The immunological response to solid tumours is insufficient. Therefore, tumour specific antigens have been explored to facilitate the activation of the immune system. The cancer/testis antigen class of MAGE-A antigens is a possible target for vaccination. Their differential expression profiles also modulate the course of the cancer disease and its response to antineoplastic drugs.The expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in five own oral squamous cell carcinoma cell lines were characterised by rt-PCR, qrt-PCR and immunocytochemistry with a global MAGE-A antibody (57B) and compared with those of an adult keratinocyte cell line (NHEK).All tumour cell lines expressed MAGE-A antigens. The antigens were expressed in groups with different preferences. The predominant antigens expressed were MAGE-A2, -A3 and -A6. MAGE-A10 was not expressed in the cell lines tested. The MAGE-A gene products detected in the adult keratinocyte cell line NHEK were used as a reference.MAGE-A antigens are expressed in oral squamous cell carcinomas. The expression profiles measured facilitate distinct examinations in forthcoming studies on responses to antineoplastic drugs or radiation therapy. MAGE-A antigens are still an interesting aim for immunotherapy. Tumour cells express specific antigens. Despite the fact that the protein products of these genes are absent or only partially found on healthy cells, the immunological response is insufficient,2. The gThe adult Normal Human Epidermal Keratinocytes (NHEK-adult) cell line was obtained from PromoCell GmbH, 69126 Heidelberg, Germany. The cell line was established using adult keratinocytes. The culturing was carried out according to the manufacturer's instructions.The PCI-13-1 cell line was established from a male patient who suffered from low grade oral squamous cell carcinoma of the retromolar triangle. The tumour stage was pT4pN1M0G3.The origin of this cell line was a larynx carcinoma of the glottis. It was harvested from a male patient. The grading was moderately differentiated and the tumour staging was pT2N0M0G2.This tumour originated from the aryepiglottic fold of a male patient. It was a primary carcinoma with moderate differentiation. The tumour staging at the time of harvesting was pT2N0M0G2.This cell line was established from a primary tongue carcinoma of male patient. The carcinoma was well differentiated. The tumour staging at time the cells were harvested was pT4N0M0G1.This cell line was established from a primary carcinoma of the base of the tongue of a male patient. It was moderately differentiated. The tumour staging was pT4N3M0G2.Superscript II . cDNA was incubated with 1 μl RNase A  for 60 min at 37°C. The cDNA was stored at -20°C until rt-PCR analysis. RNA integrity was tested by rt-PCR of the housekeeping gene beta-actin. Specific rt-PCR detection of MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6 and MAGE-A10 was performed with the primers listed in Table Total RNA from the tumour cell lines examined was extracted using RNeasy Mini Kits  according to the manufacturer's instructions. The isolated RNA was stored at -20°C until reverse transcription. CDNA was created from isolated total RNA using dN6-random-primers  and reverse transcription with The expression profiles of MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6 and MAGE-A10 were quantitatively measured by qrt-PCR. To validate the values measured for each MAGE-A gene, three measurements were performed and the mean value calculated. The measurements were performed with a LightCycler 2.0 qrt-PCR-System  using FastStart DNA Master Plus SYBR-Green I . The reaction volume of each measurement was 20 μl, consisting of 1 μl cDNA, 1 μl forward primer (20 μM), 1 μl reverse primer (20 μM), 4 μl LightCycler DNA Master SYBR-Green I (Roche), and 13 μl water. The steps for the qrt-PCR program for the LightCycler were: an initial denaturation at 95°C for 7 minutes, 45 cycles of amplification with denaturation at 95°C for 10 minutes, primer annealing for 10 seconds (for specific temperatures see Table 5 adherent cells per chamber) on a four chamber slide  and incubated. Afterwards the cells were washed three times  and fixated with acetone. Endogenous peroxidase was blocked with for ten minutes  and the cells were washed again three times. The antibody ligands were blocked with 5% goat serum  for one hour. The monoclonal antibody (anti-MAGE 57B) was diluted 1:10 and incubated with the fixed cells for one hour. The cells were washed three times again. Staining was performed with a secondary antibody (radish peroxidase) with the Envision™/HRP-System  according to the manufacturer's instructions and additional counterstaining was done with hematoxylin.For immunocytochemistry, the monoclonal global MAGE-A antibody 57B was used . This monoclonal antibody binds to a common epitope of MAGE-A antigens and facilitates simultaneous detection. The cells were seeded . This should cause no bias regarding the amplification in qrt-PCR.Some splicing variants of MAGE-A2 and MAGE-A4 were found. They can be seen in figure For further analysis, immunocytochemical staining and hematoxylin counterstaining of cultured cells was performed Figures , 10, 11.In rt-PCR all MAGE-A antigens could be amplified. This cell line showed a splicing variant for MAGE-A2. In qrt-PCR these cells expressed like cell line PCI 9-1, MAGE-A antigens -A2, -A3 and -A6 significantly. The expression levels were also comparable to cell line PCI 9-1. MAGE-A2 showed an expression of 11.46 a.u., MAGE-A3 9.94 a.u.  and MAGE-A6 had an expression of 62.79 a.u.The staining revealed a relatively equal pattern with some additional strong (about 15% of the cells) staining Figure . The staIn rt-PCR all MAGE-A antigens could be amplified. This cell line showed also a splicing variant for MAGE-A2. PCI 1-1 also has a similar multi-MAGE-A expression profile in qrt-PCR compared to PCI 13-1. This cell line expresses 3 different MAGE-A antigens . MAGE-A2 and MAGE-A6 were expressed at a moderate level  and MAGE-A6 was expressed at a high level (64.54 a.u.). A MAGE-A4 expression was absent.The cells of this tumour cell line were stained by mAb 57B in a very similar pattern compared to PCI 13-1 Figure . StaininThe rt-PCR all MAGE-A antigens could be amplified and PCI 52 had also a splicing variant for MAGE-A2 and an additional splicing variant for MAGE-A4.In qrt-PCR the expression profile of this cell line showed the most increased MAGE-A antigens. MAGE-A2, MAGE-A3, MAGE-A4, and MAGE-A6 were increased. PCI 52 is also the only cell line examined that showed a significant expression of MAGE-A4 in rt-PCR as well as in qrt-PCR (15.96 a.u.). This is remarkable because it was the only cell line with a MAGE-A4 splicing variant. The expression of MAGE-A2 was the highest of the cell lines examined and reached 15.39 a.u. Expression of MAGE-A6 was at the same level and reached 18.31 a.u. The value of MAGE-A3 expression was the lowest, at 3.49 a.u.In this cell line, a very strong staining was seen. All cells showed a strong cytosolic and sometimes an even stronger cell membrane staining with mAb 57B Figure . The staDespite the amplification of all MAGE-A antigens in rt-PCR PCI 68-1 only MAGE-A3 was significantly increased in qrt-PCR. But an expression of 2.97 a.u. was weak. In rt-PCR this cell line was the only one with the same splicing variant of MAGE-A2 compared with the reference cell line NHEK.Within these tumour cells, no significant staining could be achieved by mAb 57B Figure . The staIn rt-PCR all MAGE-A antigens could be amplified. This cell line showed the same splicing variant for MAGE-A2 like PCI 13-1, PCI1-1 and PCI 52.This cell line also expresses a group of MAGE-A antigens. As with PCI 1-1, this cell line highly expresses MAGE-A2, MAGE-A3 and MAGE-A6 in qrt-PCR. MAGE-A2 showed an expression of 10.67 a.u., MAGE-A3 of 8.88 a.u. and MAGE-A6 of 85.86 a.u. .In this cell line, there was a significant staining by mAb 57B Figure . Nearly The single qrt-PCR measurements listed in Table The results are in accordance with reports in literature on the multiple expression of MAGE-A antigens in tumour cells that were harvested from primary solid cancers,12. It wA higher expression of MAGE-A3 reduces the responsiveness of tumour cells to doxorubicin. This anA contradictory result is achieved by an elevated expression of MAGE-A4. Its gene product binds to the tumour protein gankyrin, which has a destabilising effect on retinoblastoma protein (pRb). Gankyrin binding to pRb results in its hyperphosphorylation, release of the E2F transcription factors, activation of DNA synthesis genes and unscheduled entry into the cell cycle,22. FurtIn contrast to other investigators, no incrIn conclusion, oral and pharyngeal squamous cell carcinoma cell lines were found to express MAGE-A antigens. The MAGE-A antigens are expressed in groups of different antigens. This finding is supported by the literature,12. The The authors declare that they have no competing interests.UMR: study design, drafting of manuscript; AD: laboratory studies; TR: manuscript revision; JK: manuscript revision; TR: manuscript revision; OD: study design, manuscript revisionThe authors would like to thank Dr. Michael Kochel and Professor Alexander C Kübler for their assistance revising the manuscript.


However, the replacement of the knocked down GHRH expression by exogenous GHRH (1–29)NH2 re-established the proliferation of the silenced cancer cell lines. Furthermore, the proliferation rate of untransfected cancer cell lines could be stimulated by GHRH (1–29)NH2 and inhibited by GHRH antagonists MZ-5-156, MZ-4-71 and JMR-132. These results extend previous findings on the critical function of GHRH in tumorigenesis and support the role of GHRH as a tumour growth factor.Splice Variant 1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor, found in a wide range of human cancers and established human cancer cell lines, is a functional receptor with ligand-dependent and independent activity. In the present study, we demonstrated by western blots the presence of the SV1 of GHRH receptor and the production of GHRH in MDA-MB-468, MDA-MB-435S and T47D human breast cancer cell lines, LNCaP prostate cancer cell line as well as in NCI H838 non-small cell lung carcinoma. We have also shown that GHRH produced in the conditioned media of these cell lines is biologically active. We then inhibited the intrinsic production of GHRH in these cancer cell lines using si-RNA, specially designed for human GHRH. The knocking down of the GHRH gene expression suppressed the proliferation of T47D, MDA-MB-435S, MDA-MB-468 breast cancer, LNCaP prostate cancer and NCI H838 non-SCLC cell lines  Growth hormone-releasing hormone is secreted by the hypothalamus and upon binding to the specific GHRH-Receptors (GHRH-R) on somatotrophs regulates the release of Growth Hormone (GH) from the anterior pituitary gland. In turn, GH stimulates the production of insulin-like growth factor-I (IGF-I), which functions as a cell cycle stimulator (Dysfunction of cell differentiation and cell cycle regulation define and promote carcinogenesis. Growth hormone-releasing hormone (GHRH) was first isolated from human pancreatic tumours and only subsequently identified in human and animal hypothalami (reviewed in imulator .Growth hormone-releasing hormone-receptors (GHRH-R) is a class II G protein-coupled receptor and contains seven transmembrane domains . Growth Splice variant 1 of GHRH receptor is a functional receptor, which differs from the pGHRH-R only in the N-terminal extracellular domains. The first 89 amino acids of the pGHRH-R are replaced in SV-1 receptor by a different 25-amino acid sequence , prostate cancer (LNCaP) and non-SCLC (NCI H838) cell lines. We detected the GHRH produced in the conditioned medium of these cell lines. To further elucidate the role of GHRH in carcinogenesis, we knocked down the gene expression of GHRH. In addition, we examined the effect of GHRH and GHRH antagonists MZ-4-71, MZ-5-156 and JMR-132 at two dose levels on the proliferation rate of the cited cancer cell lines. Our results demonstrate the critical function of GHRH and its receptors in tumorigenesis.0, DArg2, Phe (4-C)6, Ala8, Har9, Tyr (Me)10, His11, Abu15, His20, Nle27, D-Arg28, Har29] human GHRH (1–29)NH2, MZ-5-156  hGHRH (1–28) Agm, where Abu is a-aminobutyric acid, Agm is agmatine, Har is homoarginine, Nle is norleucine, PhAc is phenylacetyl and Tyr(Me) is O-methyltyrosine were synthesized in our laboratory by solid phase methods . Growth hormone-releasing hormone (1–29)NH2 and GHRH antagonists were dissolved in 0.1% DMSO and diluted with incubation media.Growth hormone-releasing hormone antagonists JMR-132 -Tyr)-GHRH-]) (human)  as the labelled peptide and a rabbit antibody against GHRH (1–44)NH2 . This antibody crossreacts 100% with human GHRH (1–44)NH2 and 30% with human GHRH (1–29)NH2. Growth hormone-releasing hormone (1–44)NH2  was used as a standard. The range was 1–128 pg per tube.Tumour cells  without serum and 5 μl lipofectamine 2000 were diluted in an equal volume of Opti-MEM I. After incubation for 5 min at room temperature, the diluted oligomers were combined with the lipofectamine 2000 and incubated for 30 min also in room temperature. The oligomer-lipofectamine complexes were added to each well, which contained 2 ml of medium without antibiotics. The medium was changed 8 h after transfection. The cells were incubated at 37°C in an atmosphere of 5% CO2 and 95% air for 48–72 h after transfection and then assayed by western blot for gene knockdown.Small interfering (si) RNA designed specially for the inhibition of the human GHRH was used to knock down the GHRH gene expression of the T47D, MDA-MB-435s, MDA-MB-468 breast cancer cell lines, LNCaP prostate cancer cell line and NCI H838 non-SCLC cell line. A pool of three oligonucleotides especially designed for the inhibition of the human GHRH  and cationic liposomes  were used to knock down the gene expression of the GHRH. The sequence of the sense strand (a) was GGUAUGCAGAUGCCAUCUUTT and the mRNA location is 92. The sequence of the sense strand (b) was CCAGUUAAUCCUCUCAUUUTT and its mRNA location is 376. The sequence of the sense strand (c) was CCAGUUAAUCCUCUCAUUUTT and its mRNA location is 434. RNA that does not lead to any specific degradation of any known cellular mRNA  was used as control. The sequence of the sense strand was UUCUCCGAACGUGUCACGU?. One day before the transfection, the cells were plated in six-well plates in 2 ml of growth media without antibiotics to be 40% confluent on the day of transfection. For the transfection 40–60 nThe rate of the cell proliferation was calculated by seeding 10 000 cells in six-well plates and after an incubation for 4 days counting them under light microscope using the trypan blue assay.t-test (two-tailed). P-values shown are against the control group.These data are expressed as the mean±s.e.m. Statistical evaluation of the results was performed by the Student's A band of 45 KDa which reflects the production of pre-pro GHRH (−1), NCI H838, MDA-MB-435s , LNCaP, and MDA-MB-46 ng ml−1  cell lines after 24, 48 and 72 h respectively as shown in The concentration of the GHRH in samples from culture medium was measured by RIA. Significant amounts of GHRH were detected in the medium from T47D NH2 the proliferation rate of the cells was strongly stimulated by 16, 132, 87, 97, 119% respectively. The increase on the proliferation rate in response to 1 μM GHRH was even greater, being 32, 143, 112, 134, 140% respectively. The GHRH did not affect the proliferation rate of MCF-7 breast cancer cell line, which was also transfected with siRNA for GHRH. The results are shown in When knocked down T47D, MDA-MB-486, MDA-MB-435s breast, LNCaP prostate and NCIH-838 lung cancer cell lines were exposed to 0.1 in vitro were exposed to two concentrations of GHRH(1–29)NH2 and GHRH antagonists MZ-5-156, JMR-132 and MZ-4-71. At the dose of 1 μM GHRH (1–29)NH2 did not appreciably influence the proliferation rate of the cells, producing a change only of 1–5%. However GHRH (1–29)NH2 at 0.1 μM concentration stimulated of the proliferation rate of the T47D, MDA-MB-468, MDA-MB-435s, LNCaP and NCI H838 cells by 15, 17, 14, 14, 16% respectively. The proliferation of MCF-7 cells (negative control) was not affected by GHRH at 0.1 and 1 μM concentrations. The results are shown in T47D, MDA-MB-486, MDA-MB-435s breast, LNCaP prostate and NCIH-838 lung cancer cell lines cultured μM decreased the proliferation of T47D, MDA-MB-468, MDA-MB-435s, LNCaP and NCI H838 cancer cell lines by 37, 29, 32, 29, 28% respectively. The results are presented in μM MZ-4-71 produced a somewhat greater inhibition of proliferation of T47D, MDA-MB-468, MDA-MB-435s, LNCaP and NCI H838, the decreases being 39, 35, 36, 35 and 33% respectively. MZ-4-71 did not affect the proliferation of MCF-7 cells (negative control) (Growth hormone-releasing hormone antagonist MZ-4-71 at the dose of 0.1 control) .μM reduced the proliferation of T47D, MDA-MB-468, MDA-MB-435s, LNCaP and NCI H838 cancer cell lines by 30–35, 28–34, 33–40, 30–37, and 30–36% respectively. Higher doses of the GHRH antagonist again caused a greater inhibition, indicated by the second set of numbers. The results are illustrated in Similar inhibitory effects on proliferation were obtained with the other two antagonists. Thus GHRH antagonist MZ-5-156 at doses of 0.1 and 1 μM. Higher doses of JMR-132 produced a greater inhibition, indicated by the second set of numbers. The results are shown in The proliferation of T47Ds, MDA-MB-468, MDA-MB-435, LNCaP and NCI H838 cancer cell line was also inhibited by 31–37%, 26–31%, 31–38%, 34–41%, 37–42% respectively after exposure to GHRH antagonist JMR-132 at doses of 0.1 and 1 Growth hormone-releasing hormone and the major SV1 of the full length GHRH receptor are expressed in surgical specimens of diverse human cancers as well as in a various human cancer cell lines (reviewed in 2 by doing western blots for synthetic GHRH (1–44) NH2, but no corresponding signal was detected. Biologically active GHRH was detected by RIA in the conditioned medium of all the cancer cell lines, except for MCF-7.The precise role of the production of GHRH in the process of tumorigenesis and tumour progression has not been investigated previously. One way to elucidate the role of GHRH in the pathogenesis of cancer is to inhibit its gene expression. In the present study, we first examined the expression of pre-pro GHRH by western blot in human cancer cell lines. The band that reflects the production of the pre-pro GHRH appeared to have a molecular size of 45 kDa on the blots. The facts that this band disappeared in the knocked down cells and could not be detected in the negative control (MCF-7), together with the stimulation of the knocked down cells by exogenous GHRH, indicate that this band represents a precursor of GHRH , possibly because the ligand independent activity of the SV1 continued to enhance the proliferation rate of the T47D cells NHμM GHRH stimulated the proliferation rate of the cancer cell lines by 15–17%. However, at a dose of 1 μM GHRH did not influence significantly the proliferation rate of cell lines. Thus, because of the possible presence of GHRH or other related peptides in the medium, as well as of GHRH secreted by the cells, it is likely that the corresponding signalling pathways were saturated after exposure to 1 μM GHRH.We also investigated the effect of GHRH and GHRH antagonists at two concentrations in MCF-7, MDA-MB-468, T47D, MDA-MB-435s, LNCaP and NCI H838 cancer cell lines. The proliferation rate of the MCF-7 breast cancer cell line was not influenced by the presence of the GHRH or its antagonists, since MCF-7 does not express specific receptors for GHRH. At the dose of 0.1 in vitro to two concentrations of GHRH antagonists MZ-4-71, MZ-5-156 and JMR-132. At concentrations of 0.1 and 1 μM, the proliferation rate of these cells was decreased by 26–37% and 31–42% respectively.These breast, lung and prostate cancer cell lines were also exposed The present study demonstrates the tumorigenic effect of GHRH in the human experimental tumour cell lines representative of leading cancers. Our work supports the concept that GHRH functions as growth factor in human cancers.


The initiation of a cultured human colon carcinoma line on a feeder layer of confluent fibroblasts is described. Attempts to initiate cultures without fibroblast feeder layers were not successful. Two sub-lines (designated HCT C and HCT C Col) were isolated and weaned from cells growing on the surface of the feeder layer. The sub-lines had different morphologies, secreted different levels of carcinoembryonic antigen (CEA) into the medium of confluent cultures and had slightly different karyotypes. Both sub-lines grew in semi-solid medium and formed xenografts when injected s.c. into athymic nude mice. Analysis of radioiodinated cell membrane components indicated small, but significant differences between the sub-lines.


The ultrastructure and in vitro growth properties of 5 tumorigenic mouse submandibular-gland epithelial cell lines were studied. In all lines, in vitro acinus formation occurred, and well differentiated epithelial cells showing epithelial microvilli and desmosomes and cytoplasmic tonofilaments were present. None of the cells showed specific ultratructural features of the normal differentiated submandibular-gland ducts. All the lines formed colonies in semi-solid agar and on confluent monolayers of BALB/c 3T3 cells, and all lacked density-dependent inhibition of growth, as demonstrated by a random distribution of [3H]TdR labelling throughout growing colonies. These 3 growth properties appear to be reliable in vitro markers for epithelial neoplastic transformation in this system, but colony-forming efficiency in agar is lower than that reported for many transformed mesenchymal cells.


Overexpression of urokinase-type plasminogen activator and its receptor correlates with metastatic capacity in breast cancer. In this study we show that the urokinase/urokinase receptor-overexpressing, metastatic human breast cancer cell line MDA-MB-231 (1) bound significantly more cell-surface plasminogen in a lysine-dependent manner and (2) was capable of generating large amounts of plasmin compared with the non-metastatic cell lines MCF-7 and T-47D. In addition, distinct plasminogen binding proteins were detected in the plasma membranes of the cell lines, suggesting heterogeneity of binding proteins. Plasminogen binding was analysed using a combination of dual-colour fluorescence flow cytometry and ligand histochemistry , and fluorimetry . Apart from revealing the greater plasminogen binding capacity of MDA-MB-231 cells, flow cytometry and histochemistry also revealed that, in all three cell lines, non-viable or permeabilized cells bound significantly more plasminogen in a lysine-dependent manner than viable or non-permeabilized cells. Viable MDA-MB-231 cells bound plasminogen with moderate affinity and high capacity . Our results indicate that differences in cell surface-specific plasminogen binding capacity between cell lines may not be detectable with binding techniques that cannot distinguish between viable and non-viable cells.


To obtain information regarding the growth-inhibitory effect of 1,25-dihydroxyvitamin D3 and its non-calcaemic analogue 22-oxa-1,25-dihydroxyvitamin D3 on pancreatic cancer cell lines, differences in the effects of G1-phase cell cycle-regulating factors were studied in vitamin D-responsive and non-responsive cell lines. Levels of expression of cyclins , cyclin-dependent kinases (2 and 4) and cyclin-dependent kinase inhibitors (p21 and p27) were analysed by Western blotting after treatment with these compounds. In the responsive cells , our observations were: (1) marked up-regulation of p21 and p27 after 24 h treatment with 10(-7) mol l(-1) 1,25-dihydroxyvitamin D3 and 22-oxa-1,25-dihydroxyvitamin D3; and (2) marked down-regulation of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors after 7 days' treatment. In non-responsive cells (Hs 766T and Capan-1), no such changes were observed. In conclusion, vitamin D analogues up-regulate p21 and p27 as an early event, which in turn could block the G1/S transition and induce growth inhibition in responsive cells.


The establishment in culture and characterization of 4 human neuroblastoma (NB) cell lines and 1 human ganglioneuroblastoma cell line are described. Each cell line fulfilled at least 2 of 4 criteria for malignant or transformed cells: viz., subcultured more than 70 times, high saturation density with absence of contact inhibition, population-doubling time within a range of 10-40 h, and tumour formation in nu/nu mice. Each cell line also fulfilled at least 2 of 3 criteria for neuroblastoma cells: viz., humoral and cell-mediated immune reactivity toward NB-associated cell-surface antigen, intracellular storage and extra-cellular secretion of catecholamines, and characteristic neuroblast and ganglion-cell morphology.


But we found that the routinely used breast cancer cell lines were mostly derived from Caucasians or African-Americans. There were few standard models to study the pathogenic mechanism at molecular level and cell signaling pathway of breast cancer for Asian patients. It is quite necessary to establish new breast cancer cell lines from xanthoderm to study the pathogenic mechanism and therapeutic methods.Breast cancer is a major malignancy affecting females worldwide. It is the most common cause of death from cancer in women. Cell lines are widely used in laboratory research and particularly as in vitro, and they were ER-, PR- and C-erbB-2-positive. Their chromosomes showed high hyperdiploidy and complex rearrangements, and they displayed aggressive tumorigencity in tumorigenesis test.Three new breast cancer cell lines, designated BC-019, BC-020 and BC-021, were successfully established and characterized from breast invasive ductal carcinoma tissues of three Chinese female patients. These new cell lines growing as adherent monolayer with characteristic epithelial morphology could be maintained continuously The three newly established breast cancer cell lines from Chinese patients were tested for a number of, and the results indicate that the cell lines were in good quality and could be served as new cell models in breast cancer study. Apart from MCF-7, the most commonly used breast cancer cell line in the world derived from a pleural effusion in the Michigan Cancer Foundation [in vitro studies using breast cancer cells are based on a few well-characterized cell lines, such as MCF-7, ZR-75-30, T-47D and MDA-MB-231, which have been established in culture for over 30 years[Breast cancer is a major malignancy affecting females worldwide. It is the most common cause of death from cancer in women -3. Cell undation , a numbeundation . Moreove 30 years,7. FurthWe also found that these routinely used breast cancer cell lines were mostly derived from Caucasians or African-Americans, such as MCF-7, ZR-75-30, T-47D and MDA-MB-468, but very rarely cell lines were derived from xanthoderm, particularly from Asians. In American Type Culture Collection (ATCC), there were more than 70 breast cancer cell lines, but only one of them was derived from Asian and one from East Indian . And there were few standard models to study the pathogenic mechanism at molecular level and cell signaling pathway of breast cancer for Asian patients. Even in some studies on tumor pathologic features in Chinese patients, only the cell lines derived from Caucasians or African-Americans were used, so there were few experimental systems to study the pathogenic mechanism of breast cancer for Asian patients ,11. So iIn this report, we established and characterized three new breast cancer cell lines BC-019, BC-020 and BC-021 from three Chinese patients. These cell lines which are positive for estrogen receptor (ER), progesterone receptor (PR) and C-erbB-2, could provide us with new experimental materials to study the pathogenic mechanism and to screen for new therapeutic reagents against breast cancer.3. The small tissue fragments were placed in three 25-cm2 culture flasks and cultured at 37°C in a humidified incubator containing 5% carbon dioxide in Eagle's minimum essential medium (MEM) with non-essential amino acids supplemented with 10% fetal bovine serum , penicillin (100 U/mL), and streptomycin (100 μg/mL). Half of the medium was replaced every 3–4 days. Sparsely outgrowth colonies of polygonal epithelial cells from the tissue pieces were observed within 5 days. On the 12th day after initiation of the primary culture, the epithelial cells were transferred selectively to fresh culture flasks. Cells were harvested after treatment with 0.25% trypsin and 0.02% EDTA solution and subcultured with a 1:3 split ratio. During the subsequent period of continuous propagation by subculture, the cells were sampled at intervals, resuspended in a freezing medium , and stored in liquid nitrogen every ten passages. After quickly thawing at 37°C, the frozen cells could be propagated in culture without noticeable change in growth and morphology. The cell lines were named BC (breast cancer)-019, BC-020, BC-021 and deposited in China Center for Type Culture Collection (CCTCC) with designation number CCTCC-GDC0198, CCTCC-GDC0199 and CCTCC-GDC0200, respectively.Three breast cancer cell lines were established from three Chinese female breast cancer patients who underwent mammary gland excision in Zhongnan Hospital of Wuhan University. The study was approved by the hospital ethics committee and patient's informed consent was obtained. The tumor specimen from primary mammary glands was removed at the excision operation. Standard blocks were then taken for pathological examination Table . Tissue 3 cells per well and 7 plates per cell line. One plate of each cell line was assayed every 24 hours. The absorbance was measured in a microtiter plate reader at 570 nm as the test wavelength and 690 nm as the reference wavelength. The number of cells was calculated by the result of MTT compared with the cell number at the first day. The growth curves were plotted and the population doubling time of these 3 cell lines were calculated during the exponential growth phase of the cells.BC-019, BC-020 and BC-021 cells at the same passage 21 (P21) were used to plot the growth curve by using the 3--2,5-diphenyl tetrazolium bromide (MTT) method -16. The Detection of the contaminants in cell lines followed the standard procedures of American Type Culture Collection (ATCC). The celThe cell lines were examined for mycoplasma using both direct and indirect assays . In the 5 cells/well. After 24 hours, 0.2 ml to 0.5 ml of each test cell line were added to the wells of Vero cells, 2 wells/cell line. 0.5 ml supernatant of B6yH4 cells was added to 2 wells as positive control and 1.0 ml culture medium was added to 2 wells as negative control. The 12-well microplates were incubated at 37°C in a humidified incubator containing 5% carbon dioxide. After 6 days, cells were washed with PBS twice, fixed with fixative (methanol: acetic acid = 3:1) for 15 minutes twice and stained with Hoechst 33258 in the dark for 45 minutes. The cover slips were then washed with distilled water three times, dried in the dark at room temperature, placed in a drop of mounting medium (10% glycerin in PBS) on glass slides, and observed under fluorescence microscope with 330–380 nm excitation filter. Extracellular fluorescence indicated mycolpasma contamination.In the indirect assay, mycoplasma DNA was examined by staining the cells with Hoechst 33258. Vero cell line was used as the indicator cell in the DNA staining procedure. Vero cells were inoculated on a cover slip in 12-well microplates with 1.0 × 10To verify that the three breast cancer cell lines were derived from humans without cross-contamination, we compared the profiles of lactate dehydrogenase (LD) and malate dehydrogenase (MD) from the 3 breast cancer cell lines. HeLa cell line and mouse fibroblast L929 cell line were used as standard references. LD and MD were analyzed using AuthentiKit™  following the manufacturer instructions.ER, PR and C-erbB-2 had been used as marker for the detection of breast cancer in clinical diagnosis -21. The 7 cells/ml in PBS. The same procedure was done to MRC-5 cell line and MCF-7 cell line, the negative and positive controls. MRC-5 and MCF-7 were cultured in MEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). 10 μg/mL bovine insulin was added to the medium for MCF-7. 4 weeks old nude mice were injected subcutaneously with 0.2 ml cell suspension at left groin. Each cell line was used to inject 10 mice. All the mice were examined within 8 weeks [Large-scale cultures were prepared at P21 for BC-019, P22 for BC-020 and P20 for BC-021. Cells were washed twice with MEM without serum and antibiotics and adjusted to 5~10 × 10 8 weeks .BC-019, BC-020 and BC-021 cells at passage 23 were used for chromosome analysis. Metaphase cells were collected after 3-hour colcemid (0.2 μg/mL) exposure and incubation in 0.075 M KCl solution at 37°C for 40 min. After fixing with a mixture of methanol and glacial acetic acid , cell suspensions were spread onto cold slides. The slides were stained with 3% Giemsa for morphological examination and chromosomes analysis . G-bandi2 culture flasks and formed sparse colonies within 5 days of primary culture. The first successful subculture of 3 cell lines was performed on about 14th day and the second one 3 days later. 5 passages later, cells were subcultured at a split ratio of 1:3 every 3–4 days. The breast cancer cells grew as adherent monolayer with characteristic epithelial morphological features  may arise from fracture of the chromosomes.in vivo models for studying the biology of breast cancer.The three breast cancer cell lines had been proved to have severe tumorigenicity when injected into nude mice, and their tumorigenicity was higher than that of MCF-7 cell line. Metastasis could be occasionally observed in nude mice injected with BC-019 cells and BC-021 cells, but not in mice injected with BC-020 cells and MCF-7 cells. It was well known that breast cancer cell lines tend to be less tumorigenic and less metastatic than other cell lines derived from lung, renal and colon carcinoma when injected subcutaneously in nude mice. InteresMost of the previously established breast cancer cell lines used in research did not derive from primary breast tumors, but from tumor metastases, especially ascites or pleural effusions. While primary cultures are indispensable for direct comparison to the tissue of origin, immortalized cell lines are necessary for long-term studies ,32. TherMoreover, most of the published breast cancer cell lines were derived from Caucasians, which limited the study of pathogenic mechanism of breast cancer among Asian patients. To resolve this problem, we established three breast cancer cell lines which were derived from Chinese and provided a research resource and tool to compare the difference of cells and tumors between the two races.We established and characterized three new human breast cancer cell lines designating BC-019, BC-020 and BC-021 from three Chinese patients. These three cell lines would provide us new experimental models to study the pathogenic mechanism, to investigate the biological behavior and to test new therapeutic reagents for breast cancer.ATCC: American Type Culture Collection; CCTCC: China Center for Type Culture Collection.The authors declare that they have no competing interests.CS, MG, DL, LM and LH carried out all experimental work. CS and CZ conceived ideas, analyzed data and wrote manuscript. JC contributed in collecting tissues of the patients and pathology assay. CS, LH and CZ analyzed results and evaluated manuscript. All authors read and approved the final manuscript.


Gallium-67 is a radionuclide that accumulates in haematological malignancies and is used for diagnostic purposes. Uptake of 67Ga into the cell occurs via the transferrin receptor, which is differentially expressed during the various cell cycle phases. With the aim of selectively increasing 67Ga uptake, we studied whether the transferrin receptor (TfR) expression could be modulated in the U937 and U715 lymphoma cell lines by cytostatic drugs inducing cell cycle phase accumulation. We tested clinically relevant drugs such as 1-beta-D-arabinofuranosylcytosine (Ara-C), hydroxyurea and methotrexate. Cytotoxicity was determined by testing the clonogenic capacity of the lymphoma cell lines. All three drugs induced an increase in S-phase content, TfR expression and 67Ga uptake in U937 and U715 single cells. The combinations of drugs and 67Ga resulted in an additive effect on the clonogenic capacity. In U937 spheroids, cultured by the fibrin clot technique, we found an accumulation in the S-phase too as well as an increase of the transferrin receptor expression after Ara-C preincubation. As in single cells 67Ga uptake was increased without synergistic effects on the clonogenic capacity. In conclusion, priming with drugs induces increased transferrin receptor expression and 67Ga uptake. Inhibition of clonogenic capacity was additive rather than synergistic.


The growth inhibitory effect was associated with a dose-dependent inhibition of DNA synthesis, cell cycle progression and α fetoprotein expression. © 2001 Cancer Research Campaign http://www.bjcancer.comPeroxisome proliferator-activated receptor γ (PPARγ) ligands have been implicated in the growth inhibition and differentiation of certain human cancers with diverse tissue origin. In this study, expression of PPARγ in human hepatocellular carcinoma (HCC) and the effect of PPARγ ligands on HCC cells were investigated in vitro using Hep G2, HuH-7, KYN-1 and KYN-2 cell lines. All cell lines were found to express functionally active PPARγ and a marked growth inhibition was induced by thiazolidinedione ligands troglitazone, and pioglitazone as well as with its natural ligand 15-deoxy-Δ


All-trans retinoic acid (ATRA) has been previously shown to inhibit the proliferation of some human ovarian carcinoma cell lines, and this inhibition was accompanied by cellular changes that were indicative of differentiation . In this work, a pretreatment of these adenocarcinoma cells with ATRA, for their respective doubling time, enhanced cisplatin (CDDP) cytotoxicity in the cell ines that were sensitive to its antiproliferative effect, but not in the ATRA-resistant ones. Results were assessed using median effect analysis in two ATRA-sensitive cell lines (OVCCR1 and NIHOVCAR3 cells) and in one ATRA-insensitive cell line (IGROV1 cells). Synergy between these two agents was observed only in cells sensitive to ATRA, regardless of their relative sensitivity to CDDP. Potential mechanisms for this synergy were investigated. ATRA did not increase the cellular platinum content, did not decrease the cellular glutathione and had no influence on the metallothionein IIA mRNA levels in NIHOVCAR3 cells. Moreover, the protein kinase C (PKC) activity was modulated by this differentiating agent in all cell lines tested, indicating that this activity was not directly involved in this potentiation. However, an ATRA inhibition of glutathione-S-transferase activity associated with an increase in the total DNA adducts formation could explain the potentiation of the CDDP cytotoxicity observed in NIHOVCAR3 cells. Finally, the ATRA modulation of the epidermal growth factor (EGF) receptor mRNA level could also be implicated in this synergy.


RAP-5, a monoclonal antibody raised against a p21ras peptide, has been claimed to show immunohistochemical localisation of cells with infiltrative properties in human tumours. We confirmed that this antibody reveals pronounced cellular heterogeneity in human colonic neoplasms but could find no obvious relationship to infiltrative activity. RAP-5 bound to many different cell types, neoplastic and normal. In order to clarify the specificities of RAP-5 we applied it to two cell lines: nontumorigenic hamster fibroblasts in which ras expression is barely detectable, and a vigorously tumorigenic line derived from these fibroblasts by insertion of the human mutated Ha-ras oncogene in a high expression vector. Another antibody to p21ras, Y13-259, clearly distinguished between these cell lines both on immunoblots and immunocytochemically, but RAP-5 did not. Rather, it bound to proteins of a variety of molecular weights in both cell lines. The results show that RAP-5 is unlikely to be a useful reagent for detection of ras associated proteins in human tissues.


The monoclonal antibody (MAb) A7 has been used to treat patients with colorectal or pancreatic carcinoma with encouraging results. We therefore determined if MAb A7 would also react with gastric carcinoma cell lines. MAb A7 reacted with seven of eight gastric carcinoma cell lines tested. The intensity of the reaction, measured by flow cytometry, was equal to that of WiDr (colon) and HPC-YS (pancreas) cell lines. In nude mice bearing xenografts of the MAb A7-reactive gastric cancer line MKN45, the percentage injected dose of MAb A7 per g of tumour tissue on day 7 was 9.79; this value was 77% of that on day 1. The in vivo tumour-to-blood ratio of MAb A7 was 2.77 on day 7. Therefore, MAb A7 has long-term retention at binding sites as well as a high probability, high intensity and high specificity of reactivity against gastric cancer, which make it an ideal drug carrier for immunotargeted chemotherapy and immunodiagnosis.


The pattern of surface proteins of different types of normal and transformed rat liver cells have been studied in culture by means of lactoperoxidase-catalysed iodination procedures, followed by SDS-gel electrophoresis. The cells examined were primary cultures of epithelial liver cells, long-term cultures of epithelial liver cells, in vitro transformed epithelial liver cell lines and liver tumour-cell lines; mesenchymal cells from liver and skin were also examined. The principal surface proteins of primary cultures of epithelial cells from adult or neonatal rats had components with mol. wts of 140,000-160,000, 100,000 and 40,000-70,000. A band that had the same position as fibronectin from mesenchymal cells was also present and this band, as well as other iodinated components, were less sensitive to trypsin than fibroblastic fibronectin. A similar pattern of iodinated proteins was seen in long-term cultures of epithelial liver cells, with a great reduction in the number and intensity of the bands in the mol. wt region below 100,000. Almost all the in vitro transformed and tumour epithelial cell lines contain a protein with a mol. wt 135,000 as one of the major iodinated bands, and in contrast to the observation in transformed fibroblasts, the fibronectin was retained by most of these transformed cell lines.


L-lysine chlorine6 conjugates with either cationic or anionic charges in two cell lines, human ovarian cancer cells and mouse macrophages. The cationic conjugate after pegylation became less aggregated, consumed less oxygen and had reduced cellular uptake. However, the phototoxicity corrected for cellular uptake increased three- to five-fold. In contrast, the anionic succinylated conjugate on pegylation became more aggregated, consumed similar amounts of oxygen, and had higher cellular uptake. The anionic conjugate showed the highest relative phototoxicity towards both the cell lines (compared to the other three conjugates) and it decreased most towards the macrophages after pegylation. Pegylation reduced the amount of oxygen consumed per chlorine6 molecule when photosensitised cells were illuminated. These in vitro studies suggest that pegylation alters the phototoxicity of PS conjugates depending on the effect produced on the aggregation state.Conjugates between photosensitisers (PS) and charged polymeric carriers are under investigation for photodynamic therapy of cancer and may allow targeting to certain cell types or compartments in tumours. Covalent attachment of polyethylene glycol to macromolecules (pegylation) may alter their pharmacokinetics, cell type targeting, and photophysical properties. Macrophages may take up large amounts of aggregated PS, thus lessening the selectivity for cancer cells in tumours. We investigated the effect of pegylation on the uptake and phototoxicity of poly- L-lysine (pL) may be used as the macromolecular backbone for attaching PS such as chlorine6 (ce6) to some of the ɛ-amino groups of the polyamino-acid backbone, and in addition the unaltered ɛ-amino groups remain available to first attach PEG, and then the overall charge can be made anionic by succinylation  is a novel approach for destruction of malignant or other unwanted tissue. It involves the administration of nontoxic dyes known as photosensitisers (PS) either systemically or topically, followed by illumination of the lesion with visible light  . Our lab−1 penicillin, and 100-μg ml−1 streptomycin, and maintained in an incubator at 37°C in a humidified atmosphere of 5% carbon dioxide.NIH:OVCAR-5 (OVCAR-5) cells were obtained from Dr T Hamilton  and J774. A1 (J774) mouse macrophage-like cells were from ATCC . Cells were grown in RPMI-1640 media containing HEPES, glutamine, 10% heat-inactivated fetal bovine serum, 100 U mle6-ac and pL-ce6-PEG-ac . Fluorescence calibration curves  were constructed for each conjugate so that the fluorescence could be converted into mol ce6 equivalent. Pegylation was confirmed as previously described (μM–110 nM) in RPMI-1640 (containing 10% FCS). One set of dilutions was then centrifuged at 16 000 g for 15 min at 4°C, while the other set was gently agitated. The amount of conjugate in solution in each tube was determined by fluorescence and the fraction aggregated was calculated from the difference between the fluorescence in the supernatant of the centrifuged and that in the agitated samples.This was carried out essentially as previously described for pL-cμM solutions in RPMI-1640 containing 10% FCS) were illuminated by 75-mW cm−2 666 nm light in a 1-cm cuvette. After calibrating the Clarke electrode with pure N2 and pure O2, the conjugate solutions were oxygenated to approximately 500 mmpO2, the cuvette was then sealed and stirred by a Hellma Cuvet-O-Stir  and illumination commenced. The signal from the electrode was captured by LICOX software on a PC via a RS232 interface. The pO2 showed an exponential decline, and fluorescence measurements before and after illumination gave a measure of photobleaching. The experiment was repeated three times by adding fresh aliquots of each conjugate and reoxygenating. The initial rate of oxygen consumption was determined by drawing a tangent to the exponential pO2 decay curve.A LICOX oxygen partial pressure monitor  was used to monitor the consumption of oxygen when the conjugates  to give fluences ranging from 0 to 10 J cm2 at an irradiance of 50 mW cm2. Cells were then incubated with fresh medium for 24 h when the MTT-microculture tetrazolium assay was used to measure viability  is likely to play a role in their photochemical and biological behaviour, the synthetic scheme used ensures that all four conjugates are directly comparable. The substitution ratio in this study was estimated by absorption spectroscopy to be 4 ce6 per chain of 46 lysine residues (8.7%). Two-phase partition in PEG 8000/dextran 500 mixtures showed increased partition coefficient after pegylation for both pairs of charged conjugates . There were no differences in absorption spectra between any of the conjugates whether measured in PBS or in NaOH/SDS (data not shown). In order to attempt to characterise differences in aggregation behaviour that could be visually observed between the various conjugates, experiments were performed in medium containing serum over three logs of dilution. The results are shown in Figures 1A and BM. By contrast, the succinylated conjugate is hardly aggregated at any concentration, but when it is pegylated, the degree of aggregation is increased at all concentrations.Since the substitution ratio . Both the cell lines took up many times more ce6 from the nonpegylated cationic compared to the non-PEG succinylated (7–12 times); however, this difference was much less pronounced in the pegylated conjugates (1.6–1.8 times). For both the cell lines, pegylation reduced the uptake from the cationic conjugate . However, pegylation of the succinylated conjugates gave an opposite effect, increasing the cellular uptake in both the cell lines .The cellular uptakes were compared between cell lines, between charges, and between PEG and non-PEG . The results are shown in 2 consumption per mole of ce6 taken up for all the conjugates in both the cell lines, and in addition reduces the number of molecules of oxygen consumed by each ce6 molecule for all the conjugates in both the cell lines. Note that the number of molecules of O2 consumed per ce6 when loaded into cells is significantly higher (up to 10 times) than the same number found when the conjugates were illuminated in solution for all the conjugates in both the cell lines except for pL-ce6-PEG-succ where it is similar .Pegylation e6-ac conjugate , in the present study no consistent differences were observed between pegylated and nonpegylated conjugates in either cell line (data not shown). Pegylation reduced both the initial rate and the total number of oxygen molecules consumed for both the charges in both the cell lines. The cationic conjugates consumed more oxygen in J774 cells than OVCAR cells, while the opposite was true for the anionic conjugates that consumed more oxygen in OVCAR cells than J774 cells. The drop in oxygen consumption was much steeper after pegylation for the anionic conjugates than for the cationic conjugates. However, the changes in phototoxic efficacy did not seem to correlate with either the rate or amount of oxygen consumption inside cells as shown in in vivo and this is also likely to apply to macromolecular–PS conjugates (Pegylation of the cationic conjugate led to increased killing of OVCAR cells, but slightly less killing of J774 cells compared to the non-PEG cationic conjugate. However, pegylation of the succinylated conjugate decreased the killing for OVCAR cells and decreased it even further for macrophages . When thnjugates .In conclusion, pegylation of a cationic pL–PS conjugate reduced its tendency to aggregate, decreased its cellular uptake, decreased the oxygen consumption both in solution and when loaded into cells, and increased the relative phototoxicity. These results were similar to those previously found with pegylation of the neutral acetylated conjugate in our previous study (


Permanent cell lines (UCT-Mel 1 through 7) were established from biopsies of metastatic tissue taken from seven patients with malignant melanoma. Cells from these lines were all aneuploid and all grew as non-contact-inhibited, adherent monolayers. All of the lines, with the remarkable exception of UCT-Mel 6, formed tumours in nude mice, expressed the melanoma M-18 antigen and synthesized plasminogen activators exclusively of the tissue-type. UCT-Mel 6 cells were non tumourigenic, they lacked the M-18 antigen and they synthesized plasminogen activators exclusively of the urokinase type. UCT-Mel 1 and UCT-Mel 2 formed pigment in vitro and both of these lines showed an increase in pigment content and tyrosinase synthesis with increasing cell density. The rate of plasminogen activator released by UCT-Mel 1 and UCT-Mel 3 declined strikingly as the cells became confluent. Assuming that proteolytic activity is required for cell migration in vivo; that tyrosinase synthesis reflects expression of the differentiated phenotype and that melanoma cells retain some of the characteristics of neural crest cells, we suggest that the effects of confluence and close cell-cell contact provide a useful experimental counterpart for the study of normal neural crest all behaviour that is characterized by an inverse relationship between migration and a protease secretion on the one hand and pigmentation on the other.


Mammary epithelial cells (MECs) were isolated and cultured from mammary glands of healthy women undergoing reduction mammoplasty. Normal MECs were infected with the transforming hybrid virus adeno-5/SV40. Two transformed epithelial cell lines, M1 and M2, were obtained, characterised phenotypically and studied for the production of and the response to cytokines and growth regulators. In both cell lines, expression of the SV40 large T antigen was associated with loss of interleukin 6 (IL-6) production and responsiveness as well as with down-regulation of IL-8 and transforming growth factor (TGF)-alpha production. Both M1 and M2 cell lines were capable of forming colonies in semisolid media, but upon injection into severe combined immunodeficient (SCID) mice only M2 cells were tumorigenic. DNA synthesis in M1 cells was partially inhibited by serum or TNF-alpha and weakly stimulated by hydrocortisone (HC) and IL-8. In contrast, M2 cells were totally unresponsive to a variety of growth regulators. Both lines overexpressed the p53 protein at levels about 20-fold higher than those observed in primary MEC cultures, but no mutations of the p53 gene could be detected. The date confirm the view that the expression in human mammary cells of different oncogenes - including the SV40 T antigen - is frequently associated with alterations of cytokine production and responsiveness.


The potent mitogenic activity of insulin-like growth factor I (IGF-I) on breast epithelium is inhibited by retinoic acid in oestrogen receptor-positive (ER+) breast cancer cell lines. We studied and compared the effects of N-(4-hydroxyphenyl)-retinamide (4-HPR) in terms of growth inhibition and modulation of the IGF-I system in ER+ (MCF-7) and oestrogen receptor-negative (ER-) (MDA-MB231) breast cancer cell lines. Treatment with 1-10 microM 4-HPR for up to 96 h induced a dose- and time-dependent inhibition of proliferation in both breast cancer cell lines. Induction of apoptosis was much more evident in MCF-7 than in MDA-MB231 cells (30-40% compared with 0-5% respectively at 5 microM for 48 h). Exogenous human recombinant IGF-I (hr-IGF-I)-stimulated cell proliferation was abolished by 1 microM 4-HPR in MCF-7 cells. Immunoreactive IGF-I-like protein concentration in conditioned medium was reduced by 38% in MCF-7 and by 90% in MDA-MB231 cell lines following treatment for 48 h with 5 microM 4-HPR. Western ligand blot analysis showed a reduction of IGF-binding protein 4 (BP4) and BP5 by 67% and 87%, respectively, in MCF-7, whereas IGF-BP4 and -BP1 were reduced by approximately 20% in MDA-MB231 cells. Exposure to 5 microM 4-HPR for 48 h inhibited [125I]IGF-I binding and Scatchard analysis revealed a decrease of more than 50% in maximum binding capacity (Bmax) and a reduced receptor number/cell in both cancer cell lines. Steady-state type I IGF-receptor mRNA levels were reduced by approximately 30% in both tumour cell lines. We conclude that 4-HPR induces a significant down-regulation of the IGF-I system in both ER+ (MCF-7) and ER- (MDA-MB231) breast cancer cell lines. These findings suggest that, in our model, interference with the ER signalling pathway is not the only mechanism of breast cancer growth inhibition by 4-HPR.


The level of expression of the multidrug resistance-associated protein (MRP1) in a panel of human ovarian carcinoma cell lines and their variants with acquired cisplatin resistance was determined using Western blotting. No overexpression of MRP1 was detected in any of the cell lines. In addition, we have transfected the MRP1 gene into an intrinsically cisplatin-resistant cell line SKOV3, previously shown to have elevated levels of glutathione (GSH). The MRP1-transfected line SKOV3-S2 was shown to be cross-resistant to doxorubicin, vincristine and etoposide but not to paclitaxel, vinblastine and platinum agents, such as cisplatin, JM216 [bis-acetato-ammine-dichloro-cyclohexylamine platinum (IV)] and AMD473 [cis-ammine dichloro (2-methyl-pyridine) platinum (II)]. No cross-resistance to any of the platinum agents was observed in a MRP1-overexpressing human lung cancer cell line with acquired doxorubicin resistance. Reduction of GSH levels (80-90%) by buthionine sulphoximine (BSO) produced significant potentiation in cisplatin sensitivity in the parental SKOV3, the vector-alone control SKOV3-puro and the MRP1-transfected line SKOV3-S2. The degree of sensitization was similar in all cell lines (1.6-fold). However, selective sensitization by BSO to vincristine was observed in the MRP1-transfected line (4.1-fold) but not in the vector control. No significant differences were observed in cisplatin accumulation in the SKOV3-puro and the SKOV3-S2 cells, although both these transfected lines accumulated significantly more than the parental line. Our results suggest that MRP1 does not play a significant role in platinum resistance in the human tumour cell lines investigated in this study.


P-glycoprotein gene (mdrl) amplification and expression were examined in murine leukaemia P388/DX and melanoma B16VDXR cell lines, which exhibit a high level of resistance to a selecting agent, doxorubicin, and express a multidrug-resistant phenotype because they are cross-resistant to multiple cytotoxic drugs. The multidrug-resistant phenotype was obtained in different conditions of selection . In both multidrug-resistant cell lines, an increased expression of P-glycoprotein gene (5 kb transcript detected in Northern blots) was observed and the level of P-glycoprotein mRNA correlated with the degree of resistance. In addition, high molecular weight mRNAs homologous to mdrl gene sequence were consistently detected only in P388/DX cells. Overexpression was associated with a high level of gene amplification only in resistant melanoma cells, whereas it occurred in P388/DX cells with a marginal increase in gene copy number. These results, suggesting that different genetic mechanisms could be responsible for P-glycoprotein overexpression, emphasise the complexity of genetic regulation that may affect tumour cell sensitivity to cytotoxic agents.


Ionising radiation is commonly used in the treatment of colorectal cancer. Tumour cells with mutant p53 undergo cell cycle arrest at G2/M after ionising radiation and evidence suggests that abrogation of this G2 arrest can lead to a premature, aberrant mitosis, thus enhancing ionising radiation-induced cell killing. The G2 checkpoint inhibitor UCN-01 was thus investigated to determine whether it would abrogate the G2 checkpoint induced by 5 Gy ionising radiation in a range of colorectal tumour cell lines. Data presented show that, at doses that are alone non-toxic to the cells, UCN-01 inhibits the ionising radiation-induced G2 checkpoint in five colorectal tumour cell lines with mutant p53. The ability of UCN-01 to sensitise cells to ionising radiation-induced growth inhibition and apoptosis was also investigated and UCN-01 was found to radiosensitise two out of five cell lines. These results were confirmed by long-term colony forming efficiency studies. These results demonstrate that abrogation of the ionising radiation-induced G2 checkpoint is not necessarily associated with sensitisation to ionising radiation, however, some colorectal tumour cell lines can be radiosensitised by UCN-01. Although the mechanism of radiosensitisation is not clear, this may still be an important treatment strategy.British Journal of Cancer (2002) 87, 352–358. doi:10.1038/sj.bjc.6600492www.bjcancer.comCancer Research UK© 2002  Mutations in the p53 tumour suppressor gene have been observed in many epithelial tumours and leukaemias .Abrogation of the G2 checkpoint has been associated with sensitisation of tumour cells to DNA damaging agents. For example, agents such as the methylxanthines caffeine and pentoxifylline have been shown to abrogate the G2 checkpoint and increase sensitivity to IR, presumably due to the ability of these agents to cause premature, and hence lethal, mitosis after IR (Streptomyces species as an inhibitor of protein kinase C (PKC) (7-hydroxystaurosporine (UCN-01) was originally isolated from a  C (PKC) . Potenti C (PKC) . In shor C (PKC) . UCN-01 Colorectal cancer is the second most common cause of cancer death in the Western world and hence improvement of existing treatments will significantly reduce the morbidity and mortality associated with this disease. Previous work by HT29 is derived from a human sporadic colon carcinoma  and has 2 (T25) tissue culture flasks and maintained at 37°C in 5% CO2 LEEC dry incubators.The carcinoma  and adenoma (S/RG/C2) cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented as described previously . All cel6 (carcinomas) or 2×106 (adenoma S/RG/C2). Cells in the exponential phase of growth were exposed to 5 Gy γ IR using a 137Cs generator (Gravatom) at a dose rate of 0.33 Gy per min  which was prepared as a 10 mM stock solution in DMSO. Cells were grown for 7 days and the medium was replaced with fresh medium containing UCN-01 every 3–4 days, or with fresh medium after 24 h, depending on experiment.Cells were seeded in duplicate flasks in standard growth medium at a density of either 1×10Cell cycle arrest after IR was determined using a FACScan (Becton Dickinson), as described previously . Mitotic cells were scored for the characteristics of mitosis, that is visible, evenly stained chromosomes as described previously . At specific timepoints after treatment mitotic index was determined as described above.The mitotic inhibitor colcemid (Gibco) was added to exponentially growing cells (grown on slides as described above) immediately after IR and/or UCN-01 treatment at a dose of 0.01–0.04 μg mlAttached cell yield was calculated in order to determine cell survival after treatment with IR and/or UCN-01. Duplicate flasks were trypsinised and attached cells were quantified using a counting chamber.We have previously shown that 5 Gy IR can induce apoptosis in colorectal adenoma and carcinoma cell lines . Cells tThe attached cell population was also analysed for the presence of apoptotic cells by acridine orange staining; in all cell cultures less than 1% of the attached population showed morphological features of apoptosis. The attached population was also examined for the formation of giant, polyploid cells. These are cells that are proposed to arise from checkpoint disruption and the completion of multiple rounds of DNA synthesis without mitosis (3 cells from each treatment were then re-seeded in T25 flasks with fresh (untreated) medium. The flasks were incubated for a further 14 days and medium was replaced every 3–4 days during this time. The attached cells were then fixed in 10% formaldehyde  overnight and then stained with methylene blue (Sigma) overnight. The methylene blue dye was then washed off with water and colonies of greater than 50 cells were counted and colony forming efficiency (CFE) was calculated as a proportion of the original cell number seeded.After 7 days of treatment with IR and/or UCN-01 attached cell yield was determined (as described above) and 1×10t-test was performed on combined treatments of IR and UCN-01, to determine whether there was a significant difference between treatment with a combination of UCN-01 and IR, compared to IR alone. A P value of <0.05 was taken as significant and *=P<0.05, **=P<0.01, ***=P<0.001.A two-tailed, paired, students Colorectal tumour cell lines with mutant p53 treated with 5 Gy IR arrest at the G2 phase of the cell cycle and this is maximal 12 to 24 h after IR, depending on cell line (M) alone had very little effect on cell cycle distribution. The colorectal carcinoma cell lines HT29, SW480, SW620, S/KS and the adenoma cell line S/RG/C2 all undergo a delay at the G2 phase of the cell cycle after 5 Gy IR.Preliminary studies were carried out to determine a dose of UCN-01 that alone was non-toxic to the cell lines over a 7 day period. As shown in Table 1Treatment with IR and UCN-01 resulted in a clear abrogation of the G2 arrest . In all Table 2We next examined whether the combined treatment of IR and UCN-01 results in a decrease in cell yield  and/or an increase in the proportion of apoptotic cells . Apoptosis was examined in the floating and attached cell populations.P<0.05, Attached cell yield was used as a determinant of cell survival 7 days after IR. In all cell lines investigated 5 Gy IR caused a reduction in cell yield . These cells were also investigated for the presence of giant polyploid cells. These are cells which are proposed to have undergone multiple rounds of DNA synthesis in the absence of mitosis which may result from checkpoint disruption (M UCN-01 (M UCN-01 (a non-toxic dose in S/RG/C2) abrogated the IR-induced G2 checkpoint in S/RG/C2 (M) for a shorter treatment period (24 h) . However, despite using this high dose of UCN-01 and following a previously published protocol . Furthermore the CFE of S/RG/C2 was not significantly reduced in those cells treated with IR and UCN-01 combined, compared to IR alone , thus these long-term studies confirm the observations of 7 day cell survival.The colorectal cell lines HT29, SW480 and S/RG/C2 were further investigated to determine whether UCN-01 had any effect on long-term survival after treatment with 5 Gy IR. After 7 days of treatment with IR and/or a non-toxic dose of UCN-01, cells were trypsinised and re-seeded at a known density in control medium without UCN-01. After a further 14 days of growth, cells were fixed, stained and CFE was calculated. Results presented in Figure 3a,bThe present study set out to determine whether abrogation of the IR-induced G2 checkpoint by UCN-01 would lead to sensitisation of colorectal tumour cells to 5 Gy IR. Previous work has shown that abrogation of the G2 checkpoint can potentiate cell death induced by a range of DNA damaging agents  and in the adenoma cell line (S/RG/C2), abrogation of the G2 arrest after IR did not result in sensitisation to IR. Cell survival and apoptosis were equal in cells treated with IR and UCN-01 compared to cells treated with IR alone. This contrasts with previous data published by our group which has shown that abrogation of the IR-induced G2 checkpoint by caffeine is associated with radiosensitisation in all colorectal cell lines investigated with mutant p53 (Thus it appears, at least in colorectal tumour cell lines, that inhibition of the IR-induced G2 checkpoint is not necessarily associated with sensitisation to IR. This in contrast to other reports which have shown that UCN-01 is able to sensitise tumour cells to a range of DNA damaging agents, including IR (The observation in the present study that some colorectal tumour cell lines cannot be radiosensitised by UCN-01, despite abrogation of the G2 checkpoint, is intriguing. These data suggest, firstly, that lack of both the G1 and G2 arrest in some cell lines with mutant p53 has a no more detrimental effect on the cells than lack of a G1 arrest after IR. Secondly, these data suggest that the G2 checkpoint may be redundant or ineffective in those cells lines where radiosensitisation was not seen. That is, if the DNA repair mechanisms were not functional or impaired in the cell lines where radiosensitisation was not demonstrated, then this would explain why loss of the G2 checkpoint has no adverse effect on these cells. In this scenario, the cells would be unable to adequately repair IR-induced double-strand breaks and therefore would be expected to undergo apoptosis regardless of the delay at G2. The observation by In summary, this investigation demonstrates that inhibition of the IR-induced G2 arrest is not necessarily synonymous with sensitisation to IR. In two out of five cell lines, however, radiosensitisation was demonstrated and thus treatment with UCN-01 may still be an important strategy for colorectal cancer. To our knowledge this is the first report of this nature in a range of colorectal tumour cell lines and the observation of cell line-specific radiosensitisation by UCN-01 may be clinically valuable.


We describe here a murine monoclonal antibody of the IgG2a isotype which was generated against the SW2 human small cell carcinoma cell line. The antibody, SWA11, was shown to bind to a partially defined antigen preferentially expressed on cell lines of small cell carcinoma origin. In vitro binding studies revealed 6.1 x 10(5) antigenic sites on the SW2 cell line and the Ka to be 1.2 x 10(9)M-1. Following injection into mice bearing 1-2 cm3 SW2 xenografts, SWA11 showed strong selective accumulation in the small cell heterotransplants with a tumour to blood ratio of 7.5:1 at day 4. Other tumour to organ ratios were similarly high at 19:1, 22:1 and 12:1 for liver, kidney and spleen respectively. The absolute amount of SWA11 which localised was 10.5% of total injected material per gram tumour at day 2 and this level did not markedly decrease by day 4. The high ability of SWA11 to localise to SCC xenografts was confirmed by external gamma scintigraphy. The potential application of SWA11 as a model system for in vivo radioimmunotherapy is discussed.


Two cell lines (2-10-1 and 8-10-2) derived by exposure to primary tracheal explants to MNNG in vitro were not tumorigenic in syngeneic F-344 rats or athymic BALB/c (nu/nu) mice at early passage, but became tumorigenic at late passage. These cell lines are therefore suited to study the expression of neoantigens during neoplastic development. Transplantation resistance to late-passage, tumorigenic cells was indicated in syngeneic rats using an immunization protocol of repeated cell inoculation and tumour ablation. Spleen cells from such animals were reactive in 20h microcytotoxicity assays against neoplastic cell lines, but unreactive to normal tracheal epithelial cells. Similarly, immune spleen cells co-cultivated in vitro for 6 days with irradiated neoplastic cell lines before assay for microcytotoxicity were strongly reactive, whereas co-cultivation with normal epithelial cells did not stimulate reactivity. Antibody to these neoplastic cell lines was demonstrated in sera of tumour-resistant rats by an indirect radiolabelled-antibody binding test and by indirect immunofluorescence. There was no significant binding to normal tracheal epithelial cell outgrowths.


A hypothesis that m-AMSA may have greater cytotoxicity in melanin-containing tumour tissues, because it may reversibly bind to melanin, leading to prolonged drug exposure, was examined. Clonal lines of B16 melanoma which differed widely in pigmentation level were selected by isolating artificial lung colonies and in vitro soft-agar colonies, and implanting them into mice. Excision cell-survival assays performed 24 h after drug administration showed that in vivo sensitivity to m-AMSA progressively increased as pigmentation level decreased, but that m-AMSA drug levels measured 24 h after treatment were much lower in amelanotic than in melanotic lines. In dose-survival studies the reduced sensitivity of melanotic cell lines was revealed as a large shoulder (Dq = 27 mg/kg) though the terminal slopes for melanotic and amelanotic cell lines were similar (D10 approximately 31 mg/kg). Time-course studies indicated that there was no significant loss of drug from a melanotic cell line for 72 h after drug administration, though in an amelanotic cell line drug levels fell 10-fold in 10 h. There was, however, no evidence for prolonged drug cytotoxicity in the melanotic cell line. Using a fractionated drug-treatment regime, the greater cytotoxicity of m-AMSA to amelanotic tumour tissue was confirmed in a non-invasive regrowth-delay assay.


The expression of c-myc protein was studied in primary cultures of rat hepatocytes and rat liver-derived epithelial cell lines. The levels of the protein were determined by flow cytometry using a monoclonal antibody to the c-myc protein. Freshly isolated hepatocytes from normal adult male Fischer F344 rats had low but detectable levels of the protein which were similar in the different ploidies. Higher levels were detected in immortalised but untransformed rat liver cell lines, and increased expression was observed during passage through the cell cycle. Following in vitro transformation of one of the immortalised epithelial cell lines by ras genes, similar levels of c-myc expression to those present in the untransformed cells was maintained. Transformation by activated aflatoxin B1 (AFB1) resulted in lower levels of expression. The cell cycle related level of expression was also seen in the transformed cells. Similar results to those observed in the in vitro ras transfected liver-derived cell lines were obtained from in vivo AFB1-induced rat hepatoma cell lines. These results demonstrate that continuously dividing rat liver-derived cell lines have higher levels of expression of c-myc protein than non-dividing, freshly isolated hepatocytes, and that there is no further elevation in the levels observed when these cell lines are transformed. In some cases decreased levels can result from malignant transformation.


Thesemolecular and functional characteristics suggest that the 4F1-Ag is a novel adhesionmolecule that may be involved both in intrathymic T lymphocyte differentiation and inperipheral T-cell function.The rat monoclonal antibody (mAb) 4F1, raised against mouse thymic stromal cells,recognizes cortical epithelium in tissue sections of mouse thymus; however, in flowcytometry, activated leucocytes  and transformedthymocytes are also positive for the 4F1-antigen (4F1-Ag). Western blotting, under bothreducing and nonreducing conditions, demonstrates that the molecule to which 4F1binds is expressed in four forms, 29, 32, 40, and 43 kD, all of which carry N-linkedcarbohydrate; and that the structure is identical on epithelium and lymphocytes. The4F1-Ag on cortical epithelium is partially sensitive to PI-PLC treatment, whereas ontransformed epithelial and lymphoid cell lines, it was resistant to this enzyme. Themolecule, therefore, may exist in both transmembrane and phosphoinositol-linkedforms. In functional blocking experiments, mAb 4F1 gave inhibition of both T-cellproliferation in MLR and of cytotoxic T-cell killing of alloantigenic targets; it alsoblocked adhesion of transformed thymocytes to thymic epithelial cells


We have examined the ability of eight compounds to enhance adriamycin (ADM) sensitivity of two human tumour cell lines  and their multidrug-resistant variants. The resistant cell lines (H69AR and HT1080/DR4) do not overexpress P-glycoprotein. Verapamil, nicardipine, perhexiline maleate, chloroquine, tamoxifen, clomiphene, prenylamine and trifluoperazine were tested alone and in combination with ADM for their cytotoxic effects. No major differences in sensitivity between the parent and resistant cell lines were noted when these agents were tested alone, except for HT1080/DR4 cells which exhibited a slight collateral sensitivity to nicardipine and H69AR cells which showed cross-resistance to chloroquine and clomiphene. When the chemosensitisers were combined with ADM no enhanced cytotoxicity of either parent cell line was observed. In HT1080/DR4 cells, verapamil showed only a modest dose-dependent chemosensitising effect while the other compounds had no effect. Verapamil and nicardipine enhanced ADM cytotoxicity in H69AR cells slightly but these effects were not dose-dependent. These results demonstrate that the reversal of drug resistance by verapamil and other calcium antagonists in a dose-dependent fashion is not an invariable property of multidrug-resistant tumour cells.


A malignant cell line derived from the s.c. inoculation of Adenovirus 12 into a CBA mouse has been isolated in vitro, cloned, and within 10 passages the clones have been investigated for their karyotype, morphology, growth rate, saturation density and response to plant lectin in vitro, and their tumorigenicity and growth rate in vivo. The cell lines rapidly acquired a highly heterogeneous karyotype, but remained homogeneous with respect to more complex physiological parameters. Examination of the cellular characteristics has indicated that the rate of growth of the cell lines in vivo, but not their tumorigenicity, may be related to their in vitro potentials. The clones responded differently to the cytotoxic effects of concanavalin A, but there was no correlation between the effect of the lectin and the malignant potential of the cells.


Characterisation of altered glycosylation of P-glycoprotein (P-gp) found associated with the absence of a multidrug resistance (MDR) phenotype in cell lines prompted an investigation to assess the role of post-translational processing in establishing P-gp efflux pump functionally. The clone A cell line used in this study displays a strong MDR phenotype mediated by high constitutive levels of expression of P-gp. Incubation of clone A cells with tunicamycin for different periods resulted in a time-dependent increase in daunorubicin accumulation, reflecting a reduction in P-gp function. Parallel experiments conducted with verapamil resulted in no loss of P-gp functionality in clone A cells. Reduction in surface-associated P-gp following exposure to tunicamycin was established by FACS analysis, Western blot analysis and immunoprecipitation of surface-iodinated P-gp. In addition, immunoprecipitation of P-gp from 32P-orthophosphate-labelled cells demonstrated reduced phosphorylation of P-gp associated with tunicamycin exposure. From these studies we conclude that glycosylation of P-gp is required to establish the cellular MDR phenotype.


Immunotoxins that carry two toxin molecules to the target cell should in theory have a greater anti-tumour effect than those that carry just one. We have investigated the therapeutic efficacy of two anti-CD7-saporin immunotoxins constructed with one saporin (HB2-Sap 1-mer) or two saporin molecules (HB2-Sap 2-mer) per immunotoxin molecule. In vitro, the 2-mer immunotoxin was 5.6 times more effective than the 1-mer immunotoxin at inhibiting protein synthesis in the CD7+ human T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2 and was also more effective at inhibiting HSB-2 cell proliferation. Flow cytometry revealed that the 2-mer immunotoxin had a reduced binding capacity to HSB-2 cells compared with the 1-mer immunotoxin or native HB2 antibody. In therapy studies in SCID mice with disseminated HSB-2 human leukaemia, the 2-mer immunotoxin performed marginally better than the 1-mer immunotoxin, but log-rank analysis did not reveal any significant differences between the two therapy groups. We therefore conclude that, although the 2-mer immunotoxin performed better than the 1-mer immunotoxin against target HSB-2 cells in vitro, this improved performance was not reflected as an improved in vivo therapeutic outcome in the SCID mouse model.


Midkine (MK) is a heparin-binding growth factor and is frequently expressed at high levels in many human carcinomas. To investigate further the roles of MK in the regulation of cell growth, we introduced MK expression in NIH3T3 cells. A mixture of transfectants of an MK expression vector, but not a control vector, formed colonies in soft agar, showed an elevated cell number at confluence, and formed tumours in nude mice. An interesting characteristic of the transformed cells was that they became spontaneously detached from the culture dish substratum. In the transformed cells, MK was not only secreted, but also localized, in the perinuclear region as spots. The present data indicate that MK has the potential to transform NIH3T3 cells and suggest that overexpression of the MK gene may promote unregulated cell growth in vivo.


The effects of several members of the family of lamellarins, polyaromatic alkaloids isolated from tunicates belonging to the genus Didemnum, on the growth of several tumour cell lines and on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR), were investigated. Cytotoxicity experiments of lamellarins were performed on a panel of tumour cell lines, including two multidrug-resistant cell lines. Some lamellarins showed good anti-tumour activity, with similar levels of cytotoxicity against both the resistant and their corresponding parental cell lines. Two lamellarins displayed a high potency against lung carcinoma cells. Studies of the resistance modifier activity of the different lamellarins at non-toxic concentrations were also carried out in cells exhibiting MDR, and lamellarin I was selected for the highest chemosensitising activity. At non-toxic doses, verapamil and lamellarin I effectively increased the cytotoxicity of doxorubicin, vinblastine and daunorubicin in a concentration-dependent manner in multidrug-resistant cells, but the potency of lamellarin I as a MDR modulator was 9- to 16-fold higher than that of verapamil. In vitro measurements of rhodamine 123 accumulation in the multidrug-resistant Lo Vo/Dx cells suggest that lamellarin I reverses MDR by directly inhibiting the P-gp-mediated drug efflux. This work underscores the possibility of using these marine-derived compounds as a potential new source of anti-tumoral drugs active on resistant cells as well as of non-toxic modulators of the MDR phenotype.


Glutathione levels were measured in 30 human lung cancer lines. Lower levels were detected in cell lines derived from small cell lung cancer specimens compared to non-small cell lines . However, no difference were detected between cell lines derived from previously untreated patients, compared to those derived from patients who had received chemotherapy. Non-small cell lines were found to have increased activity of 4 detoxification enzymes compared to small cell lines, although these differences did not reach statistical significance: glutathione transferase activity , glutathione reductase , gamma-glutamyl transpeptidase  and superoxide dismutase . As the cell lines exhibit a similar chemosensitivity pattern to that observed in clinical practice, these differences in glutathione and detoxification enzyme levels may prove to be important indicators of intrinsic drug resistance often seen in patients with non-small cell lung cancer.


BCNU (carmustine), VM26 (teniposide) and ARA-A5'P (vidarabin-monophosphate) were compared in their activity against 30 cell lines of primary (N = 21) and metastatic (N = 9) human brain tumours, which were characterized in tissue culture by cytochemical, immunological and cytogenetic criteria. In vivo achievable concentration-time products c X t were correlated with in vitro pharmacokinetic data in order to evaluate in vitro drug sensitivity at relevant exposure doses. A microcytotoxicity assay was employed to screen for drug toxicity in individual tumour cell lines. Following drug exposure and 5 to 8 population doubling times of untreated controls, RNA-synthesis - as a parameter of cell metabolism and proliferation - was determined by incorporation of -uridine into cellular RNA (liquid scintillation counting protocol). The cytotoxic effect of each drug on individual cell lines was expressed in terms of a sensitivity index (SI); by these means effects of different drugs on individual tumour cell lines could be compared. Mean sensitivity indices of ARA-A5'P, BCNU and VM26 for primary brain tumour cell lines were 0.59, 0.82 and 0.54. ARA-A5'P and VM26 had almost similar activities against brain tumour cell lines, whereas BCNU was significantly (P less than 0.001) less active. High grade gliomas were less sensitive to all three agents than low grade and infratentorial gliomas. ARA-A5'P was also able to effectively reduce colony formation in brain tumour cell lines. A cross-resistance of ARA-A5'P to either BCNU or VM26 could not be observed. Clearly, ARA-A5'P is an effective drug in treatment of brain tumour cells in vitro.


The sensitivity of eight cell lines established from treated and untreated patients with small cell carcinoma of the lung (SCCL) was tested in the clonogenic assay with 1 h and continuous exposure to aclarubicin (ACLA), adriamycin (ADR), daunorubicin (DAU) and mitoxantrone (MITO). The sensitivity to ADR, DAU and MITO covariated, and varied with a factor of five. The sensitivity to ACLA was independent of the sensitivity to ADR and varied only within a factor of two. Only ACLA showed pronounced increased potency with continuous incubation, and ACLA was the most potent drug in the three cell lines least sensitive to ADR. Two resistant cell lines were selected by treating NCI-H69 in vitro with DAU. One cell line (9-fold resistant to DAU) expressed large amounts of P-glycoprotein, the other cell line (4-fold resistant to DAU) had barely detectable glycoprotein. Both lines acquired resistance to ADR, ACLA and MITO. The cross-resistance to ACLA and MITO was only partial and ACLA was still the most potent drug on these lines. The sensitivity to ACLA of the cell lines least sensitive to ADR suggest that ACLA partially circumvents mechanisms of multidrug resistance. Together with the pronounced increase in potency with prolonged exposure, these results suggest that ACLA has a mechanism of action different from the 'classical' anthracyclines. In this context mitoxantrone is more similar to the classical anthracyclines although its structure is more dissimilar.


VCR, UKF-NB-2DOX, UKF-NB-2CDDP, UKF-NB-3VCR, UKF-NB-3CDDP, UKF-NB-6VCR, UKF-NB-6CDDP) to an endothelial cell monolayer, accompanied by downregulation of the adhesion receptor neural cell adhesion molecule (NCAM). Based on the UKF-NB-3 model, N-myc proteins were enhanced in UKF-NB-3VCR and UKF-NB-3CDDP, compared to the drug naïve controls. p73 was diminished, whereas the p73 isoform deltaNp73 was upregulated in UKF-NB-3VCR and UKF-NB-3CDDP. Valproic acid blocked adhesion of UKF-NB-3VCR and UKF-NB-3CDDP, but not of UKF-NB-3DOX, and induced the upregulation of NCAM surface expression, NCAM protein content and NCAM coding mRNA. Valproic acid diminished N-myc and enhanced p73 protein level, coupled with downregulation of deltaNp73 in UKF-NB-3VCR and UKF-NB-3CDDP. Valproic acid also reverted enhanced adhesion properties of drug-resistant UKF-NB-2, UKF-NB-6 and SKNSH cells, and therefore may provide an alternative approach to the treatment of drug-resistant NB by blocking invasive processes.Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). In pursuit of alternative treatments for chemoresistant tumour cells, we tested the response of multidrug-resistant SKNSH and of vincristine (VCR)-, doxorubicin (DOX)-, or cisplatin (CDDP)-resistant UKF-NB-2, UKF-NB-3 or UKF-NB-6 NB tumour cell lines to valproic acid (VPA), a differentiation inducer currently in clinical trials. Drug resistance caused elevated NB adhesion (UKF-NB-2 Multiple-agent chemotherapy is the conventional therapy for patients with advanced stages of neuroblastoma (NB) and disseminated NB. However, drug resistance arises in the majority of stage IV and relapsed NB, often leading to treatment failure (Histone deacetylases (HDAC) represent one of the most important intracellular targets, as these molecules modulate a wide variety of cellular functions. Abnormal histone acetylation status can result in undesirable phenotypic changes, including developmental disorders and cancer. Indeed, aberrant histone acetylation may be an aetiological factor in several types of cancer by derepressing gene transcription. Hence, HDAC inhibitors may be useful for cancer prevention, due to their ability to ‘reactivate’ the expression of epigenetically silenced genes, including those involved in differentiation, invasion and metastasis. Most notably, recent data indicate that HDAC inhibition may be successful in treating refractory or relapsing tumours after conventional chemotherapy. Histone deacetylase inhibition has been demonstrated to block cell growth of drug-resistant small-cell lung cancer lines  expression play an essential part in the progression of NB, we investigated NCAM expression  has been shown to possess HDAC inhibitory properties and to affect the growth and survival of tumour cells sistance , closely−1 medium, respectively. The resistant sublines were grown for more than 6 months in Iscove's modified Dulbecco's medium  supplemented with 10% fetal calf serum  and either 10 ng ml−1 VCR , 20 ng ml−1 DOX , or 1000 ng ml−1 CDDP . The clinical relevance of drug-resistant UKF-NB-2 and UKF-NB-3 sublines has been examined before in a xenograft setting , VCR  and DOX  were prepared in accordance to the manufacturer's instructions. The initial CDDP, VCR and DOX concentrations were 50, 0.2 and 2 ng ml setting . Multidr50.Chemoresistance of the indicated cell lines was verified by the MTT test , 0.1% heparin, 100 ng ml−1 gentamycin and 20 mM HEPES buffer (pH 7.4). Subcultures from passages 2–6 were selected for experimental use.Human endothelial cells (HUVEC) were isolated from human umbilical veins and harvested by enzymatic treatment with chymotrypsin. Human endothelial cells were grown in Medium 199 , supplemented with 10% FCS, 10% pooled human serum, 20 M for 3 or 5 days. Tumour cell adhesion, NCAM, p73, deltaNp73 and N-myc expression were then measured in VPA-treated cells. Results were compared to untreated controls. Viability of tumour cells in presence of VPA was assessed by propidium iodide dsDNA intercalation.Parental NB tumour cells and their drug-resistant sublines were treated with VPA  at a final concentration of 1 m6 parental NB tumour cells or their drug-resistant sublines (VPA treated vs non-treated) were carefully added to the HUVEC monolayer for 60 min. Subsequently, non-adherent tumour cells were washed off using warmed (37°C) M199. The adherent cells were fixed with 1% glutaraldehyde and counted in five different fields (5 × 0.25 mm2) using a phase contrast microscope (20 × objective) to calculate the mean cellular adhesion rate.Human endothelial cells were transferred to six-well multiplates  in complete HUVEC medium. When confluency was reached, 0.5 × 10−1) for 20 h at 60°C. Thereafter, the fluorescent dye PicoGreen , which shows high specificity for dsDNA, was added (1 : 200 dilution) for 10 min at 20°C. Fluorescence intensity was determined using a computer-controlled fluorescence reader  at λex=485 nm and λem=530 nm.Proliferative activity of NB tumour cells and HUVEC was estimated by the PicoGreen assay as described elsewhere (4 cells/scan) and expressed as mean fluorescence units (MFU). A mouse IgG1-FITC was used as the isotype control.Neuroblastoma cells were disaggregated mechanically, washed in blocking solution  and then incubated for 60 min at 4°C with an FITC-conjugated monoclonal antibody anti-CD56 which detects the NCAM 120, 140 and 180 kDa isoform (clone 16.2). Neural cell adhesion molecule expression of NB cells was then measured using a FACscan  channel histogram analysis; 1 × 102+ and Mg2+) and then fixed in cold (−20°C) methanol/acetone (60/40 v/v). Subsequently, cells were washed again with PBS (without Ca2+ and Mg2+), and later once with blocking buffer (0.5% BSA in PBS without Ca2+ and Mg2+). After removing the washing buffer, cells were incubated for 60 min with FITC-conjugated anti-NCAM monoclonal antibody. To prevent photobleaching of the fluorescent dye, cover glasses with stained cells were taken out of the wells and the residual liquid was removed. The cells were then embedded in an antifade reagent/mounting medium mixture  and mounted on slides. The slides were viewed using a confocal laser-scanning microscope  with a plan-neofluar × 100/1.3 oil immersion objective.To explore NCAM localisation, tumour cells were transferred to round cover slips, which were placed in a 24-well multiplate. Upon reaching confluency, cell cultures were washed two times with PBS . HRP-conjugated goat anti-mouse IgG  served as the secondary antibody. The membrane was briefly incubated with ECL detection reagent  to visualise the proteins and exposed to an x-ray film .μg) was reverse transcribed and the resulting cDNA directly added to the PCR. Amplification reactions (20 μl) were performed in the presence of 1/10 (2 μl) of the cDNA reaction, with an initial incubation step at 94°C for 1 min. Cycling conditions consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min over a total of 30 cycles. The reaction was completed by another incubation step at 72°C for 10 min. The PCR products were subjected to electrophoresis in 2% agarose gel and visualised by ethidium bromide.Total RNA was extracted and purified with Trizol reagent according to the manufacturer's instructions and treated with RNase-free DNase. The NCAM primer sequences were as follows: for NCAM-180: 5′CGAGGCTGCCTCCGTCAGCACC 3′ and 5′CCGGATCCATCATGCTTTGCTCTCG 3′; for NCAM-140: 5′GAACCTGATCAAGCAGGATGACGG 3′ and 5′CCGGATCCATCATGCTTTGCTCTCG 3′  before the antibodies were added. Monoclonal anti-N-myc antibody was from Calbiochem . p73 was measured using monoclonal anti-p73 . To identify deltaNp73, the monoclonal antibody anti-deltaNp73 (clone 38C674) was purchased from Active Motif . Primary antibodies were labelled with goat anti-mouse IgG-FITC. To evaluate background staining, goat anti-mouse IgG-FITC was used.β-Actin  served as the internal control.N-myc was also explored by Western blot analysis using monoclonal antibodies against N-myc . To investigate N-myc coding mRNA, RT/PCR has been carried out as described above. The N-myc primer sequences were as follows: forward: 5′GACCACAAGGCCCTCAGTAC 3′; reverse: 5′GTGGATGGGAAGGCATCGTT 3′.U-test. Differences were considered statistically significant at P<0.05.All experiments were performed 3–6 times. Statistical significance was investigated by the Wilcoxon–Mann–Whitney CDDP, UKF-NB-3VCR or UKF-NB-3DOX was quantified 60 min after plating the cells on to an endothelial cell monolayer . The amount of adherent cells increased fourfold when UKF-NB-3 became resistant to CDDP or VCR. Doxorubicin resistance did not induce any effects on tumour cell binding to HUVEC. Adhesion of UKF-NB-3, UKF-NB-3onolayer . Nearly M VPA to UKF-NB-3CDDP or UKF-NB-3VCR significantly blocked the cellular adhesion process .The application of 1 m process . A 5-dayThe PicoGreen assay did not reveal any proliferative activity during the experiment, which rules out the possibility that adhesion differences between drug resistant, VPA-treated and control NB cells may be caused by different cell growth capacity.vs VPA-treated (5-day treatment)-resistant tumour cells. Results of parental UKF-NB-3 and of resistant tumour cells treated for 3 days with VPA were not inserted, because the histograms overlapped, making the figures unclear. However, the complete experimental data set  is given below the histogram.CDDP and UKF-NB-3VCR, when compared to the parental control cell line. However, no changes were seen with NCAM expression on UKF-NB-3DOX, compared to the controls. Treatment of CDDP- or VCR-resistant cell lines with VPA led to a significant increase in NCAM, which exceeded the control values after a 5-day incubation period.Reduction of NCAM surface expression was observed on UKF-NB-3n=5).Valproic acid also acted on the parental cell lines, as evidenced by a significant upregulation of NCAM by +56.9±22.8%  UKF-NB-3CDDP, UKF-NB-3VCR and UKF-NB-3DOX. The complete experimental data set  is given below the histogram.N-myc proteins were enhanced in UKF-NB-3ell line . ModeratCDDP, UKF-NB-2VCR and UKF-NB-2DOX cell adhesion, compared to the parental controls. Adhesion correlated inversely with the NCAM surface level  reverted the increased adhesion phenotype and restored NCAM levels, it did not re-sensitise the drug-resistant NB cells to VCR, DOX or CDDP (data not shown).To strengthen the relevance of our findings, further NB tumour cell lines were included in the study. Analysis of UKF-NB-2 and drug-resistant sublines indicated a significant increase in UKF-NB-2ce level . A similCDDP, UKF-NB-6 and UKF-NB-6CDDP, compared to the controls. However, no differences were seen between VPA-treated and non-treated VCR or DOX-resistant tumour cell lines.N-myc was significantly diminished by VPA in UKF-NB-2, UKF-NB-2vs chemoresistant NB cells were analysed in final experiments. To better interprete our data, antiproliferative effects of VPA on the chemosensitive Based on this cell culture model, we have demonstrated that drug-resistant NB cancer cells develop an increased malignant phenotype as evidenced by enhanced adhesion to vascular endothelial cells, accompanied by significant downregulation of the adhesion receptor NCAM. Valproic acid reverted this process by downregulating cell adhesion and upregulating NCAM expression. Considering clinical utility, it is encouraging that VPA was active in NB cell lines resistant to existing chemotherapies, since overcoming resistance to anticancer agents is a major challenge in the development of novel antitumour protocols.In fact, several cases have been documented showing antineoplastic effects of VPA in patients with relapsed tumours. When VPA was given as maintenance therapy for childhood malignant glioma after postoperative combined chemotherapy and irradiation, about 10% of these patients were maintained in continuous complete remission and an equal number of patients showed at least partial responses  and MDR-associated protein 1  signalling has been observed in NB cells with acquired resistance to DOX (μM significantly diminished UKF-NB-3CDDP or UKF-NB-3VCR proliferation and cell adhesion to HUVEC in our own experiments (data not shown). Histone deacetylase inhibitors different from VPA may therefore be considered to become additional options for the treatment of drug-resistant NB. However, detailed studies are necessary to explore their clinical value.Other HDAC inhibitors have also been shown to inhibit tumour growth and to overcome multidrug resistance. Notably, suberoylanilide hydroxamic acid (SAHA) has been demonstrated to act on VCR-resistant leukaemia cell lines (


We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen. This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F10 murine melanoma or MDA-MD-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16F0 cell line. Replacement of tumour cell CM by 10 ng ml-1 basic fibroblast growth factor (bFGF) also results in capillary-like structure formation by BAE cells. An anti-bFGF antibody blocks this effect, showing that bFGF is one of the factors responsible for the angiogenic response induced by B16F10 CM in our assay. Addition of an anti-laminin antibody reduces significantly the formation of capillary-like structures, probably by blocking the attachment of BAE cells to laminin present in Matrigel. The anti-angiogenic compound suramin inhibits in a dose-dependent manner (complete inhibition with 100 microM suramin) the migration and differentiation of BAE cells on type I collagen in response to B16F10 CM. This assay represents a new model system to study tumour-induced angiogenesis in vitro.