High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells. CST6 gene  thymidine (Amersham) for 24 h. Cells were detached with trypsin and 5 × 104 [3H]-labeled cells in 100 μl medium were added to each well in a 24-transwell permeable plate  coated with Matrigel basement membrane matrix . For migration studies the wells were left uncoated. The bottom wells received 600 μl medium with the same concentration of serum as the top well, either 1% or 10% FBS. Cells were removed from the upper and lower sides of the filter after 72 h incubation and [3H]-thymidine was counted in a liquid scintillation analyzer . The ratio of the counts obtained from the bottom versus total counts from both sides was used to determine migration and invasiveness.Cells were seeded at 2 × 10Legumain activity was measured by cleavage of the substrate Z-Ala-Ala-Asn-NHMec  as previously described ,45. CellInhibitory activity towards legumain was measured in the culture media from all the cell lines and from the pCST6-transfected cells (A375 and MCC11). Inhibitory activity was evaluated by measuring the residual enzyme activity in a preparation of legumain after the addition of conditioned media. As a source of legumain, homogenized rat kidney was applied to a cation exchange column  and eluted with a gradient of sodium chloride as described previously . Peak let-test, whereas two-way ANOVA was used to evaluate protease activities.Data are shown as the mean ± standard error of the mean (SEM). Statistical significance of the Matrigel invasion assays was evaluated using the two-tailed Student's Although it is known that cystatin E/M is secreted by keratinocytes and is present in the skin , little Another cysteine protease inhibitor, cystatin C, was detected as one band (15 kD) in media from most of the cell lines examined .The conditioned media from all cell lines was analyzed by fluorometry for their ability to inhibit the activity of a partially purified legumain fraction from rat kidney Fig. . LegumaiAlthough the activities of cathepsin B and L in melanoma and other tumor types are well documented, the expression and activity of legumain in melanoma is relatively unknown. To determine expression of legumain, cathepsin B and L, whole cell lysates were prepared from the same panel of cell lines as used for cystatin E/M analysis Fig.  followinBoth cathepsin B and L were detected in most of the cell lines, except those predominantly secreting the glycosylated form of cystatin E/M  was detected. When cells lacking cystatin E/M were transfected with an expression vector containing cystatin E/M cDNA (pCST6), a significant decrease in cellular legumain activity was observed, and conditioned media collected from the transfected cells had high inhibitory activity against exogenously added legumain. Furthermore, following transfection with pCST6, invasiveness was significantly decreased as measured in a Matrigel invasion assay. Collectively, our data strongly suggests that cystatin E/M inhibits melanoma progression by suppressing legumain activity.CST6 gene expression progressively decreases during disease progression [CST6 gene expression was observed. Furthermore, there was a significant decrease in CST6 expression between primary and metastatic melanoma when analyzed by microarray and quantitative RT-PCR. These data were the first to suggest that expression of CST6 decreases during progression to metastatic melanoma, a finding that is in agreement with previous studies in breast cancer [In a previous study comparing the gene expression profiles of primary and metastatic melanoma, it was shown that gression . When met cancer .CST6 functions as a tumor suppressor gene in melanoma, further studies are needed to determine the extent of cystatin E/M expression and the glycosylation status in cultured melanocytes and thin primary tumors.We have identified several cell lines derived from both primary and metastatic melanoma that secrete cystatin E/M. However, despite data suggesting that cystatin E/M has tumor suppressive capabilities, we did not detect lower levels in the metastatic versus the primary cell lines. This was probably due to the relatively small number of cell lines at our disposal originating from primary tumors, and furthermore, that most of these lines were derived from rather large lesions possibly having a phenotype more closely resembling metastatic rather than primary melanoma. Interestingly, the glycosylated 17 kD cystatin E/M form predominated in the melanoma samples, whereas the unglycosylated 14 kD form predominated in HaCaT keratinocytes. To confirm that in situ or primary melanoma.Although the expression of cystatin E/M in melanocytes has not been demonstrated, it has previously been shown that the protein is secreted from keratinocytes and thus present within the tumor microenvironment of the primary melanoma . Herein,By immunoblotting, we detected cystatin E/M secretion from four melanoma cell lines , and the conditioned media from these cells demonstrated the highest inhibitory activities against legumain. Surprisingly, we detected cystatin E/M by ELISA in media from only one of these melanoma cell lines. There are several potential explanations for this discrepancy, but the most likely being that there are fundamental differences between the applied methods. Immunoblotting measures denatured proteins while the ELISA attempts to detect the native cystatin E/M, possibly in complex with the interacting proteases. It is also possible that the amount of cystatin E/M in the media collected from the melanoma lines is below the limit of detection in the ELISA assay, as some cell lines showing strong expression on immunoblotting  had low amounts detected by ELISA. Furthermore, we utilized different antibodies in the two assays in this study and differences in the antibody affinity for the various forms of cystatin E/M are probable. The polyclonal antiserum used in the ELISA has, however, been demonstrated to recognize both the glycosylated and non-glycosylated forms of cystatin E/M (data not shown), making this explanation less likely. Nevertheless, there was a statistical positive correlation between the ELISA results for cystatin E/M and both molecular forms observed by immunoblotting.Some interesting relationships were observed between the different expression profiles of cystatin E/M, cystatin C, legumain, cathepsin B and L. First, in most melanoma cell lines examined, we identified an inverse correlation between secreted cystatin E/M and C. The only exception was the ocular melanoma cell line 92.1, in which neither of the cystatin isoforms was detected. Likewise, a similar inverse correlation between secreted cystatin E/M and both pro- and active forms of the examined proteases in lysates was observed. Legumain was expressed at very low levels in cell lines secreting cystatin E/M , while immunobands representing active legumain were high in all cells lacking the protease inhibitor. Similarly, with one exception (MCC70), there was no detectable expression of neither cathepsin B or L in the cells secreting cystatin E/M. Interestingly, this pattern was observed only in the melanomas and not in the cell lines derived from other cancer types. Although the expression levels of cystatin E/M and the proteases inversely correlated, an alternative explanation could be that cystatin E/M and protease expression levels are counter-regulated by some unknown mechanism. If so, the lower metastatic potential of the cystatin E/M expressing cells might be due to lower total expression levels of legumain, cathepsin B and L, respectively, and should be investigated further.Although legumain has been shown to impart metastatic properties in several different types of cancer, the present study is the first to address the expression and activity of the protease in melanoma. In general, high legumain expression and activity was revealed in the examined panel of melanoma cell lines. Two immunobands were repeatedly observed, one migrating at approximately 52 kD corresponding to the inactive proform of the enzyme, and the other at approximately 36 kD corresponding to the active form . The latTo determine the influence of cystatin E/M on protease activity and invasion, two cell lines (A375 and MCC11) with no basal secretion of cystatin E/M were transfected with pCST6. Cystatin E/M over-expression resulted in significantly increased ability to inhibit exogenously added legumain by the conditioned media, as well as decreased cellular legumain activity. The percentage decrease in legumain activity was greater in lysates from A375 than MCC11, possibly due to the lower basal level of legumain activity in A375. Surprisingly, cathepsin B was not affected by over-expression of cystatin E/M in these cells. Furthermore, a significant inhibition of invasive capacity was observed in both cell lines following transfection with pCST6. Since enhanced expression of cystatin E/M did not influence on the cell ability to migrate, the observed effect on invasion is most likely due to cystatin E/M-mediated inhibition of legumain activity. However, it is possible that the decreased invasiveness could in part result from effects on proteases other than legumain.in vitro motility and Matrigel invasiveness [in vitro growth whereas over-expression increased growth rate and tumor burden in vivo in pancreatic cancer [Although the majority of reports on cystatin E/M have indicated that it suppresses tumor growth and metastasis, there have been some conflicting data regarding the role of the protease inhibitor in cancer. It has been reported that cystatin E/M is up-regulated during the progression from primary to metastatic oropharyngeal squamous cell carcinoma . In addisiveness . A studyc cancer . The autc cancer -14.in vivo. The data presented provide new insight into the role of cystatin E/M in protease regulation and invasion in malignant melanoma.In summary, we have shown that cystatin E/M acts as a regulator of proteolytic activity of legumain and suppressor of invasion in metastatic melanoma. Further studies are needed to elucidate the role of both cystatin E/M and legumain in melanoma, including their expressions during different stages of the disease and their roles in tumor formation and metastasis Cys: cystatin; MCC: Moffitt Cancer Centre; pCST6: cystatin E/M (CST6)-encoding plasmidThe authors declare that they have no competing interests.JJB carried out cell culturing and harvesting, immunoblotting of cystatins, transfections, Matrigel invasion assays and statistical analysis, and participated in design of the study. MHH carried out immunoblotting and densitometry scanning of cystatins and proteases, participated in the statistical analysis and performed the final design of the figures. HTJ performed chromatography of rat legumain, participated in protease activity measurements and study design. AIR established several of the used melanoma cell lines and participated in the original conception and design of the study. MA performed ELISA measurements. ØF participated in the original design of the study. GMM participated in design and progression of the study. RS participated in protease activity measurements and statistical analysis, as well as in design and coordination of the study. JJB, GMM and RS drafted the manuscript, whereas all authors have contributed to, read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/17/prepub


Oncogene16: 933–934). When they were injected intravenously into athymic nude mice, KoTCC-1/BH formed more than three times as many tumour nodules in the lungs as did KoTCC-1/C. In addition, tumour progression, including lymph node metastasis and haemorrhagic ascites, was observed to be more advanced after the implantation of KoTCC-1/BH cells into the bladder wall of nude mice than after implantation of KoTCC-1/C cells. These enhanced malignant progression of KoTCC-1/BH cells were well correlated with anti-apoptotic activity under anchorage-independent conditions in in vitro experimental models. In contrast, there were no significant differences among these cell lines in their growth rates both in vitro and in vivo, invasive ability and cell motility. These findings suggest that, if it is overexpressed, Bcl-2 prolongs cell survival under unfavourable conditions encountered in the metastatic process, resulting in the enhanced metastatic potential of bladder cancer. © 1999 Cancer Research CampaignWe investigated the effect of Bcl-2 expression on the metastatic process of bladder cancer cells by using the Bcl-2-transfected human bladder cancer cell lines (KoTCC-1/BH) and the control vector only-transfected cell line (KoTCC-1/C), which were generated in our previous study


Histamine has recently been shown to be a growth factor for some gastric and colorectal cancer cells. Previous studies have shown that cimetidine blocks in vitro and in vivo histamine-stimulated growth and cAMP release from the human colonic cancer cell line, C170. In this study, ranitidine, another H2 receptor antagonist, did not affect either basal or histamine-stimulated in vitro proliferation of C170, and failed to prevent cAMP release in vitro. Ranitidine did not inhibit in vivo growth of C170 at a dose of 1, 10, 25, 50 or 100 mg/kg, in contrast to 50 mg/kg/day cimetidine, which produced 39.3% inhibition of tumour volume (p<0.01) after 23 days' treatment. Ranitidine did not inhibit in vivo histamine-stimulated growth of C170 cells . LIM2412, another colonic cancer cell line, was significantly stimulated by both cimetidine and ranitidine in vivo. Ranitidine had no effect on in vitro cell proliferation.


CDDP acts by formation of platinum (Pt)–DNA adducts; dFdC by dFdCTP incorporation into DNA, subsequently leading to inhibition of exonuclease and DNA repair. Previously, synergism between both compounds was found in several human and murine cancer cell lines when cells were treated with these drugs in a constant ratio. In the present study we used different combinations of both drugs (one drug at its IC25 and the other in a concentration range) in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its dFdC-resistant variant AG6000 and two NSCLC cell lines, H322 (human) and Lewis lung (LL) (murine). Cells were exposed for 4, 24 and 72 h with a total culture time of 96 h, and possible synergism was evaluated by median drug effect analysis by calculating a combination index . With CDDP at its IC25, the average CIs calculated at the IC50, IC75 IC90 and IC95 after 4, 24 and 72 h of exposure were < 1 for all cell lines, indicating synergism, except for the CI after 4 h exposure in the LL cell line which showed an additive effect. With dFdC at its IC25, the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the Cls after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all Cls were < 1. CDDP did not significantly affect dFdCTP accumulation in all cell lines. CDDP increased dFdC incorporation into both DNA and RNA of the A2780 cell lines 33- and 79-fold (P < 0.01) respectively, and tended to increase the dFdC incorporation into RNA in all cell lines. In the AG6000 and LL cell lines, CDDP and dFdC induced > 25% more DNA strand breaks (DSB) than each drug alone; however, in the other cell lines no effect, or even a decrease in DSB, was observed. dFdC increased the cellular Pt accumulation after 24 h incubation only in the ADDP cell line. However, dFdC did enhance the Pt–DNA adduct formation in the A2780, AG6000, ADDP and LL cell lines . This increase in Pt–DNA adduct formation seems to be related to the incorporation of dFdC into DNA (r = 0.91). No increase in DNA platination was found in the H322 cell line. dFdC only increased Pt–DNA adduct retention in the A2780 and LL cell lines, but decreased the Pt–DNA adduct retention in the AG6000 cell line. In conclusion, the synergism between dFdC and CDDP appears to be mainly due to an increase in Pt–DNA adduct formation possibly related to changes in DNA due to dFdC incorporation into DNA. © 1999 Cancer Research Campaign2′,2′-Difluorodeoxycytidine  and


ErbB-2 and EGFR  are expressed in lung adenocarcinomas and associated with a poor prognosis. Immunocytochemical analysis revealed erbB-2 and EGFR coexperession as a characteristic feature of most lung adenocarcinomas, and at levels of receptor expression present in bronchial epithelial cells. In primary lung tumours and cell lines, erbB-2 detected using Western blot analysis demonstrated low-level phosphotyrosine staining of the 185 kDa band, as compared with breast cancer cell lines. A549 and A427 lung adenocarcinoma cells treated with neu differentiation factor (NDF) showed increased erbB-2 phosphotyrosine staining, but to a much lesser extent than breast cancer cells. The lung cells were examined for expression of the potential autocrine growth factors NDF and transforming growth factor alpha  by Northern blot analysis. Both NDF and TFG-alpha mRNA were abundantly expressed in the A549 cells. NDF mRNA was highest during active cell proliferation and decreased in confluent cells or after treatment with the growth-inhibitory steroid dexamethasone. Primary tumours and cell lines expressed EGFR, showing higher basal level phosphotyrosine staining than erbB-2. Treatment with NDF and EGF  stimulated cell growth, and in A549 cells the presence of both factors provided an additive increase in cell growth. The growth stimulus that ligand-activated erbB-2 and EGFR provides to lung adenocarcinoma cells may establish a background of continued cell proliferation over which other critical transforming events may occur.


The cellular pharmacology of a series of C8-linked pyrrolobenzodiazepine dimers with polymethylene linkers of n = 3-6 (compounds 1-4) has been studied in a range of human tumour cell lines. The four compounds showed the same pattern of relative activity in five ovarian carcinoma cell lines and one cervical carcinoma cell line with the order of IC50 values of 1 < or = 3 < 4 < 2, which correlated with the previously demonstrated DNA interstrand cross-linking ability of the compounds in plasmid DNA. In human leukaemic K562 cells the agents produced a block in the G2/M phase of the cell cycle characteristic of cross-linking drugs, and extensive interstrand cross-linking was observed in cells by alkaline elution with no evidence of single-strand breaks. Cross-links continued to increase up to 24 h following a 1 h exposure to drug, and no repair was evident by 48 h. In a series of ovarian and cervical carcinoma cell lines with acquired resistance to cisplatin no cross-resistance to the most potent compound 1 was observed in two lines whose major mechanism of resistance to cisplatin was reduced platinum transport. Cross-resistance to 1 was observed in a cell line (A2780cisR) possessing elevated glutathione, and depletion of intracellular glutathione using D,L-buthionine-S,R-sulphoximine (BSO) from 10.25 nmol to 2.8 nmol 10(-6) cells reduced the level of resistance from 11-fold to 2-fold compared with sensitive cells. Cross-linking in the resistant cells was restored to 80% of the level in the parent line by BSO pretreatment. There was also a correlation between glutathione levels and sensitivity to 1 measured in several other ovarian cell lines. Compound 1 also showed cross-resistance in the doxorubicin-resistant cell line 41MdoxR and partial cross-resistance in CH1doxR cells. Both these lines possess elevated levels of p170 glycoprotein. Following treatment with 6 microM verapamil, the resistance in these lines decreased almost 2-fold and 8-fold respectively.


Taxol is a substrate for this transporter system and is used in the treatment of a wide range of human malignancies including lung, breast and ovarian cancer. We have generated a series of ovarian cell lines resistant to this compound, all of which overexpress MDR-1 through gene amplification. We present novel evidence that a constitutive activation of the ERK1/2 MAP kinase pathway was also observed although the level of active JNK and p38 remained unchanged. Inhibition of the ERK1/2 MAP kinase pathway using UO126 or PD098059 re-sensitised the Taxol resistant cells at least 20-fold. Importantly, when Mdr-1 cDNA was stably expressed in the wild-type cell line to generate a highly Taxol-resistant sub-line, 1847/MDR5, ERK1/2 MAP kinases again became activated. This result demonstrated that the increased activity of the signalling pathway in the Taxol-resistant lines was directly attributable to MDR-1 overexpression and was not due to the effects of Taxol itself. Additionally, we demonstrated that inhibition of the P13K pathway with LY294002 sensitised the MDR-1-expressing 1847/TX0.5 cells and 1847/MDR5 cells at least 10-fold but had no effect in the wild-type cells. This finding suggests a possible role for this pathway, also, in the generation of resistance to Taxol. © 2001 Cancer Research Campaign


The effect of sodium butyrate on mouse and human melanoma cell lines was evaluated. Sodium butyrate (0.1-2mM) is shown to reduce the clonogenic potential of several melanoma cell lines. The antiproliferative effect of sodium butyrate is accompanied by a marked increase in the activity of the plasma-membrane bound enzyme gamma-glutamyl transpeptidase. Sodium butyrate treated cells acquire a well developed rough endoplasmic reticulum and accumulate fat droplets. The development of the endoplasmic reticulum is associated with a marked increase in the activity of the enzyme marker NADPH cytochrome c reductase. It is suggested that the phenotypic alterations induced by sodium butyrate may serve as markers for the action of this agent on melanoma cells and other tumours.


The study of blocking factors requires in vitro assay of cell mediated immunity that parallels the in vivo response. By microcytotoxicity testing, progressor and immune peripheral blood lymphocytes caused significant target cell reduction. The cytotoxicity was specific as no cytotoxic effect was detected against unrelated normal as well as a malignant target cell lines. No anti-tumour effect was noted when progressor peripheral blood lymphocytes were evaluated in the Winn assay. In marked contrast, immune peripheral blood lymphocytes were capable of preventing tumour growth in the Winn assay. Furthermore, hamsters repeatedly immunized with irradiated SV40 tumour cells could resist a live cell challenge. Thus immune peripheral blood lymphocytes were chosen as the effector population to evaluate the abrogation ability of serum in the microcytotoxicity assay.


The main results of these studies can besummarized as follows: (1) exogenous or endogenous catecholamines suppress PBL functions through alpha-2-receptor-mediatedmechanisms, lymphocytes of the spleen are resistant to adrenergic in vivo stimulation,(2) direct or indirect cholinergic treatment leads to enhanced ex vivo functions of splenic and thymic lymphocytesleaving PBL unaffected, (3) cholinergic pathways play a critical role in the “talking back” of the immune system to the brain,(4) acetylcholine inhibits apoptosis of thymocytes possibly via direct effects on thymic epithelial cells, and maythereby influence T-cell maturation, (5) lymphocytes of the various immunological compartments were found to beequipped with the key enzymes for the synthesis of both acetylcholine and norepinephrine, and to secrete theseneurotransmitters in culture supernatantsFor several years, our group has been studying the


An in vitro osteolysis assay with 45Ca-labelled mouse calvaria has been used to investigate mechanisms of direct bone invasion by squamous carcinomas of the head and neck. Short-term (3-day) organ cultures of 8 fresh squamous carcinomas showed varying degrees of in vitro bone-resorbing activity which was blocked by indomethacin, an inhibitor of prostaglandin synthesis. Supernatant media from 6 established cell lines also induced bone resorption in vitro and evoked an osteoclastic response in the cultured calvaria. Osteolysis by supernatant media was not blocked by indomethacin in all the tumour-cell lines, and the production of non-prostaglandin osteolysins by the indomethacin-resistant lines is postulated. The two principal findings that emerge are: (1) Stimulants for osteoclastic activity are derived from both squamous-carcinoma cells and from host cells in the tumour stroma. (2) These stimulants are diverse. Indomethacin-sensitive agents, presumed to be prostaglandins, are most convincingly demonstrated in the fresh tumours. Indomethacin-resistant agents, presumably not prostaglandins, are more characteristic of the carcinoma cell lines.


The properties of a new tumour cell line (NB1-G) derived from human neuroblastoma by xenografting in nude rats followed by adaptation to tissue culture are described. Studies using a panel of monoclonal antibodies demonstrate the neuro-ectodermal nature of the cells and support the diagnosis of the primary tumour as neuroblastoma. Cytogenetic studies have revealed a human karyotype with several chromosomal abnormalities. Genetic analysis by in situ DNA hybridization has demonstrated the presence of multiple copies of the N-myc gene. Approximately 20-30 fold amplification of the gene is observed on Southern blot analysis. The cell line has been adapted to growth as multicellular tumour spheroids as well as monolayer culture. Radiobiological studies on spheroids show the cells to be radiosensitive with low capacity for sub-lethal damage accumulation and repair. The cell line should be useful for fundamental studies of human neuroblastoma as well as experimental therapy in vitro.


MDR1, MRP1 or MRP2 cDNA, and 2008 ovarian carcinoma cells stably expressing MRP1 cDNA, we have investigated in this study the specificity of the reversal agents V-104 (a pipecolinate derivative), GF120918 , and Pluronic L61 (a (poly)oxypropethylene and (poly)oxypropylene block copolymer). Transport experiments with cytotoxic drugs with polarized cell lines indicate that all three compounds efficiently inhibit MDR1 Pgp. Furthermore, V-104 partially inhibits daunorubicin transport by MRP1 but not vinblastine transport by MRP2. V-104 reverses etoposide resistance of 2008/MRP1 cells, whereas GF120918 does not reverse resistance due to MRP1. V-104 partially inhibits the export of the organic anion dinitrophenyl S -glutathione by MDCKII-MRP1 but not by MDCKII-MRP2 cells. Unexpectedly, export of the organic anion calcein by MDCKII-MRP1 and MDCKII-MRP2 cells is stimulated by Pluronic L61, probably because it relieves the block on entry of calcein AM into the cell by endogenous MDR1 Pgp. © 2000 Cancer Research CampaignThe human multidrug transporter MDR1 P-glycoprotein and the multidrug resistance proteins MRP1 and MRP2 transport a range of cytotoxic drugs, resulting in multidrug resistance in tumour cells. To overcome this form of drug resistance in patients, several inhibitors  of these transporters have been isolated. Using polarized cell lines stably expressing human


Sir,British Journal of Cancer (2007) 97: 934–940, we indeed did not check the authenticity of the cell lines MCF-7/ADR and KB, so, it is likely that MCF-7/ADR is not an authentic variant of the MCF-7 breast carcinoma cell line and that KB is not derived from oral carcinoma, although there are numerous papers to mention that MCF-7/ADR is a variant of the MCF-7 breast carcinoma cell line and KB is derived from oral carcinoma. However, the authenticity of these cell lines does not compromise the conclusion and scientific significance of this paper. In our article, it is very clear that these cell lines (MCF-7/ADR and KBV200) are drug resistant and their drug resistances are mediated by P-glycoprotein. These characteristics of these cell lines are enough to justify them to be used to study the pharmacologic effects of ZD6474 and the mechanisms involved.In the article ZD6474 reverses multidrug resistance by directly inhibiting the function of P-glycoprotein in


We have produced adriamycin (ADM)-resistant variants of the human lung cancer cell lines NCI-H69 , MOR (adenocarcinoma) and COR-L23 (large cell) but have failed to produce resistant variants of two other small cell lines. In each case, the derivation protocol took 7-9 months and included a period of drug-free growth. All three resistant lines show reduced cellular content of ADM after 1 h exposure when compared with their controls. During prolonged incubation of control and resistant NCI-H69 cells in 0.4 microgram ml-1 ADM, the ADM content of resistant cells was 6-7 times lower than that of control cells. The ratio of ADM doses to suppress growth of the two lines, however, was in the range of 40-200X. The ADM-resistant variant of NCI-H69 was also resistant to vincristine, colchicine, VP16, mitozantrone, 4' epiadriamycin and 4' deoxyadriamycin, somewhat resistant to melphalan but not resistant to aclacinomycin A, bleomycin of CCNU. The resistance to ADM could be partially overcome by the use of verapamil, an inhibitor of calcium transport.


Additional cell lines expressing both Neu and S100A4 have also been derived by transfection of rat S100A4 cDNA into tumour cell lines cloned from neu single transgenic mice. Using these cells in transfilter migration assays, it has been shown that increases in either motility or invasive properties correlate with each other and with the level of S100A4 protein. Injection of three of the cell lines separately into the mammary fat pads of nude mice showed that elevated levels of S100A4 correlated with the degree of metastasis to the lungs. In contrast, changes in cell proliferation and cell–substrate adhesion did not correlate with S100A4 levels. Neither motility nor invasiveness correlated with proteolytic degradation of gelatin as measured by zymography. Thus, the results suggest that the main effect of increases in S100A4 levels in metastasis is to generate increased cell motility and invasion and that this latter change is not dependent upon an increased ability to degrade the intercellular matrix.Elevated levels of the calcium-binding protein S100A4 are associated with poor patient survival in breast cancer patients and induce metastasis in rodent models. To investigate the effects of S100A4 on different components of the metastatic process, epithelial cells lines have been isolated from nonmalignant tumours in  The processes by which solid primary tumours, such as those of the breast, are able to disseminate and establish growth at a secondary site are still poorly understood. Tumour metastasis is complex, probably requiring both gain and loss of functions, enabling escape from the primary tumour, and growth at a secondary site. This has led to the search for metastasis-associated genes, which, unlike oncogenes, are unable to initiate tumour formation but are able to induce a metastatic phenotype in previously tumorigenic cells  foetal calf serum (FCS), 100 ng ml−1 insulin, 50 ng ml−1 hydrocortisone). The cells were tested for the expression of peanut lectin-binding protein and cytokeratin 18  (−1 Geneticin (G418). Cells were cultured in this Selective Medium for 2 weeks, the medium being replaced every 2–3 days and the resulting colonies for any one cell line pooled and expanded. Incubation of control nontransfected cells with the Selective Medium caused death of all the cells within 5 days.For transfection, cell lines grown to approximately 60% confluence in 3 cm diameter dishes were transfected with 0.5 μl of 0.125 M Tris-HCl (pH 6.8), 4% (w v−1) sodium dodecyl sulphate (SDS), 20% (v v−1) glycerol; scraped with a rubber policeman, collected in 1.5 ml tubes and protein quantitated using Bradford's reagent  following the manufacturer's instructions. Samples (10 μg) of protein from each cell line in gel loading buffer (0.125 M Tris-HCl (pH 6.8), 4% (w v−1) SDS, 20% (v v−1) glycerol, 0.04% (w v−1) bromophenol blue, 10% (v v−1) 2-mercaptoethanol), heated to 100°C, were separated by electrophoresis on 10 or 15% (w v−1) polyacrylamide gels in the presence of SDS. The separated polypeptides were transferred to nitrocellulose membranes and incubated with a primary antibody to either c-erbB-2  or S100A4 , lysed in 100 5 cells ml−1. Cell suspension (1 ml) was added to each well of a 24-well plate and at time periods of 0.5, 1, 1.5, 2.0, 2.5 h, the number of adherent cells was found by washing the wells three times with PBS to remove any cells in suspension. Any cells adhering to the well were trypsinised and counted using the Coulter counter. Each experiment was carried out in triplicate.Cells were trypsinised and counted using a Coulter counter, and resuspended at 2 × 104 cells ml−1 and 1 ml was added to each well of a 24-well plate. The cells were incubated for 24, 48, 72 h periods, and at the end of each time period the number of cells was found by trypsinising the cells and counting them in a Coulter counter. Each experiment was carried out in triplicate.Cells were trypsinised and counted as described previously, and resuspended at 1 × 10μm pores were measured using a Boyden chamber in a 24-well plate assay system . Chemotactic-induced motility in response to a FCS concentration gradient was measured by adding 400 μl of Routine Medium containing 10% (v v−1) FCS to the lower compartment, and 2 × 105 cells in 200 μl of Routine Medium, but with 2% (v v−1) FCS to the upper compartment of the Boyden chamber. The cells were incubated for 24 h, the upper side of the filter was wiped with a cotton swab to remove any nonmotile cells, and the motile cells on the lower side of the filter were fixed and stained using the Diffquik histochemical stain  according to the manufacturer's instructions. The lower compartment of the Boyden chamber was checked for cells and the number of stained cells/field of 0.50 mm2 on the lower side of the filter was counted using a Dynascope with a × 20 objective . For each cell line, four experiments were carried out, each experiment consisting of three filters and 10 fields per filter were counted. In controls, fixed numbers of cells incubated for different times up to 24 h showed a linear increase in the number of motile cells on the lower side of the filter. Thereafter, there was an appreciable number of cells present in the lower chamber.The motile and invasive abilities of the cell lines through a filter containing 8 μg Matrigel  and the experiments were carried out as for the chemotactic motility assays. The number of invasive cells/field on the lower side of the filters were determined as for the motility assays for three experiments for each cell line.For cell invasion, the filters were coated with 50 −1) FCS, 200 ng ml−1 insulin and 50 ng ml−1 hydrocortisone for 48 h. The media were harvested from each of the cell lines, grown to either 40–50% confluency (sparse cultures) or 80–90% confluency (dense cultures) and any protein concentrated into a 1 ml volume or less using an Ultrafree-15 centrifugal filter unit . The concentration of protein in each sample was then determined by measuring the absorbance at 595 nm using Bradford's reagent  following the manufacturer's instructions and relating it to the absorbance found for standards containing a known concentration of bovine serum albumin.Cell lines were washed twice with PBS and incubated in 8 ml of growth medium consisting of phenol-red-free DMEM, 2% (v v−1) SDS polyacrylamide resolving gels containing 1 mg ml−1 of gelatin, with 4% (w v−1) polyacrylamide stacking gels using the Bio-Rad Miniprotean system, as described previously (μg of conditioned medium in loading buffer (0.4 M Tris-HCl (pH 6.8), 5% (w v−1) SDS, 20% (w v−1) glycerol, 0.03% (w v−1) bromophenol blue) were loaded onto the gel, without being heated and in the absence of 2-mercaptoethanol. Gels were run at 4°C until the dye front reached the anode end of the gel. The resolving gel was washed for 2 × 30 min in 2.5% (v v−1) Triton X-100 to remove the SDS from the gel. Following which the gels were incubated for 18 h at 37°C in substrate buffer (50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 75 mM NaCl). The gel was subsequently incubated in Coomassie blue stain for 1 h and then incubated in destain (45% (v v−1) methanol, 5% (v v−1) glacial acetic acid) for 1 h, until the digested bands in the gel became clear. The gels were dried on gel drying frames between cellophane sheets .Conditioned medium was analysed on 10% . The tumours were selected from mice representative of the general phenotype of the transgene status, as demonstrated by haematoxylin and eosin-stained tumour sections. Three permanently growing cell clones were established from the mammary gland tumours of a neu transgenic mouse and termed 4Neu, 5Neu and 8Neu. Two permanently growing cell clones were established from the mammary gland tumours of a bitransgenic neu/S100A4 mouse and termed PN1 and PN2. The stable transfection of the 4Neu and 8Neu clonal cell lines isolated from the neu transgenic mouse with the rat pSV2neo-p9Ka (S100A4) expression vector yielded two further cell lines obtained from the pooled transfectant clones, termed 4Neup9Ka and 8Neup9Ka, respectively, which grew in Geneticin-containing medium. Parallel mock transfections without the expression vector yielded no colonies after incubation of cells in Geneticin-containing medium. All of the isolated cell lines were epithelial in nature as determined by staining for epithelial lineage markers such as Cytokeratin 18 and peanut lectin-binding protein (not shown)  .t-test, P<0.05). The correct sized protein of apparent molecular weight 9 kDa was seen for all cell lines . The correct sized protein of 185 kDa was seen for all the cell lines, but an additional band at 135 kDa was sometimes seen; this may have been due to crossreactivity with the EGF receptor   protein levels    , althoug data in .t-test, P<0.03)  were higher than those isolated from neu transgenic mice   . Moreoveu, 8Neu) .t-test, P<0.05). When S100A4 protein levels are plotted against motility, there is a significant linear relationship when assessed by a least-squares regression analysis   (Comparison of S100A4 levels with cell motility for the different cell lines showed that S100A4 levels and motility were closely associated . This diP=0.012) . In the  P=0.88) .t-test, P<0.03)  were higher than those isolated from neu transgenic , that for PN2 was significantly so .To allow for direct comparison between cell invasion and cell motility, the invasive abilities of the cell lines were measured using the assay conditions optimised for the motility assay but with a coating of Matrigel on the filter separating the upper and lower compartment of the Boyden chamber. Using this assay, cell invasion for the different cell lines varied from about three to nearly 26 invading cells per field . The inv P<0.03) . Moreovet-test, P<0.01) and demonstrated a significantly higher invasive ability when compared individually to 8Neu . A significant linear correlation between S100A4 protein levels and invasive ability was demonstrated by least-squares regression analysis for each cell line    . However P=0.96) .R2=0.95; P=0.00096)  . Interes0.00096) . It woul−1) FCS, the same medium as that used in the upper chamber of the Boyden system during the invasion assay. To analyse any effect of cell confluency on MMP production, medium was harvested from sparse cultures of cells, grown to 40–50% confluency and from dense cultures of cells grown to 80–90% confluency.Matrix metalloproteinase (MMP) activities were measured by gelatin zymography of conditioned media harvested from cell lines grown in Routine Medium supplemented with 2%  and MMP9 (Mr 78 kDa)  (P<0.03). The incidence of metastasis in the high S100A4-expressing PN2 cell line was also greater than that in the moderate S100A4-expressing 8Neup9Ka cell line, although not statistically significant . Furthermore, when the level of S100A4 protein in the cultured cells was plotted against the percentage of tumour-bearing mice with lung metastases, a straight line could be drawn through the three points  . Metastases were also observed in the inguinal lymph nodes of some of the mice injected with the 8Neup9Ka and PN2 cell lines; however, these were not included in any statistical analyses, since lymph node tissue was not available from all of the injected mice.The primary tumours consisted of chord-like structures  . In contImmunocytochemical staining for Neu showed that all three cell lines 8Neu (Figure 5A and PN2  produced and PN2 . Lymph n and PN2 , the mic and PN2  and the  and PN2  in the l and PN2 . The tum and PN2 , which v and PN2 . Immunoc and PN2 , and all and PN2 . There w and PN2 . Althoug and PN2 . These rneu transgene or by isolation of cell lines from mice carrying both the neu and the S100A4 transgenes. The former mice produce only nonmetastatic tumours, while the latter mice produce metastatic tumours (in vitro, which may be related to metastasis in vivo. There was no correlation between the levels of S100A4 and cell-substrate adhesion to plastic substrata or proliferation rates for this particular set of cell lines; cell–cell adhesion was not measured. These data are the first to demonstrate in a transgenic murine-derived tumour system that S100A4 levels correlate positively in a linear fashion with cell invasive ability through Matrigel in response to an FCS density gradient. This result demonstrates also that S100A4 causes, either directly or indirectly, the enhanced invasive ability in at least the S100A4-transfected cell lines. In contrast, correlation of levels of Neu protein with only proliferation rates and not with substrate adhesion, motility or invasion is consistent with the expression of the neu transgene causing the largely proliferative breast lesions and noninvasive breast tumours observed in transgenic mice (neu transgenic mouse tumour. These analyses still yielded the same significant correlations as before (Cell lines expressing elevated levels of S100A4 have been obtained either by direct transfection of cell lines from mice carrying the nic mice , the anas before .in vitro of Lewis lung carcinoma cell lines (in vitro (Whether production of S100A4 correlates with invasive ability has remained equivocal. Previously, S100A4 had been shown to correlate with the invasive ability in vivo is more complex (Matrix metalloproteinase production could, however, be regulated in all of the cell lines by confluency in culture. Matrix metalloproteinase activity was observed only in the conditioned medium of sparsely plated cells. It would therefore seem that complete cell–cell contact provides an inhibitory extracellular signal, which prevents MMP production/secretion in these cells. Previous studies have also demonstrated that proteolytic activity is dependent on cell density. For example, in the media of sparse astrocytoma cells, more collagenase activity was seen than in dense cultures (in vitro, showed a strong linear correlation with levels of S100A4 protein in the different cell lines. The stimulus for motility did not need to be directional, since chemotactic and chemokinetic stimuli provided by 10% FCS, induced the same degree of motility (in vitro suggest that an appropriate and sufficient chemical stimulus is probably required for cell migration in vivo. The results demonstrated here are in accordance with previous findings in other cell systems that increased S100A4 levels results in increased cell motility (in vitro, were not invasive in vitro or in vivo, due to a lack of MMP production (In contrast to gelatinase activity, motility, a second major component of invasion examined motility . Howevermotility , suggestmotility  suggestsmotility , the thrin vitro (inter alia (The mechanisms whereby S100A4 modulates cell motility are unknown. However, S100A4 is known to bind to various components of the actin–myosin cytoskeleton (neu and neu/S100A4 transgenic mice increased the efficiency of mammary tumour production from 44 and 68% as seen after 14 months in neu and neu/S100A4 transgenic mice (neu was observed in the cell lines. There was no requirement to add hormones in vitro or to mate in vivo to stimulate the expression of neu, whereas in the original transgenic mice the neu transgene requires the hormones of pregnancy to activate its MMTV promoter (neu transgenic mice all of the epithelial cells express the neu transgene, but only some cells are able to form tumours. This latter result indicates that a second genetic change is required, in addition to expression of the activated neu transgene, to allow tumour formation. One possible explanation is the high level of the loss of heterozygosity, a fact which may be related to the loss of tumour suppressor genes, which has previously been observed in these neu transgenic mice (The tumorigenic and metastatic abilities of the cell lines were examined in the immunoincompetent, nude mouse, without the complication of an immune response. The isolation and culture of tumour cells from both nic mice . Since cneu/S100A4 tumour-derived PN2 cell line as in the neu/S100A4 transgenic mice, with only 50% of mice with mammary gland tumours developing lung metastases. The efficiency of tumour metastasis for the 8Neup9Ka cell line was lower, with only 30% of mice with mammary gland tumours developing lung metastases. The 8Neu cell line developed no lung metastases. These in vivo results follow the same pattern observed for the cell lines in in vitro assays, where the 8Neu cell line had a low level, the 8Neup9Ka cell line a moderate level, and the PN2 cell line a high level of both motility and invasion. Confirmation that these metastases are derived from the neu/S100A4-expressing mammary gland tumours is obtained by positive staining of these metastases for both Neu and S100A4. The level of metastasis expressed as a percentage of afflicted animals was found to be positively correlated with the level of S100A4, but not with the level of Neu protein in the cell lines, albeit for only three of the cell lines. Metastasis in these Neu-expressing cell lines is therefore clearly dependent, either directly or indirectly, on the level of S100A4 protein.The frequency of tumour metastasis, however, remained the same in the neu acts as an oncogene producing neoplastic transformation of the targeted mammary epithelial cells in vivo (neu these cells remain normal and cannot be grown in culture (neu transgenic mice (in vivo. Tissues of transgenic mice overexpressing S100A4, on the other hand, fail to undergo neoplastic transformation and that includes those of the mammary gland (neu-transformed neoplastic immortalised mammary cell lines, in vitro studies demonstrate that S100A4 levels correlate well with cell invasion and that S100A4 mediates, either directly or indirectly, this invasion through its ability to induce cell motility, at least in the transfected cells. It therefore seems probable that S100A4-driven cell motility mediates invasion in vivo and thereby metastasis in vivo, at least in this particular model system.In this transgenic mouse model system,  culture  without


Some tumour cell lines, notably the vast majority of melanomas and hepatocellular carcinomas, tend to be deficient in argininosuccinate synthetase (EC 6.5.4.3.), and therefore cannot recycle citrulline to arginine. Argininosuccinate synthetase is present at levels that convert enough citrulline to arginine to allow limited growth in about half of a modest range of malignant cell types analysed in this study. Attempts to rescue cells that are unable to utilise citrulline with the immediate downstream product, argininosuccinate, had very limited success in a few tumour cell lines. Particularly noteworthy is the demonstration that argininosuccinate was totally incapable of rescuing cells that utilise citrulline efficiently, consistent with tight channelling (coupling) of argininosuccinate synthetase and argininosuccinate lyase in the urea cycle. The findings suggest that an excellent opportunity exists for further exploitation of arginine deprivation in the selective killing of tumour cells.Arginine deprivation causes death of up to 80% of cancer cell lines  Having successfully reduced L1210 cells in culture to negligible viability in 3–5 days with arginase (EC 3.5.3.1), citrulline supplementation in the continued presence of the enzyme partially circumvented the arginine deficiency. The rate of conversion became a rate-limiting factor, most noticeably in such fast-growing leukaemia as L1210 . In a corresponding in vivo study, arginase treatment of DBA/2 mice injected intraperitoneally with 1210 cells increased neither their survival time nor decreased tumour burden, despite plasma arginine falling to ∼1–2 μM level. Plasma citrulline remained normal, and therefore the tumour cells must have converted citrulline to arginine fast enough to sustain relatively normal tumour growth rate  generation). Its removal from culture medium by medium formulation or arginase treatment quickly leads to death in ∼80% of tumour cell lines . Counts are representative of the normal cell population, within standard parameters, and unless otherwise stated cannot be taken as an indication of viability. All cell cultures were routinely screened, and just prior to experimental use, for four types of mycoplasma, using the Mycoplasma Screening Kit of Roche , according to the manufacturer's instructions to ensure that no cultures used for experimental work tested positive.Cells from a wide range of suppliers and sources were grown in RPMI 1640 medium  at 37°C in a humidified atmosphere of 5% COM arginine and 5% dialysed foetal calf serum (FCS) overnight. Prior to treatment, cells were washed with prewarmed arginine-free medium (hereafter AFM) and treated with fresh AFM to which arginine, argininosuccinate, citrulline and ornithine was added at 1 mM, where appropriate. Native bovine arginase  or human recombinant arginase  was added at a concentration of 1 U ml−1 to arginine-containing RPMI. In the case of L1210 cells, a suspension culture line, cells were treated immediately on being resuspended at the desired concentration in AFM to which arginine, argininosuccinate, citrulline and ornithine were added as desired.Cells were seeded at approximately 50 000 cells per well in 12-well plates and allowed to settle in RPMI medium containing 1 min vitro overtime at 37°C in a humidified atmosphere of 5% CO2 to show that it maintained activity over the course of each experiment, using the ornithine method of μmol min−1 mg protein−1. In all, 1 U of arginase was taken as the amount that broke down 1 μmol arginine min−1.Arginase was analysed  pathway  because The findings on human melanomas contrast sharply with those on mouse B16F10 melanoma cells, which efficiently handle citrulline , but unlall tumour cell types given in in situ from citrulline) is used by the lyase to form arginine. The three human leukaemic cell lines  arginine. Although rescue with argininsuccinate in AFM with traces of contaminating arginine is therefore very unlikely, it would have been pre-emptorily destroyed before the cells could make use of it where arginase had been used to create the deficiency  normally operates, thereby preventing the access of argininosuccinate to argininosuccinate lyase (EC 4.3.2.1.), which would explain the absence of Type C/AS tumours . This paRescue of cell lines incapable of utilising citrulline with argininosuccinate does occur, but with considerable differences between the cell lines. CHO is an excellent example of a cell line that cannot use it and is therefore totally dependent on arginine. This is more noteworthy because the generalisation of not exclusively show this argininosuccinate synthetase deficiency, the findings show that most cases of this dangerous and aggressive tumour can be appropriately and quickly selected for treatment. They are the preferred targets for therapy, for which screening of primary tumours has already begun.Nevertheless, the inability of a number of tumour cell lines to utilise citrulline renders them prime targets for eradication by arginine deprivation, whether achieved by leaving it out in medium formulation or using catabolic enzymes in its presence. This potential Achilles' heel in tumour metabolism deserves further exploitation in view of our previous findings (


Concentrations of methylprednisolone which have lethal effects on human lymphoblastoid cell lines in vitro cause a reduction both in the uptake of uridine and thymidine into acid-soluble material and their incorporation into acid-insoluble material. These effects are virtually instantaneous, which indicates that they do not depend on alterations in gene activity. Normal uptake of nucleosides into cells is by both simple and facilitated diffusion, and methylprednisolone appears to act directly on the cell surface to inhibit only facilitated diffusion uptake.


The expression of the gp100 antigen is generally thought to be confined to cells of the melanocytic lineage, which makes the protein a suitable melanoma-specific marker. Strikingly, after screening a panel of normal tissues, tumour samples and cell lines of non-melanocytic origin, we found transcripts encoding gp100 in virtually every tissue and cell line tested. In contrast, tyrosinase and MART-1/MelanA transcripts were detected only in cells of the melanocytic lineage. However, no gp100 protein could be detected by either Western blotting or cytotoxicity assays. Therefore, at the protein level, gp100 remains exclusive for cells of melanocytic origin despite its transcription in many cell types. The major implication of this finding is that screening of patient material for gp100 expression should preferrably be performed by antibody staining. Reverse transcriptase polymerase chain reaction (RT-PCR) can be employed, provided that it is performed in a tightly controlled, semiquantitative setting.


The Wnts can be classified into two classes based on their ability to transform cells. The Wnt5a class can antagonize the effects of transforming Wnts partly through effects on cell migration. To understand the mechanisms of regulation of Wnt5a, we investigated its expression in human normal and breast cancer cell lines. Elevation of Wnt5a in HB2, a normal breast epithelial cell line, was linearly correlated with cell density, but this did not occur in cancer cell lines. We examined intracellular events responsible for the regulation of Wnt5a by cell to cell contacts, using various metabolic agents known to affect signal transduction pathways. Agents that selectively blocked protein kinase C  or protein tyrosine kinases (genistein) reduced the level of Wnt5a expression markedly. Protein kinase C activation by phorbol 12-myristate 13-acetate up-regulated Wnt5a partly through prolongation of Wnt5a mRNA half-life. Cytoskeleton reorganization following cytochalasin D treatment caused an induction of Wnt5a, which was associated with changes in cell morphology. Calphostin C did not block these effects, showing that protein kinase C is acting upstream of cytoskeletal modulation. However, the cancer cell lines treated with cytochalasin D showed no changes in cell morphology or Wnt5a induction, suggesting disruption of this regulatory pathway in cancer.


Bryostatin 1 is a novel macrocyclic lactone activator of protein kinase C (PKC) which has clinical potential as an anti-cancer agent. The mechanism of action of this agent is unknown, but protein kinase C has been implicated. In order to investigate this possibility, we have developed P388 sublines resistant to bryostatin 1, by continuous challenge of the parent cell line with increasing incremental concentrations of the drug over 4 months. Cell lines were established at monthly intervals yielding four sublines: P388/BR/A, which were removed at 1 month; P388/BR/B, obtained after 2 months; P388/BR/C, obtained after 3 months; and P388/BR/D, which were established after 4 months. All four P388/BR sublines show an equal degree of resistance to the growth inhibitory effects of bryostatin 1, with a relative resistance ratio (RR) IC50 of approximately 4,000. The ability of the cytosol of cells to phosphorylate PKC-specific substrate is decreased by 41% for BR/A, 57% for BR/B 80% for BR/C and 94% for BR/D compared with the parental cell line, even when grown in the absence of bryostatin 1 for up to 4 weeks. Similar decreases are seen for cytosolic phorbol ester binding and whole-cell PKC isoenzyme expression. All four P388/BR sublines show high and equal levels of cross-resistance to the PKC activatory phorbol ester, phorbol 12-myristate 13-acetate (PMA). There is no loss of resistance to either bryostatin 1 or PMA up to 3 months after termination of exposure of the sublines to bryostatin 1. There was no significant degree of cross-resistance to daunorubicin in the bryosatin 1-resistant cell lines, P388/BR/A, B, C or D, when compared with the parent cell line, P388.


SP-G inhibits mitogenic neuropeptide signal transduction and small cell lung cancer (SCLC) cell growth in vitro and in vivo. Using the SCLC cell line series GLC14, 16 and 19, derived from a single patient during the clinical course of their disease and the development of chemoresistance, it is shown that there was an increase in responsiveness to neuropeptides. This was paralleled by an increased sensitivity to SP-G. In a selected panel of tumour cell lines , the expression of the mitogenic neuropeptide receptors for vasopressin, gastrin-releasing peptide (GRP), bradykinin and gastrin was examined, and their sensitivity to SP-G tested in vitro and in vivo. The tumour cell lines displayed a range of sensitivity to SP-G . The expression of the GRP receptor measured by reverse transcriptase–polymerase chain reaction, correlated significantly with growth inhibition by SP-G. Moreover, introduction of the GRP receptor into rat-1A fibroblasts markedly increased their sensitivity to SP-G. The measurement of receptor expression from biopsy samples by polymerase chain reaction could provide a suitable diagnostic test to predict efficacy to SP-G clinically. This strategy would be of potential benefit in neuropeptide receptor-expressing tumours in addition to SCLC, and in tumours that are relatively resistant to conventional chemotherapy.[Arg Despite recent advances in cytotoxic drug development, it is a universal goal to develop novel cancer therapies that are more specific to cancer cells and produce minimal damage to nonmalignant cells. One approach to realising this goal is the development of drugs that are able to block the growth-promoting effects of cancer cell mitogens.in vitro and also as xenografts in nude mice  (SP-G), were initially identified as antagonists of substance P-mediated cellular effects and were subsequently found to also antagonise the cellular effects of bombesin . SCLC cells proliferate in response to a range of neuropeptide growth factors, and in many cases these neuropeptides are involved in autocrine and paracrine growth loops that fuel unrestrained proliferation , gastrin-releasing peptide (GRP), bradykinin (BK2) and gastrin by reverse transcriptase–polymerase chain reaction (RT–PCR), and show that GRP-receptor expression predicts sensitivity to SP-G in a variety of tumours , colorectal, ovarian and pancreatic) in vitro and in vivo. Our results suggest that an antineuropeptide growth factor strategy may be effective in a wider range of tumours and may also be of benefit in these neuropeptide-expressing tumours, which have acquired relative resistance to conventional chemotherapeutic agents.In this study, we measured the expression of the mitogenic neuropeptide receptors for vasopressin (VRat-1a cells and rat-1a cells stably expressing the mouse bombesin/GRP receptor (GRPR) (BOR-15) were established by the Imperial Cancer Research Fund . RPMI-1640, Dulbecco's essential Eagle's medium (DMEM), bombesin and vasopressin were from Sigma . SP-G was a kind gift from Peptec . All other reagents were of the purest grade available.−1) fetal bovine serum (heat-inactivated at 57°C for 1 h) 50 U ml−1 penicillin, 50 μg ml−1 streptomycin and 5 μg ml−1L-glutamine in a humidified atmosphere of 5% CO2 : 95% air at 37°C. For experimental purposes, cells were either grown in SITA medium ) or with 0.1% (v v−1) fetal bovine serum in DMEM (nontumour cell lines). Rat-1A fibroblasts stably transfected with the mammalian bombesin receptor (BOR-15 cells) were cultured in the presence of 400 μg ml−1 G418-sulphate.Stocks were maintained in RPMI-1640 (tumour cell lines) or DMEM (nontumour cell lines) supplemented with 10% . Colony growth was determined in 0.3% agarose in SITA medium for 21 days as described by 2+concentration was determined using the fluorescent indicator Fura-2-tetraacetoxymethylester AME (FURA-2-AM 1 μM) as described (Intracellular Canu/nu mice (6 weeks old)  were maintained in negative pressure isolators . All xenografts used throughout were initially established by subcutaneous injection of 107 cells from their respective cell lines and then maintained by serial passage of fragments of viable tumour as described previously  was then calculated by the formula V=π/6LW2, where L is the longest diameter and W the diameter perpendicular to L. Results were expressed as a relative tumour volume (RTV) which is defi-ned simply as: V on day x/V on day 0, with RTV=1 on day 0. Xeno-graft experiments were repeated on two or three separate occasions.Mice were allocated into control and treatment groups containing six to eight mice. Treatment started when the xenografts reached a diameter of 3–10 mm with the first day of peptide administration designated as day 0. SP-G was dissolved in sterile distilled water at a concentration of 10 mg ml1AR sense 5′-TACCTGCTACGGCTTCATCTGC-3′, antisense 5′-ACACAGTCTTGAAGGAGATGGC-3′; BK2R sense 5′-CCTGAGTGTCATCACCTTCTGC-3′, antisense 5′-TTGATGACACGGCAGAGG-3′; receptor for gastrin (gastrinR) sense 5′-CCTATCTCCTTCATTCACTTGC-3′, antisense 5′-AGTGTGCTGATGGTGGTGTAGC-3′; γ-actin sense 5′-ATGGCATCGTCACCAACTGG-3′, antisense 5′-ATGACAATGCCAGTG-GTGGC-3′. Polymerase chain reaction products were run on a 1.5% agarose gel containing 1 μg ml−1 ethidium bromide. Stored images of the gels were analysed by densitometry using Gel Base/Gel Blot software. γ-Actin levels were measured as an internal control. Each product was expressed relative to the levels of γ-actin for the same cDNA batch and for each PCR reaction.RNA was extracted using Tri-Reagent  from exponentially growing cells, which had been in SITA medium for 3–5 days and was subsequently treated with DNase1 to remove any DNA contamination. cDNA was produced from this RNA using a first-strand synthesis kit . PCR primers used were: GRPR sense 5′-ATCTTCTGTACAGTCAAGTCC-3′, antisense 5′-GCTTTCCTCATGGAAGGGATA-3′; Vin vitro sensitivity to chemotherapeutic agents reflected the clinical resistance to treatment (M) in each of the three cell lines were screened for their ability to increase [Ca2+]i. 2+]i and smaller responses were seen in case of neuropeptides CCK, GRP and neuromedin B. No Ca2+ mobilisation was observed in response to neurotensin, substance P or vasopressin. However, in the GLC19 line, addition of neuropeptides bradykinin, CCK, GRP, neuromedin B and substance P caused large increases in [Ca2+]i. Vasopressin and neurotensin caused smaller but consistent increases in [Ca2+]. The GLC19 was the only cell line in which neurotensin caused a measurable increase in [Ca2+]i.The GLC14, 16 and 19 cell lines are SCLC cell lines established from one patient during longitudinal follow-up  and the HT29 colorectal cell line (IC50=18 μM). Four out of five SCLC cell lines had IC50 values below the mean IC50 value of 44 μM. Both the ovarian carcinoma and the non-small-cell carcinoma were sensitive to growth inhibition by SP-G, with IC50 values of 31 and 33.5 μM, respectively. During the phase 1 clinical trial of SP-G, plasma levels of up to 40 μM were achievable with no dose-limiting toxicity , NSCLC and colon cancer, which are relatively insensitive to standard conventional chemotherapy.Four members from the original panel of tumour cell lines were established as xenografts from their respective cell lines: H69 SCLC, HT29 colon carcinoma, PEO4 ovarian cancer and PANC-1 pancreatic cancer. Typical growth inhibition curves . Receptors for vasopressin (V1AR) were detected in four out of four lung cancer cell lines (three SCLC and one NSCLC). The WX330 SCLC cell line was the highest expresser of V1AR. The pancreatic and ovarian cancer lines expressed low levels of the V1AR and in the colorectal cancer cell lines only HT29 cells expressed V1AR. GRPRs were detected in three out of four lung cancer cell lines (two SCLC and one NSCLC). In the colorectal cell lines, the HT29 cell line showed high levels of GRPR expression and HCT116 cell line showed no detectable GRPR expression with intermediate expression in the HT18 cell line. The pancreatic cell line PANC1 had very low levels of GRPR expression. The PEO4 ovarian cancer cell line and H69 SCLC cell lines showed high expression of the GRPR (γ-actin) and sensitivity to SP-G was the most apparent  , whereas BOR15 cell growth was completely inhibited by 80 μM SP-G  was expressed at high levels . The sensitive cell lines  had IC50 values that were in the range of physiologically obtainable plasma concentrations. Moreover, in the cell lines tested in vitro sensitivity correlated with sensitivity to SP-G in vivo. The results of this screen suggest that SP-G could be used therapeutically against several tumour types in addition to SCLC.It is well known that neuropeptides can stimulate the growth of many types of cancers including pancreatic, colorectal, prostate, ovarian, breast and NSCLC (in vitro (and in vivo) are resistant to standard chemotherapeutic agents such as etoposide, are sensitive to the growth-inhibitory effects of SP-G. This raises the possibility that SP-G may be an effective anticancer agent in patients with neuropeptide-expressing tumours which are intrinsically resistant or have acquired resistance to conventional chemotherapy. A plausible extension of this hypothesis is that clinically aggressive drug-resistant SCLC cells that emerge after chemotherapy might have a more extensive network of neuropeptide regulation and therefore display increased sensitivity to neuropeptide antagonists. Further studies in longitudinal cell lines are required to investigate this hypothesis in further detail.We have previously shown that SP-G augments etoposide-induced growth inhibition and apoptosis in SCLC cells and suggested that SP-G may be of increased benefit in patients following relapse or in conjunction with conventional chemotherapy  pathways leading to growth arrest and apoptosis (The GRPR has been shown to be oncogenic when transfected into the nonmalignant NCM460 colon epithelial cell line (These findings have important implications for the design of more advanced phase human clinical trials using substance P analogues. SP-G is currently entering a phase II clinical trial where its effectiveness will be tested in SCLC patients, but ultimately, compounds of this type may also be suitable for the treatment of a wide range of other tumour types and neuropeptide-expressing tumours that have become resistant to standard conventional chemotherapeutic agents. Screening tumour biopsy samples for neuropeptide receptor expression may provide insight into the likelihood of patients responding to treatment with substance P analogues, analogous to oestrogen receptor expression conferring efficacy to tamoxifen in breast cancer. It is suggested that tumours should be biopsied to select patients for substance P-analogue trials based on the expression of the GRPR and another neuropeptide receptor. Our results suggest that these tumours should show growth inhibition regardless of intrinsic or acquired resistance to standard chemotherapeutic agents. It is therefore proposed that in the first instance, patients with tumours that express the GRPR and another neuropeptide receptor who have failed conventional treatment should be randomised into two groups – best supportive care and treatment with substance P analogues.


A cytotoxicity assay was used to study the action of bacillus Calmette-Guerin (BCG) and cytokines on four human bladder cancer cell lines. Monocytes and lymphocytes from peripheral blood were incubated with or without BCG or cytokines for 24 h, after which [3H]thymidine-labelled target cells were added and the 72 h percentage specific release determined. BCG had a direct cytotoxic effect against tumour cells and significantly enhanced monocyte/macrophage and enhanced lymphocyte cytotoxicity against one cell line (UCRU-BL-17). Supernatants (SNs) from BCG-activated monocytes/macrophages and lymphocytes increased the percentage specific release of [3H]thymidine from UCRU-BL-17 cells. Interferon alpha  and interleukin 2 (IL-2) were cytotoxic towards UCRU-BL-17. No synergy occurred between BCG and cytokines at the concentrations tested. The results suggest that BCG is superior to IFN-alpha, interferon gamma (IFN-gamma) and IL-2 in enhancing cell-mediated cytotoxicity.


Thymidine (1 μM) completely prevented growth inhibition at the MTA IC50in all cell lines. At 10 × IC50, growth inhibition was only partially reversed by thymidine (≤ 10 μM); both thymidine and hypoxanthine (30 μM) being required for complete reversal, reflecting the multi-targeted nature of MTA. Growth inhibition by MTA was not affected by hypoxanthine alone. A non-toxic concentration (1 μM) of DP prevented thymidine/hypoxanthine rescue of MTA indicating that DP may potentiate MTA activity by preventing nucleoside and/or base salvage. Thymidine transport was inhibited by ≥ 89% by 1 μM DP in all cell lines, whereas hypoxanthine transport was inhibited only in A549 and MCF7 cells. Therefore, prevention of end-product reversal of MTA-induced growth inhibition by DP can be explained by inhibition of thymidine transport alone. © 2000 Cancer Research CampaignThe novel pyrrolopyrimidine-based antifolate LY231514 (MTA), inhibits multiple folate-requiring enzymes including thymidylate synthase, glycinamide ribonucleotide formyltransferase and dihydrofolate reductase. Both thymidine and hypoxanthine are required to reverse MTA growth inhibition in leukaemia and colon cancer cells. Prevention of MTA growth inhibition by thymidine and/or hypoxanthine was investigated in two human lung  and two breast  tumour cell lines, and the effect of the nucleoside/base transport inhibitor dipyridamole (DP) on thymidine and hypoxanthine rescue defined. MTA IC


The development of resistance to cisplatin (DDP) occurs rapidly both in vitro and in vivo, and constitutes a major obstacle to effective therapy. We have previously demonstrated that there is a highly synergistic interaction between tamoxifen (TAM) and DDP against cell lines representative of three different human cancers: melanoma, ovarian carcinoma and small-cell lung cancer. The purpose of these studies was to determine if TAM interferes with the development of resistance to DDP. T-289 melanoma cells and 2008 ovarian cancer cells were cultured with increasing concentrations of DDP +/- TAM in an attempt to induce resistance to DDP. At various time points the cells were removed from culture and the degree of resistance to DDP was quantitated. Concurrent exposure to TAM and DDP decreased both the rate and the absolute magnitude of resistance to DDP in both melanoma and ovarian cancer cell lines. In the T-289 cell line the rate was decreased by a factor of 3.4 +/- 1.4 (P < 0.05), while in the 2008 cell line the rate was decreased by a factor of 2.4 (P < 0.01). TAM decreases the rate as well as the absolute magnitude of in vitro resistance to DDP in both melanoma and ovarian cancer cell lines. These data suggest that the concurrent administration of TAM and DDP may result in a delay in the development of resistance to DDP which may have important clinical implications in the design of DDP-containing regimens.


The hydro-alcoholic extracts of the plants were prepared by percolation. The cytotoxic effects and IC50 of the extracts on the cell lines were studied followed by colonogenic assay after 72 h incubation. The results showed that the extract of Juniperus sabina possesses lower IC50 in comparison with Zataria multiflora extract on all 4 normal and cancer cell lines (P<0.05); but, IC50 of the Juniperus sabina extract was significantly higher than the Taxus baccata extract and Cisplatin on all 4 normal and cancer cell lines (P<0.05). As a result, it is concluded that the extract of J. sabina has almost similar cytotoxicity with the extract of Taxus baccata on cancer cells.Isolation and identification of some potent anti-tumor compounds from medicinal plants has motivated researchers to screen different parts of plant species for the determination of anti-tumor effects. In this study, cytotoxic effects and IC Isolation and identification of some potent anti-tumor compounds, such as colchicine, Vinca alkaloids, and also docetaxol and paclitaxel (taxol), as one of the most consumed natural anticancer compounds have encouraged scientists to study the toxico-pharmacological effects of different parts of plant species against cancer cell lines.–8Juniperus sabina possess cytotoxic effects on some human cancer cell lines.[Juniperus sabina  showed the presence of cytotoxic compounds or tumor necrotizing substances.[Taxus species. However, difficulty of obtaining this compound from yew trees has limited its clinical use.[It has been previously reported that all parts of the yew plant contain poisonous taxine alkaloids. Taxines save their toxic effects during the year, with itsll lines. The potebstances.–2 Taxol ical use.Zataria multiflora Boiss belongs to the family Laminaceae that geographically grows in Iran, Pakistan and Afqanistan.[Z. multiflora showed inhibitory effects on Salmonella typhimurium and Staphylococcus aureus in brain heart infusion (BHI) broth medium.[qanistan.14 This pqanistan. In a stuh medium.Cisplatin (cis-dichlorodiammineplatinum-II) gained a widespread use against various malignant tumors in experimental animals18 and va20222650 of specific concentrations of hydro-alcoholic extracts of fruits of Juniperus sabina and leaves of Zataria multiflora were compared with hydro-alcoholic extract of bark of Taxus baccata and Cisplatin, as well as known herbal and chemical anticancer compounds on normal (CHO and rat fibroblast) and cancer (HepG2 and SKOV3) cell lines.In this investigation, cytotoxic effects and ICJ. Sabina and bark of T. baccata were collected from the northern regions of the Iran  in September 2007. Also, leaves of Z. multiflora were collected from downtown of Shiraz (Fars province). The plant specimen was identified by the Department of Pharmacognosy, Sari faculty of Pharmacy, and stored at −20°C.Fruits of A measured quantity of 50 g of dried and powdered parts of each plant was chopped and soaked in 75 ml of ethanol (80% v/v) for 24 h and then percolated . The extrCHO , normal rat fibroblast, HepG2 (Human hepatocarcinoma) and SKOV3 (Human ovary carcinoma) cell lines were purchased from Pasture Institute .The completed media were sterilized by 0.22 μ microbiological filters and kept at 4°C before use.Juniperus Sabina, Zataria multiflora and Taxus baccata, and 50 μl of 0, 2.5, 5, 10, 25 μg/ml of Cisplatin for 2 h; and then washed using sterile normal saline 0.09%.In Colonogenic assay, 50 μl of DMEM/F12 including 500-700 cells were added to 3 wells of 6well/plates for each concentration of the extracts and Cisplatin. Then, they were incubated for 48 h. After incubation, the cell lines were exposed to 50 μl of 0 (phosphate buffer), 5, 25, 50, 100, 150 μg/ml of hydro-alcoholic extracts of Then, 4 ml of fresh culture media was added to the wells, and incubated for seven days. After this period, the contents of wells were excluded; the cells were fixed by formalin 9%, and dyed by trypan blue 4% (w/v) for 20 min. Then, trypan blue was excluded, and the six well/plates were washed using sterile normal saline 0.09%. At the end, the dyed colonies were counted by light microscope.P<0.05).Prism ver.3 Software was used to perform statistical analysis. One-way ANOVA method followed by Tukey test was used to determine the differences among the groups . But, IC50 of the Juniperus sabina extract was significantly higher than the extract of Taxus baccata and Cisplatin on all 4 normal and cancer cell lines (P<0.05) [50 represent the higher potency of a compound to inhibit the growth of cells and cause toxicity and death of cells.The results showed that the extract of (P<0.05) . The low50 of the Juniperus Sabina and Zataria multiflora extracts with the Taxus baccata extract and Cisplatin on normal and cancer cell lines are showed at 50 was related to cisplatin and hydro-alcoholic extract of Zataria multiflora in all cell lines. IC50 of the compounds on the 4 cell lines increased according to the rank order of cells Cisplatin < Taxus baccata < Juniperus sabina < Zataria multiflora.Comparison of the evaluated IC50 = 0.87±0.07 μg/ml) and CHO cell lines (IC50 = 5.5±0.21 μg/ml).On the other hand, the highest and lowest cytotoxicity of Cisplatin was related to HepG2 (IC50 of cisplatin on the 4 cell lines increased according to the rank order of cells CHO > Fibroblast > SKOV3 > HepG2.The IC50 of hydro-alcoholic extract of Taxus baccata on the 4 cell lines increased according to the rank order of cells CHO > Fibroblast > HepG2 > SKOV3.On the other hand, the IC50 of hydro-alcoholic extract of Juniperus sabina on the 4 cell lines decreased according to the rank order of cells CHO < Fibroblast < HepG2 < SKOV3.Also, the IC50 of hydro-alcoholic extract of Zataria multiflora on the 4 cell lines decreased according to the rank order of cells Fibroblast < CHO < HepG2 < SKOV3.The IC50 of the Juniperus sabina and Zataria multiflora extracts, and Cisplatin, as drug control positive compound and Taxus baccata extract, as plant control positive on normal cell lines were higher than that on cancer cell lines. This difference can be resulted from dysfunction of cellular organisms following cancer incidence which cause higher rate of proliferation and increased cellular intake. Also, defensive disorders and effusion insufficiency to escape toxic substances from cells can lead to lower necessity to amounts of cytotoxic compounds to inhibit the growth of cancer cells in comparison with normal cells.[According to the results, ICal cells.8Juniperus sabina and its insignificant differences with IC50 of the extract of Taxus baccata, as a common natural anticancer product, allows us to conclude that extracts of different parts of Juniperus sabina are good candidates for further studies of activity-monitored fractionation to identify their active components.Determination of viability percent of the cells treated with the extract of Juniperus Sabina and Zataria multiflora on cancer cell lines and compared their IC50 with the hydroalcoholic extract of Taxus baccata and Cisplatin. We have observed that Juniperus sabina has insignificant differences with IC50 of the extract of Taxus baccata on HepG2 and SKOV3 cancer cells. This result presented another novel approach for the treatment ofsome cancers.In this study, we have determined the cytotoxic activity of hydroalcoholic extracts of


Poly(ADP-ribose) polymerase (PADPRP), which uses NAD to synthesize ADP-ribose polymers, is activated by DNA strand breaks and mediates cellular responses to DNA damage. The consequences of low cellular NAD levels in a cell line deficient in nicotinamide mononucleotide adenylyltransferase (NMNAT), an enzyme essential for NAD biosynthesis, were investigated by assessing NAD metabolism and DNA repair after treatment with alkylating agents. A tiazofurin-resistant L1210 cell line (TZR) was isolated. NAD levels were approximately 5933 and 3375 pmol mg(-1) protein for parental  and TZR cells respectively, and NMNAT levels were reduced by > 95%. TZR cells were more sensitive to temozolomide (TM) and 1-methyl-3-nitro-1-nitroso-guanidine (MNNG), particularly at concentrations that caused > 50% NAD depletion. TM and MNNG treatment decreased NAD levels in both cell lines, but took longer to return to control levels in TZR cells. For example, MNNG (5 microM), depleted NAD levels at 6 h to approximately 4512 (WT) and 1442 (TZR) pmol mg(-1) protein; however, NAD levels had returned to control levels by 8 h in WT cells, but were not restored by 16 h in TZR cells. Both cell lines were equisensitive to the growth-inhibitory effects of NU1025 per se (IC50 370 microM). Co-exposure of the cell lines to TM (100 microM) with increasing concentrations of NU1025 led to a synergistic enhancement of cytotoxicity, with IC50 values for NU1025 decreasing to 17 +/- 4 microM (TZR) and 37 +/- 6 microM (WT). A similar enhanced sensitivity to NU1025 (approximately 2.7-fold) was obtained when TZR cells were co-exposed to MNNG + NU1025. TM-induced DNA strand breaks were increased by co-incubation with NU1025, and again the TZR cell line showed increased sensitivity to NU1025. There were no significant changes in NMNAT activity in response to MNNG treatment over 24 h, either in the presence or in the absence of NU1025. These data demonstrate that modest decreases in cellular NAD levels can sensitize cells to alkylating agents and PADPRP inhibitors.


We have investigated the mechanism of resistance to adriamycin (ADR) of 3 human glioma cell lines in culture. The cell lines had different inherent sensitivities to ADR. Verapamil increased the ADR sensitivities of the 2 most resistant cell lines (G-UVW and G-CCM) by up to 5-fold. This effect was not seen in a sensitive cell line (G-MCF). Although the accumulation of ADR in the 3 cell lines was not related to inherent sensitivity, energy deprivation or the addition of verapamil produced an increase (up to 46%) in net uptake for both G-UVW and G-CCM, but not for G-MCF. For G-UVW the ADR efflux data were consistent with an energy-dependent ADR efflux mechanism which could be inhibited by verapamil. A similar mechanism was not found for G-CCM. In this cell line verapamil may act by increasing intracellular ADR binding. These data indicate that, while inherent resistance to ADR may be multifactorial, one possible mechanism of resistance in human glioma may involve changes in drug accumulation and/or binding as has been seen in animals models. A potential clinical role for verapamil in overcoming drug resistance in human solid tumours is also indicated.


A panel of human lung carcinoma lines representing the four main histological types , and derived from both primary and metastatic sites, has been established in xenograft and in tissue culture. The highest take rates were achieved when biopsy specimens were obtained from large tumour masses and cultured lines were most readily established after preliminary passages as xenografts. The established lines exhibited an overlapping spectrum of biochemical and morphological characteristics, and showed a tendency to change from one cell type to another, in keeping with the concept of a common endodermal cell of origin. Radiation resistance appeared to be related to the large-cell phenotype.


Eicosapentaenoic acid (EPA) has been shown to have an inhibitory effect on the growth of several pancreatic cancer cell lines in vitro. This study investigates the mechanism of growth inhibition and cytotoxicity of EPA on the pancreatic cancer cell line MIA PaCa-2. Cells were analysed for cell count, viability, cell cycle distribution and ultrastructural changes. There was a time- and dose-dependent decrease in cell count and viability in cultures of pancreatic cancer cells supplemented with EPA. Flow cytometric DNA analysis of MIA PaCa-2 cells incubated with EPA demonstrated the presence of sub G1 populations corresponding to the presence of apoptotic cells and the blockade of cell cycle progression in S-phase and G2/M-phase. The presence of apoptosis in EPA-supplemented cultures was further confirmed by DNA fragmentation and ultrastructural changes associated with apoptosis. Therefore, we conclude that EPA mediates its effect on the pancreatic cancer cell line MIA PaCa-2, at least in part, via cell cycle arrest and the induction of apoptosis.


Up regulation of genes which contribute to store operated channel (SOC) activity may plausibly be necessary for these processes to take place efficiently. TRPC proteins constitute a family of conserved Ca2+-permeable channels that have been shown to contribute to SOC activity.CaIn breast cancer biopsy tissues, TRPC3 and TRPC6 were the predominant TRPC genes expressed with TRPC3 and TRPC6 being significantly up regulated compared to normal breast tissue. In the lowly metastatic breast cancer cell line MCF-7, TRPC6 was the chief TRPC gene expressed while in the highly metastatic breast cancer cell line MDA-MB-231 both TRPC3 and TRPC6 were the predominant TRPC genes expressed. Western blotting, immunoconfocal analysis and immunoprecipitation experiments confirmed that the MDA-MB-231 cell line expressed both TRPC3 and TRPC6 protein with the majority of protein being intracellular. TRPC3 and TRPC6 were found to be in an immunoprecipitatble complex and co-localize within the cell. To demonstrate the potential of targeting TRP channels in breast cancer, hyperforin reportably a specific activator of TRPC6 significantly reduced the growth and viability of the breast cancer cell lines but had no effect on the non-cancerous breast cell line. Silencing of TRPC6 in MDA-MB-231 cells resulted in a significant reduction in cell growth but not viability.TRPC channels may be potential future targets for breast cancer diagnosis and therapy and deserve further investigation to evaluate their role in cancer cell physiology. Both are likely to have a significant role in the cancer process and Ca2+ is a regulator of proliferation and apoptosis  were added to the wells and incubated at 37°C for 2 h. The solution was removed and replaced by 890 μl of DMSO and 110 μl of Glycine buffer  for 10 mins. The numbers of viable cells were then quantified by measuring the absorbance at 570 nm with a spectrophotometer.P < 0.05.Data were analyzed by one-way ANOVA. Differences were taken as statistically significant when Our aims were to determine which TRP channel sub-types are expressed in normal and cancer breast biopsy tissues. mRNA was prepared from seven breast tissue samples obtained by biopsy of which three were found to be normal and four were diagnosed as cancerous. The mRNA was converted to cDNA and TRPC sub-type specific primers table  were utiBreast cell lines derived from cancer tissues are useful tools to understand the molecular factors involved in cancer and metastasis. In this study we chose three breast cell lines to investigate TRPC expression: MCF-10A (a non cancerous/transformed cell line), MCF-7 (lowly-metastatic cancer cell line) and MDA-MB-231 (highly-metastatic cancer cell line). mRNA was prepared from these cell lines and converted to cDNA. PCR amplification using TRPC sub-type specific primers was utilized and the PCR products were separated on agarose gels and visualized with a DNA stain Fig . PCR privisa versa. No TRPC3/TRPC6 complexes were observed in the absence of an immunoprecipitating antibody or with isotype control antibodies.Extracts of protein were made from MCF-10A, MCF-7 and MDA-MB-231 breast cell lines and separated by SDS-PAGE. Following blotting to nitrocellulose the blots were probed with antibodies to TRPC6 Fig  and TRPCOur PCR and protein results suggested that TRPC6 and/or TRPC3 were the predominant TRPC sub-types expressed as mRNA in the MCF-7 and MDA-MB-231 cell lines. To identify the localization of these TRPC channels in these cell lines we utilized immunochemistry staining with TRPC6 and TRPC3 specific antibodies. The MCF-7 and MDA-MB-231 cells were fixed and subsequently stained with a concanavalin-FITC conjugate. Following permeabilization the cells were probed with antibodies specific to TRPC6 or TRPC3 and visualized by confocal microscopy Fig , n = 4. Hyperforin was reported as a specific activator of TRPC6 compared to other TRPC channels . Since T2+ signalling to regulate pathways such as proliferation and apoptosis suggests that therapies that modulate Ca2+ signalling in cancer cells might be a therapeutic option [2+ channels and pumps that are aberrantly expressed in cancer cells might sufficiently disrupt Ca2+ homeostasis to selectively target cancer cells [2+channels are expressed in cancer cells? To date there is incomplete information as to the expression of TRP channels in normal and cancer breast tissues. Our major conclusions from this research are listed below:The ability of Cac option . Modulater cells . The pri1. Our experiments suggested that normal breast tissues expressed the TRPC1 and TRPC3 genes. Comparison of normal and breast cancer biopsy tissues indicated a significant alteration in TRPC channel expression both in terms of channel type expressed and levels of channel expression. In breast cancer biopsy tissues TRPC6 was expressed and appeared to be the predominant TRPC channel expressed. Additionally TRPC3 appeared to be significantly up regulated in breast cancer tissues. Of course our experiments could not distinguish which cell types these TRPC channels were expressed in, since the breast tissue could contain a variety of cell types.2. In the epithelial non-cancerous cell line MCF-10A only relatively very low amounts of TRPC1 expression was observed. In lowly metastatic breast cancer cell line MCF-7 the predominant channel expressed was a short splice variant of TRPC6. In the highly metastatic breast cancer cell line MDA-MB-231 the predominant TRPC channels expressed were TRPC3 and TRPC6 (of longer splice form variants). The amounts of TRPC3 and TRPC6 expressed in the MDA-MB-231 cell line were comparable in level, which leads us to speculate that they may form heteromultimers. The sizes of PCR products for TRPC3 and C6 found in the cancer tissues appeared to be consistent to that found in the MDA-MB-231 cell line.3. Protein expression of TRPC3 and TRPC6 in breast cancer cell lines appeared to be consistent with the PCR results. TRPC6 and TRPC3 expression was observed in the MDA-MB-231 cell line with the majority of protein being expressed in an intracellular location.4. Our data provides strong evidence that TRPC3 and TRPC6 form heteromutimeric channels in breast cancer epithelial cell line MDA-MB-231, which is consistent with previous reports of TRPC3/6 expression .2+ signalling hence affecting cell proliferation. In an earlier study, hyperforin induced apoptosis and inhibited the growth of various human and rat tumour cell lines in vivo, [5. Hyperforin a specific activator of TRPC6 produced a significant reduction in cell growth for the breast cancer cell lines, while the non-cancerous breast cell line was unaffected. Viability was significantly reduced in the breast cancer cell lines. TRPC6 activation is known to promote cell proliferation. This is an opposite result which is obtained here when using hyperforin however it may be expected that overactivation of TRPC6 by hyperforin could cause a disruption in Cain vivo, . Our res6. Complete silencing of TRPC6 in MDA-MB-231 cells using transfected SiRNA resulted in a significant reduction in cell growth but not viability.et al [et al [2+ current described in these cells. We propose that TRPC6 is the major TRPC channel expressed in MCF-7 cells. However correlation of the permeation and pharmacological profiles of the cationic current in MCF-7 cells as described by El Hiani et al, [et al [2+-permeable cation channels which are activated in response to agonist-stimulated PIP(2) hydrolysis and subsequent studies have provided substantial evidence that some TRPCs contribute to SOC activity [2+ is known to be involved in a number of metastatic processes including motility and proliferation [On the whole our results with the MCF-7 cell line confer with the recent report by Bolanz et al  who founl [et al  who suggi et al,  with tho, [et al  demonstrferation  these prThe authors declare that they have no competing interests.EA contributed to the siRNA and growth curves and helped to draft the manuscript. MD participated in its design and coordination. SK performed the PCR and immunoflurescence. CPP performed the remainder of assays conceived and participated in its design and coordination and drafted the manuscript.


P< 0.05), although to varying degrees, suramin and Å5 almost completely and N-acetylcysteine the least. In conclusion, this model shows the urokinase system is important for bladder invasion and can be used to investigate other mechanisms of bladder cancer invasion and also for the testing of intravesical drugs. © 2001 Cancer Research Campaign http://www.bjcancer.comAs well as being a passive support, the extracellular matrix also regulates key biological processes such as invasion, differentiation and angiogenesis. We have therefore developed an in vitro model of bladder cancer invasion using de-epithelialized rat bladder to allow for tumour cell–extracellular matrix interactions. Onto this we have seeded a panel of human bladder cancer cell lines ) representing progression from well to poorly differentiated phenotypes and used as models of superficial to invasive bladder cancer. The better differentiated cell lines RT4 and RT112 reproducibly grew as stratified epithelium, whereas poorly differentiated EJ28 cells invaded across a broad front. Invasion was not simply related to proliferation rate, measured either as doubling time on plastic (non-invasive 253J and invasive EJ28 having the same doubling time) or by Ki-67 proliferation index within the model. We used the model to test the ability of 4 compounds that interfere with tumour cell–extracellular matrix interactions  to inhibit invasion. At non-toxic concentrations, all significantly inhibited invasion (