cell are presented. This approach could be valuable in providing alow-cost multi-wavelength detection method for liquid chromatography.


The effect of the tumour promoters TPA, phenobarbitone and saccharin on the production of sister-chromatid exchanges (SCE) was studied. TPA produced a small but significant increase of SCE in Chinese hamster cell lines V-79 and CHO, and in a hybrid clone formed by fusion of CHO with rat liver epithelial cells. The enhancement of SCE by TPA was not affected by the type of culture medium, heat-inactivated bovine serum, or the batch of TPA. In the presence of exogenous L-cysteine the enhancement of SCE was reduced. TPA also increase the uptake of 2-deoxyglucose by the cells, to an extent similar to that of the SCE enhancement. This enhancement of SCE by TPA may be explained partly through the formation of free radicals, and partly through alterations in the cell-surface membrane and/or a transient delay in the cell-cycle progression. The other tumour promoters, phenobarbitone and saccharin, also enhanced both SCE and the uptake of 2-deoxyglucose in V-79 cells.


The radiation-induced genotoxic damage in three established cell lines and 15 primary cultures of human malignant melanoma and ovarian carcinoma showing different radiosensitivity was tested by the cytokinesis-block micronucleus assay. A dose-related increase in micronucleus frequency was observed in all the cell systems. The mean number of micronuclei per Gy of ionising radiation per binucleated cell was respectively 0.44 +/- 0.0075 and 0.43 +/- 0.04 for M14 and JR8 malignant melanoma cell lines and 0.19 +/- 0.013 for the A2780 ovarian cancer cell line. The number of micronuclei did not rank the cell lines in the same order of radiosensitivity as clonogenic cell survival, which showed a surviving fraction at 2 Gy of 0.38 +/- 0.02 for JR8, 0.34 +/- 0.05 for M14 and 0.22 +/- 0.007 for A2780. As regards primary tumour cultures, no correlation was observed between micronucleus induction and surviving fraction at 2 Gy. In conclusion, the discrepancy we observed between micronucleus formation and cell death raises doubts about the potential of the micronucleus assay as a preclinical means to predict radiosensitivity.


Confirmatory morphological assessment was also performed by routine resin histology and transmission electron microscopy. Cell viability was assessed over a 0.5–48 h time course. In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells. Although complete apoptosis of both cell lines was induced by 50 μM SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h). Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.The use of apoptosis-inducing agents in the treatment of malignant cancer is increasingly being considered as a therapeutic approach. In this study, the induction of apoptosis and necrosis was examined in terms of temporal dose responses, comparing a malignant and nonmalignant breast cell line. Staurosporine (SSP)-induced apoptosis and H In the normal breast, epithelial cell death by apoptosis occurs following lactation. This removal of secretory epithelial cells is necessary to remodel the breast to a quiescent organ of fat cells .Identifying and counting apoptotic cells in culture can be difficult to do with accuracy, as each method available has advantages and disadvantages. A combination of these methods can give a more accurate picture of the apoptotic processes occurring in the cultured cell population.vs necrosis, by staurosporine (SSP) and H2O2, respectively. SSP, a potent protein kinase inhibitor with a broad spectrum of activity , HThe neutral red (NR) metabolic impairment assay works simply on the principle that this dye accumulates in the lysosomes of viable cells by a combination of active endocytosis and pinocytosis until a stable equilibrium is reached. Dead cells lose their ability to accumulate and retain NR . HoweverThe methyl-thiazolyl tertrazolium (MTT) assay measures the mitochondrial function activity of mitochondrial dehydrogenases . MTT is The DNA-binding dyes Hoechst 33342 and propidium iodide were first used together in a differential dye uptake assay (Hoechst/PI) on the fluorescent-activated cell sorter (FACS) . In viab−1 sodium pyruvate and pyroxidine (Life Technologies) supplemented with 10% fetal calf serum (PAA Laboratories) and 1% penicillin/streptomycin (Life Technologies). Cells were cultured in a humidified 5% CO2 atmosphere at 37°C.The HBL-100 cell line derived from the lactating breast  and the 4 cells well−1 and for approximately 16 h before treatments were added. Experimental treatments of 50 μM SSP, 50 nM SSP and 3 mM H2O2 in growth medium were added to both cell lines for a time course ranging from 0.5 to 48 h. A control of growth medium was also run in parallel for each time period in both cell lines. After treatments, the cells were processed in accordance with each particular assay.Cells were seeded at a density of 2×10μM SSP or 3 mM H2O2 in growth media for 24 h. After treatment, cells were rinsed in calcium-free saline, harvested mechanically and then pelleted in 1.5 ml Eppendorf tubes. The cell pellets were fixed overnight in 3% glutaraldehyde in 0.1 M cacodylate buffer. They were then washed several times in 0.1 M cacodylate buffer, postfixed in 0.1 M osmium tetroxide in 0.1 M cacodylate buffer and dehydrated in a graded series of ethanol before embedding in epoxy resin. Semithin sections (1 μm) of the cell pellets were cut using a Reichert–Jung Ultracut-E microtome. The sections were stained with toluidine blue and examined using a Leitz Diaplan, microscope, and representative sections photographed. Ultrathin sections (70–90 nm) were cut and stained with uranyl acetate and lead citrate  was added and agitated for 15 min on a microtitre plate shaker. The absorbance was read at 550 nm and for each treatment six replicate wells were examined.This assay was performed according to the method of μl well−1 of a 5 mg ml−1 stock MTT solution was added to cells in a 96-well microtitre plate previously seeded at a volume of 100 μl well−1. This was incubated for 2 h at 37°C until the purple formazan crystal developed. Finally the MTT-containing medium was removed and 200 μl of dimethyl sulphoxide (DMSO) was added to each well. The absorbance was read at 550 nm and for each treatment six replicate wells were examined.This assay was performed according to the modified method of μl well−1 of a 200 μg ml−1 PI stock  and 2.5 μl of a 100 μg ml−1 Hoechst 33342 was added to a microtitre plate well volume of 100 μl, and incubated in the dark for 60 and 15 min respectively. A 100 μl of methanol : acetic acid (3 : 1) fixative was then added directly to each well. Cells were viewed under a UV microscope with DAPI filter. Six replicate wells were analysed for each treatment by quantitative and qualitative examination of four random fields in each well.This assay was performed according to a combination of methods (100) of SSP and H2O2 in both cell lines. From the results, it was concluded that in both cell lines the approximate LD100 doses for SSP and H2O2 were 50 μM and 3 mM, respectively  treatment after 24 h showed an entire cell population with no distinct nuclear staining . However, the MTT and NR assays do not detect cell death until 24 h. In contrast to the HBL-100 line, the metastatic T47D cells show only 15% apoptosis at 4 h. Although they become 100% apoptotic at 24 h, MTT and NR show only 50% cell death at that time point. At this 50 μM SSP dose, it was observed in both cell lines that a reduction in MTT correlated with end-stage apoptosis (secondary necrosis) as detected by Hoechst/PI. The onset of apoptosis as measured by Hoechst/PI was not reflected by NR either. This was perhaps not surprising, as NR indicates when membrane integrity is lost, late in the apoptotic process.We showed that the time of onset of apoptosis as judged by Hoechst/PI does not correlate with any marked cell death detected with either cytotoxicity assay. At 50 M SSP, it yielded similar results to that for 50 μM SSP, again marked changes in MTT and NR reflected end-stage apoptosis (as judged by Hoechst/PI). The T47D cell line yielded different results for the 50 nM SSP dose, as an initial reduction in MTT at 4 h correlated with the observation of apoptosis as detected by Hoechst/PI coincidentally. However, this appeared to vary over the time course and no marked increase in initial apoptosis levels, detected by Hoechst/PI), was seen even after 48 h. This can perhaps be explained by the greater number of mitotic cells observed (data not shown) and is therefore reflective of the cycling cell population. This result also highlights the low level of apoptosis induced by the 50 nM SSP dose in the metastatic breast cell line (approximately 4%) as compared with the non-malignant cell line (approximately 100%), even after 48 h of treatment.When the HBL-100 cell line was treated with 50 nM H2O2, in the HBL-100 cell line showed that by 4 h there was a decrease in the amount of viable cells as detected by NR; however, it was 24 h before this was observed by MTT. Interestingly, the percentage of necrosis detected by Hoechst/PI increased steadily from as early as 4 h.The necrotic treatment, 3 mThe T47D cell line gave a very different result with this treatment, as MTT showed a steady decrease in the number of viable cells detected (compared with control levels) from as early as 0.5 h. It was 24 h before this type of decrease was detected by NR. The MTT result may be explained by the possibility that the metastatic cell line contains a subpopulation of cells that are more resistant to necrotic cell death.M SSP dose showed that the metastatic cell line is 25 times more resistant to apoptosis than the nonmalignant cell line. The T47D cells when treated with 50 μM SSP, revealed a delay in reaching approximately 100% apoptosis (by 24 h) as compared with the HBL-100 cells (by 4 h). Results of the MTT assay in combination with the appearance of secondary necrosis (as judged by Hoechst/PI) suggest that complete mitochondrial breakdown (as opposed to membrane pore transition) occurs only when secondary necrosis has begun. The 3 mM H2O2 results also suggest that the T47D cell line is more resistant to necrotic cell death than the HBL-100 cell line. Comparing these assay methods has thus allowed for a quantitative assessment of apoptosis in these breast cell lines. In addition, this combination of assay methods, with particular emphasis on Hoechst/PI, has also shown its ability to distinguish between apoptosis and necrosis in vitro.In conclusion, the 50 nIn terms of relevance to cancer biology, the results have significance: they suggest that malignant breast cells are more resistant than nonmalignant cells to apoptotic induction, although possible differences in bcl-2 gene expression or other targets of SSP would also have an influence. Potential therapeutic approaches using drug-induced cell death  would ne


Consistent with this hypothesis, pretreatment with either quinidine or verapamil (inhibitors of Pgp-mediated transport) sensitized CHRC5 cells to the antiproliferative effects of flavopiridol. Because of concern that colony forming assays might not accurately reflect cytotoxicity, we also examined flavopiridol-treated cells by trypan blue staining and flow cytometry. These assays confirmed that flavopiridol was less toxic to cells expressing higher levels of Pgp. Further experiments revealed that flavopiridol inhibited the binding of [3H]-azidopine to Pgp in isolated membrane vesicles, but only at high concentrations. Collectively, these results identify flavopiridol as a weak substrate for Pgp. © 2001 Cancer Research Campaign www.bjcancer.comFlavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC