Acute graft-versus-host disease (GVHD) occurs after allogeneic hematopoietic stem cell transplant and is a reaction of donor immune cells against host tissues. Activated donor T cells damage host epithelial cells after an inflammatory cascade that begins with the preparative regimen. About 35%–50% of hematopoietic stem cell transplant (HSCT) recipients will develop acute GVHD. The exact risk is dependent on the stem cell source, age of the patient, conditioning, and GVHD prophylaxis used. Given the number of transplants performed, we can expect about 5500 patients/year to develop acute GVHD. Patients can have involvement of three organs: skin (rash/dermatitis), liver (hepatitis/jaundice), and gastrointestinal tract . One or more organs may be involved. GVHD is a clinical diagnosis that may be supported with appropriate biopsies. The reason to pursue a tissue biopsy is to help differentiate from other diagnoses which may mimic GVHD, such as viral infection  or drug reaction (causing skin rash). Acute GVHD is staged and graded (grade 0-IV) by the number and extent of organ involvement. Patients with grade III/IV acute GVHD tend to have a poor outcome. Generally the patient is treated by optimizing their immunosuppression and adding methylprednisolone. About 50% of patients will have a solid response to methylprednisolone. If patients progress after 3 days or are not improved after 7 days, they will get salvage (second-line) immunosuppressive therapy for which there is currently no standard-of-care. Well-organized clinical trials are imperative to better define second-line therapies for this disease. Additional management issues are attention to wound infections in skin GVHD and fluid/nutrition management in gastrointestinal GVHD. About 50% of patients with acute GVHD will eventually have manifestations of chronic GVHD. Acute graft-versus-host disease or acute GVHD.Acute graft versus host disease generally occurs after allogeneic hematopoietic stem cell transplant (HSCT). It is a reaction of donor immune cells against host tissues. The three main tissues that acute GVHD affects are the skin, liver, and gastrointestinal tract.Clinically, the diagnosis is suspected when a recipient of HSCT develops any or all of the following signs or symptoms: dermatitis (skin rash), cutaneous blisters, crampy abdominal pain with or without diarrhea, persistent nausea and vomiting, hepatitis (with elevation of bilirubin and/or liver enzymes). Typically, these symptoms occur before day 100 after the HSCT, but may occur later. Symptoms most frequently start with donor engraftment, but may happen later. Acute GVHD is a clinical diagnosis but, as many the symptoms of acute GVHD are non-specific, histologic confirmation, especially if the symptoms are atypical or involve just the liver or gut, may be extremely useful.Acute GVHD is staged by the number and extent of organ involvement. The current staging system was devised in 1994 . This sytem tries to diminish interobserver variability in GVHD grading. The system assigns one of four risk categories (A-D) to each patient with acute GVHD. It is still unknown whether one system is better at predicting outcome of patients with acute GVHD.versus unrelated donor transplants performed yearly , we can expect about 5500 patients to develop grade II-IV acute GVHD per year.In 2003, 13,700 allogeneic HSCTs were reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). The incidence of grade II-IV acute GVHD is roughly 35%–50%. The risk of GVHD increases with the use of unrelated donors, mismatched donor, older age of the donor, mutliparous female donor, older age of the recipient, graft type , and certain conditoning regimens. Given the current ratio of related etc.) and infection. For example, the symptoms of cytomegalovirus (CMV) colitis  may be very similar to those of acute GVHD. Therefore, it is recommended to obtain a biopsy to confirm clinical suspicion whenever possible. Finally, while the definition of acute GVHD is well-accepted as involving three organs, certain post-transplant complications may be related to or actually represent GVHD. For example, a few patients develop non-infectious pulmonary infiltrates upon engraftment and the outcome is generally poor. Animal models suggest that the immunologic mechanisms contributing to lung inflammation after HSCT may be similar to those responsible for GVHD [The clinical manifestations of acute GVHD represent the organs involved. The earliest and most common manifestation is skin GVHD. This is essentially a maculopapular rash that can begin anywhere in the body but often starts with palm and sole involvement. The patient may complain of pruritus or tenderness in affected areas. The onset of the rash normally correlates with engraftment of donor cells. The timing of engraftment depends on stem cell source  and intensity of preparative regimen. Patients receiving reduced intensity regimens, which do not result in marrow ablation, often have a later onset of GVHD. This is due both to the later engraftment and the damage from the preparative regimen producing cytokines that drive the immune responses resulting in clinical GVHD. As the rash progresses, it may become confluent. In severe cases, blisters may occur. The gastrointestinal manifestations include diarrhea which may become bloody, cramping, nausea, vomiting and failure to thrive . Furtherfor GVHD .The pathophysiology of acute GVHD has been described by Ferrara and colleagues as a three-phase phenomenon ,8. PleasT-cell activation pathways result in the transcription of genes for cytokines, such as IL-2 and interferon. T cells that produce IL-2 and interferon are considered to be of the Th1 phenotype, compared to T cells that produce predominantly IL-4, IL-5, IL-10, and IL-13, which define the Th2 phenotype, that are felt to modulate GVHD [α TNF-α is produced mainly by monocytes and macrophages, and secondarily by T lymphocytes and natural killer cells. TNF-α has been implicated in the pathophysiology of GVHD at several steps in the process, including induction of apoptosis in target tissues through the TNF-α receptor; activation of macrophages, neutrophils, eosinophils, B cells, and T cells; stimulating production of additional inflammatory cytokines ; increased expression of HLA; and the facilitation of T-lymphocyte lysis. High levels of TNF-α also have been associated with an increased incidence of GVHD in bone marrow transplantation (BMT) recipients. This allogeneic interaction in the setting of cytokine dysregulation leads to the tissue damage characteristic of acute GVHD [In the third phase, the effector phase, activated donor T cells mediate cytotoxicity against target host cells through Fas-Fas ligand interactions, perforin-granzyme B, and the additional production of cytokines, such as TNF-ute GVHD ,11-15.i.e., CD4 in the context of MHC class II and CD8 in class I, the presence of nonself peptide bound to the MHC molecules trigger the T cell and induction of GVHD occurs. Current research is focused on identification of additional non-MHC antigens that trigger alloreactivity.Major histocompatibility antigens (or Human leukocyte antigens – HLA) are located on the short arm of chromosome 6 at the p21 position in humans ,17. The With the ability to molecularly type the extensive HLA region, it became evident that much more of the HLA region had to be considered for optimal unrelated donor selection. This is an area of explosive development, with almost 200 new alleles identified each year. A detailed review of the intricacies of the HLA system and selection of donors is beyond the scope of this paper. There are several recent excellent reviews ,17. HoweThe risk factors for acute GVHD are well defined. As discussed above, the most important factor is HLA disparity. Among siblings, patients receiving matched grafts have lower rates of GVHD than those receiving one, two or three antigen mismatch grafts. For unrelated donor transplants, the greater the degree of HLA-mismatch, the higher the likelihood is of developing acute GVHD and the worse the overall outcome -20. ReceAs for the source of the graft, unrelated cord blood has become an important alternative stem cell source and has some unique properties. The immunologic naiveté of these stem cells allows for greater degrees of mismatch; recipients of mismatched (4/6 or 5/6 HLA group match) unrelated cord blood appear to have similar incidence of acute GVHD and similar overall outcome as compared to matched-sibling transplants . There iOther factors can also increase the likelihood of acute GVHD. Older age of both recipient and donor increase the probability of GVHD . Sex misvis a vis the duration of liver GVHD prior to the biopsy. Epithelial cells can be flattened with some missing nuclei. Other nuclei are often enlarged, irregular, and hyperchromatic. Bile duct apoptosis and endothelialitis can also be seen [Acute GVHD is usually suspected based on the clinical presentation. A biopsy can be used to confirm the diagnosis and should be used when there are competing diagnoses, such as infection and drug reaction, in the differential. Skin biopsies can show dyskeratotic keratinocytes, lymphocyte exocytosis, basal cell necrosis, depletion of Langerhans cells, and satellite lymphocytes next to the dyskeratotic keratinocytes. All findings are not necessarily present in every skin biopsy and histology is not always pathognomonic . Typical be seen .etc.), the best prophylaxis is less clearly established [ex-vivo T-cell depletion [in-vivo T cell depletion  with short course methotrexate. Although other regimens are being explored, this particular regimen has been shown repeatedly to result in a reasonable balence of GVHD and graft versus-tumor in matched sibling transplants after ablative chemotherapy ,40. For ablished . Mycopheablished . For patepletion  as well lin, ATG , alemtuzlin, ATG ) have beOnce GVHD occurs, all phases of GVHD induction are active. Successful treatment will ultimately need to work on all phases, if the process is to be stopped. Most centers treat grade II-IV acute GVHD by continuing prophylactic immunosuppression and adding methylprednisolone at 2 or 2.5 mg/kg/day. However, starting doses range from 1 to >20 mg/kg/day . SteroidThere is one randomized trial comparing high and low dose methylprednisolone for the treatment of acute GVHD . PatientPatients not responding to corticosteroids are treated with salvage therapy. There is no specific approach that is considered standard of care. Anti-thymocyte globulin has been used, and produces objective responses. However, the long-term survival of patients treated with ATG is low  given the severe immunosuppression and high incidence of infection . There aWhile the goal is not to review all potential salvage therapies, a few types will be mentioned (Deeg recently published an excellent review on therapies for steroid-refractory GVHD ). For exα antibody) in 21 patients with steroid-refractory acute GVHD. This was the only drug added to these patients. Sixty-seven percent responded, and there was significant activity in gastrointestinal GVHD. There was a high rate of infections, particularly fungal and viral. One-year survival was 38% [versus those that did not, and found a higher incidence of non-Candida invasive fungal infections in those receiving infliximab [Monoclonal antibodies such as daclizumab and infliximab have been used to treat GVHD. MD Anderson Cancer Center recently reviewed their experience using infliximab , durable responses were obtained in 245 (55%). There was a tendency to lower response if patients begun with a higher grade. Recipients of HLA-mismatched unrelated donor transplants were less likely to respond. Fifty-three percent of patients were alive at 1 year after initiation of steroid therapy, and 42% of patients had developed chronic GVHD. Deaths were mostly commonly attributed to ongoing GVHD and/or infection [MacMillan nfection . Cahn annfection .It is worth noting chronic GVHD as over half of the patients with acute GVHD later develop chronic GVHD. Chronic GVHD is a distinct clinical syndrome, although there may be an overlap period, where the pateint has symptoms of both acute and chornic GVHD. Chronic GVHD resembles many spontaneously occuring autoimmune disorders, like scleroderma. Organs affected are most typically skin (lichenoid and sclerotic rashes), mouth, joints, liver, eyes, gastrointestinal tract, and occasionally lungs . While cClearly, better immunosuppressive therapies are needed for patients with severe GVHD. In addition, as immunosuppression is increased in these patients, the risk of infection needs to be carefully weighed. Agents for prevention of acute GVHD and for treatment of steroid-refractory acute GVHD need further investigation. To be able to truly evaluate the potential clinical benefit of an agent, it needs to be studied in prospective clinical trials. Furthermore, it is imperative to continue investigations on methods of cellular manipulation  that may reduce GVHD while preserving the graft-versus-leukemia effect. Since GVHD is clearly the major barrier to successful HSCT, efforts are needed in this specific area. If GVHD outcomes begin to improve, we may see an increase in number of patients being referred for HSCT.


Moreover, treatment of cultures with bleomycin or topotecan, a novel topoisomerase I inhibitor with little substrate affinity for P-glycoprotein, led to induction of apoptosis and significant (P< 0.05) dose-dependent reduction of cell number in all RCC cell lines. Both anticancer drugs also induced upregulation of CD95 ligand expression in all cell lines. Additionally, augmentation of CD95 receptor expression was found in three RCC cell lines, including one p53-mutated cell line, whereas another p53-mutated cell line showed no or only a weak CD95 receptor upregulation after exposure to topotecan or bleomycin, respectively. Despite this upregulation of CD95 receptor and ligand, antagonistic antibodies directed against CD95 receptors or ligands could not inhibit induction of apoptosis by topotecan and bleomycin in any cell line. Thus, although a functionally intact CD95 signalling cascade is present in most RCC cell lines, the anticancer drugs topotecan and bleomycin that induce upregulation of CD95 receptor and ligand fail to effectively activate CD95-mediated apoptosis. This deficient activation of CD95-mediated apoptosis might be an important additional factor for the multidrug resistance phenotype of human RCCs. © 2000 Cancer Research CampaignThe pronounced resistance of human renal cell carcinoma (RCC) to anticancer-induced apoptosis has primarily been related to the expression of P-glycoprotein and effective drug detoxification mechanisms. Because the CD95 system has recently been identified as a key mediator of anticancer drug-induced apoptosis, we analysed the contribution of the CD95 system to chemotherapy-induced apoptosis in four newly established RCC cell lines. Here, we demonstrate that all RCC cell lines expressed CD95-receptor and -ligand. Exposure to


Human monocytes can be induced to synthesize a cytotoxin which affects certain tumour cell lines. The interaction of monocyte cytotoxin with a susceptible cell line (L929) has been studied to obtain clues to the mode of action of the cytotoxin. The cytotoxin acts directly on the cells rather than on the culture medium and is cytotoxic at higher concentrations and cytostatic at lower concentrations. First signs of cell damage appear about 20 h after contact with the cytotoxin which must be present throughout this period. The cytotoxin probably acts on the cell surface and is more effective at 40 degrees C than at 37 degrees C. For a given amount of cytotoxin the effects are inversely proportional to the target cell concentration. Treatment of the cytotoxin with phenanthroline inhibits cytotoxicity while treatment of the target cells with actinomycin D, but not cycloheximide or puromycin, enhances cytotoxicity. After 24 h cytotoxin treatment the target cells exhibit reduced respiration rate but enhanced glycolysis and glucose uptake suggesting mitochondrial dysfunction. A possible interpretation of these data is that the monocyte cytotoxin is a metalloenzyme which inactivates a cell surface receptor for a nutrient essential for mitochondrial function.


M PPPP. This treatment produced 75.8 (± 7.1)%, 73.6 (± 9.8)% and 75.3 (± 6.4)% apoptotic cells in the three MDR cell lines respectively, compared to 19.0 (± 9.8)% in the drug-sensitive cell line. A reduction in glucosylceramide level following inhibitor treatment occurred in KB-3-1 cells as well as in the MDR cell lines, suggesting that the increased apoptotic response in the MDR cells reflected a different downstream response to changes in the levels of this lipid in these cells compared to that in the drug-sensitive cells. These results suggest that the manipulation of glucosylceramide levels may be a fruitful way of causing the preferential killing of MDR cells in vitro and possibly in vivo. © 1999 Cancer Research CampaignThis study has compared the preferential killing of three multidrug-resistant (MDR) KB cell lines, KB-C1, KB-A1 and KB-V1 by two inhibitors of glucosylceramide synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP), to the killing produced by these compounds in the drug-sensitive cell line, KB-3-1. Both of the inhibitors caused much greater induction of apoptosis in each of the three MDR cell lines than in the drug-sensitive cell line, as judged by morphological assay and confirmed by poly-(ADP-ribose)-polymerase cleavage. The highest level of apoptosis was produced following 24-h exposure to 5 μ


The flux of calcium forms an important intracellular messenger system. The bcl-2 oncoprotein is thought to make cells resistant to a variety of insults, including cytotoxic drugs, by the suppression of apoptosis, which appears to involve the repartitioning of intracellular calcium. Three drugs that affect calcium pathways and may influence this repartitioning, i.e. dantrolene, azumolene  and nimodipine, were studied in cell culture, using both a transformed follicle centre lymphoma cell line and primary culture of lymphoma cells in vitro in a manner that resulted in a growth pattern closely resembling that of the malignancy in vivo. Dantrolene and azumolene were potent inducers of cell death in both systems reducing the viable cell count by 70-90% in comparison with normal controls. Nimodipine, in comparison, appeared to have no significant effect. These results obtained in an in vitro setting suggest that further evaluation of dantrolene and azumolene for the treatment of low-grade non-Hodgkin's lymphoma is warranted.


Four human melanoma xenograft lines  grown orthotopically in Balb/c nu/nu mice were characterized with respect to the fraction of radiobiologically hypoxic cells. The purpose of the study was to establish a firm radiobiological basis for future use of the lines in the development and evaluation of non-invasive assays of tumour hypoxia. The hypoxic fractions were assessed using three different assays, the single cell survival assay, the tumour growth delay assay and the local tumour control assay, and the means +/- s.e. were found to be 6 +/- 3%, 3 +/- 1% and 5 +/- 2% respectively (A-07), 26 +/- 5%, 25 +/- 6% and 22 +/- 6% respectively (D-12), 55 +/- 9%, 65 +/- 8% and 48 +/- 7% respectively (R-18) and 52 +/- 8%, 59 +/- 7% and 47 +/- 7% respectively (U-25). The three assays gave numerical values for the hypoxic fraction that were not significantly different for any of the lines. The hypoxic fraction differed significantly among the lines; the R-18 and U-25 lines showed higher hypoxic fractions than the D-12 line (P < 0.05), which in turn showed a higher hypoxic fraction than the A-07 line (P < 0.05), regardless of the assay. The wide range of the hypoxic fractions and the significant differences among the lines suggest that A-07, D-12. R-18 and U-25 tumours should be useful models in future studies attempting to develop non-invasive assays of tumour hypoxia.


Its structure was determined by a combination of spectroscopic techniques, including (+)-HRESIMS and 1D and 2D NMR spectroscopy. The compound displayed cytotoxicity at the μM level against three human tumor cell lines.A new cyclic diamine, 1,5-diazacyclohenicosane ( Mycale have been the subject of extensive research leading to the isolation and identification of a wide variety of novel structures. Some of these Mycale metabolites display interesting biological properties such as the antiviral and antitumour activities detected in the mycalamides +; (+)-HRESIMS m/z 297.3266 [M+H]+ .l-glutamine and 100 U/mL penicillin and streptomycin, at 37 ºC and 5% CO2. Triplicate cultures were incubated for 72 h in the presence or absence of test compounds (at ten concentrations ranging from 10 to 0.0026 μg/mL). For quantitative estimation of cytotoxicity, the colorimetric sulforhodamine B (SRB) method was used [50, the concentration that causes 50% inhibition in cell growth after correction for cell count at the start of the experiment . Doxorubicin and DMSO (solvent) were used as the positive and negative controls in this assay. Prism 3.03 from GraphPad was used for the statistical analysis of the cell growth inhibition results.A549 (ATCC CCL-185), lung carcinoma; HT29 (ATCC HTB-38), colorectal carcinoma and MDA-MB-231 (ATCC HTB-26), breast adenocarcinoma cell lines were obtained from the ATCC. Cell lines were maintained in RPMI medium supplemented with 10% fetal calf serum (FCS), 2 mM was used . Briefly


In the present study we have used fluorocytometry and immunoprecipitation to characterize the expression of alphav-containing integrins in a panel of eight human breast cancer cell lines and one normal human mammary epithelial line. We show that the classical vitronectin receptor alphavbeta3 is expressed in only one cell line (MDA-MB-231), whereas alphavbeta5 is expressed on all breast cancer cell lines and alphavbeta1 is expressed on the majority. Using adherence assays to purified ligands in the presence and absence of function-blocking monoclonal antibodies, we have demonstrated that alphavbeta5 mediates adhesion to vitronectin in the majority of these cells. In one cell line, ZR75-1, alphavbeta1 contributes significantly to adhesion to immobilized vitronectin. The formation of focal adhesions containing the alphav and beta1 subunits on vitronectin is also demonstrated by indirect immunofluorescence.


We have studied the mitogenic, motogenic and morphogenic effects of hepatocyte growth factor (HGF), also known as scatter factor (SF), on 15 non-small-cell lung carcinoma (NSCLC) cell lines that have had their ras genotype determined. HGF/SF stimulated proliferation in only three cell lines and exerted no mitogenic activity on six lines. The growth of the remaining six lines was inhibited. The mitogenic effects were not related to the ras genotype of these cell lines, but the inhibitory effect was more commonly observed in cell lines with relatively high levels of Met/HGF receptor (HGFR) expression. HGF/SF induced or enhanced both scatter activity on monolayer culture and single-cell invasion in collagen gels in approximately half of these cell lines. Although the ras genotype of tumour cells did not influence the HGF/SF-induced motogenic activity, cell lines with the mutant ras genotype more commonly demonstrated a spontaneous motogenic activity than those with the wild-type ras genotype. When tumour cells were grown in collagen gels, HGF/SF induced irregular branching extensions of cell aggregates formed by five out of eight adenocarcinoma cell lines, but significant lumen morphogenesis was distinctly absent. The presence of autocrine HGF/SF loop in these tumour cell lines did not influence their spontaneous or HGF/SF-induced mitogenic, motogenic or morphogenic activities. Overall, our data suggest that stimulation of cell motility, rather than proliferation or differentiation, is the predominant paracrine effect of HGF/SF on NSCLC cells in vitro.


Cellular responses to hypoxia include modulation of respiration rate and up-regulation of genes which encode for angiogenesis factors. We tested whether human malignant glioma cells vary in their response to hypoxic stress over the range of oxygen concentrations which exist in tumours. In five cell lines tested, decreased oxygen availability resulted in decreased rates of oxygen utilization, however substantial differences in the magnitude of the response were observed. Northern blot analysis was used to study induction of vascular endothelial growth factor mRNA in response to hypoxia. In two cell lines, modest hypoxia increased vascular endothelial growth factor mRNA levels compared with those of aerobic controls. In two additional cell lines, vascular endothelial growth factor mRNA was constituitively expressed under aerobic conditions and was not further increased by hypoxia. These findings demonstrate that differences in the response to hypoxia exist among human malignant glioma cell lines and suggest that therapies designed to exploit tumour hypoxia may have varying effects in tumours with different hypoxic stress responses. © 1999 Cancer Research Campaign


Trans-[PtCl2(isopropylamine)] is a novel trans-platinum compound that shows cytotoxic activity in several cisplatin resistant cell lines. The aim of this paper was to analyse, by means ofmolecular cell biology techniques and total reflection X-ray fluorescence (TXRF), the cytotoxic activity, theinduction of apoptosis, the cellular uptake and the DNA binding of trans-[PtCl2(isopropylamine)] in the cisplatin resistant cell line A2780cisR. The results show that this drug is more cytotoxic and induces a higher amount of apoptotic cells than cisplatin in A2780cisR cells. However,the intracellular accumulation and extent of binding to DNA of trans-[PtCl2(isopropylamine)] is lower than that of cis-DDP. Moreover, trans-[PtCl2(isopropylaminae)] is partially inactivated by intracellular levels of glulathione. The resultsuggest that circumvention of ciplatin resistance by trans-[PtCl2(isopropylamine)] in A2780cisR cells might be related with the ability of this drug to induce apoptosis.


Cell cultures were made from 18 human bladder tumours. Three cell lines were maintained for seven transfer generations, but all had a “fibroblastic” morphology and a normal diploid karyotype. A fourth line has been maintained for over 80 transfer generations. This was derived from a well differentiated papillary tumour of bladder. Morphologically the light and electron microscopic structure of the cells resembled that of bladder tumours. The cells formed tumour nodules, with a similar structure, when transplanted into hamster cheek pouches. There is a stem line chromosome number of 48. Karyotypes of 60% of the stem line cells had one extra chromosome in Group C and one in Group D.


A soft-agar diffusion-chamber technique was used to grow colonies from human melanoma xenografts. Plating efficiencies ranged from 0-042% to 75% and increased with serial passage of some tumours. Cells in colonies were similar to human melanoma cells in morphology, histochemistry and ultrastructure, and were shown by immunofluorescence to contain human antigens. Xenograft tumours could be regrown from the colonies when re-implanted into immune-deprived mice. Cell-survival curves were constructed from 5 xenograft lines treated with 4 cytotoxic drugs. All lines were resistant to adriamycin, but each line appeared to have an individual spectrum of sensitivity to the more effective drugs. The responses were compatible with the clinical pattern of response in melanoma, and in 2 cases the objective response of lung metastases to treatment with melphalan was consistent with the xenograft cell-survival data. Dose-response curves were exponential for treatment with methyl-CCNU and melphalan, but distinct plateaux were seen for 2 xenografts treated with doses of DTIC over 100 mg/kg. These were thought to be due to resistant subpopulations of clonogenic cells within the tumours.


Ectopic production of free beta human chorionic gonadotrophin (hCGβ) by bladder carcinoma is well described and occurs in approximately 35% of cases. hCGβ secreting tumours are more aggressive, radioresistant and have a greater propensity to metastasize. We proposed that the ectopic production of hCGβ was contributing in an autocrine fashion to the radioresistance and metastatic potential of such secreting tumours. Though we demonstrated that the addition of hCGβ to the culture media of bladder, cervical and endometrial carcinoma cell lines brought about an increase in cell populations this was not accompanied by a significant increase in the rate of replication. Since a cell population size is a balance of mitosis and mortality, we proposed that hCGβ was inhibiting apoptosis. Here we have demonstrated that following incubation with recombinant hCGβ, bladder carcinoma cells refrain from undergoing apoptosis. Quantitation of apoptotic bodies was carried out by immunoassay and corrected to cell number as determined by MTT assay. In each cell line, addition of hCGβ reduced the number of apoptotic bodies dose-dependently, indicating a diminished apoptotic rate. Furthermore, TGFβ1-induced apoptosis could be dose-dependently inhibited by co-incubation with hCGβ. We propose, therefore, that such a decline in apoptosis may account for the cell population increase previously reported. It may also explain the radioresistance and aggressive nature of hCGβ-secreting tumours and the poor prognosis associated therein. © 2000 Cancer Research Campaign


The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma.


Lissodendorix sponge, inhibits GTP binding to tubulin, preventing microtubule assembly. Cell cycle perturbations and apoptosis induced by IHB were investigated on selected human cancer cell lines by using flow cytometric and biochemical techniques. Monoparameter flow cytometric analysis showed that 1 h IHB exposure caused a delayed progression through S-phase, a dramatic block in G2M phase of the cell cycle and the appearance of tetraploid cell population in LoVo, LoVo/DX, MOLT-4 and K562 cells. At 24 h after IHB exposure, the majority of cells blocked in G2M were in prophase as assessed by morphological analysis and by the fact that they expressed high levels of cyclin A/cdc2 and cyclin B1/cdc2. At 48 h, all cells were tetraploid as assessed by biparameter cyclin A/DNA and cyclin B1/DNA content analysis. Apoptotic death was detected in both leukaemic MOLT-4 and K562 cells, which express wild-type and mutated p53 respectively, when the cells were blocked in mitotic prophase. In conclusion, IHB is a novel potent anti-tumour drug that causes delayed S-phase progression, mitotic block, tetraploidy and apoptosis in cancer cell lines. © 1999 Cancer Research CampaignIsohomohalichondrin B (IHB), a novel marine compound with anti-tumoral activity, extracted from the


Forty-seven human leukaemia/lymphoma cell lines belonging to myelocytic, monocytic, non-T/non-B, T-, and B-lineage and representing different levels of maturation as well as fresh cells from normal and leukaemic subjects were examined for immunological markers and cytochemically for acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase (pH 5.8 and 8.0), alpha-naphthyl butyrate esterase (pH 5.8 and 8.0), non-specific esterase, chloroacetate esterase, chymotrypsin-like protease, deoxyribonuclease II, beta-glucuronidase, sudan black, and periodic acid Schiff's staining. Strong sudan black, nonspecific esterase, and chloroacetate esterase reaction was obtained only for myelocytic and monocytic cell lines with the reaction intensity increasing progressively in more mature cells. Focal acid phosphatase reaction like T-ALL was found in all T-ALL cell lines, whereas myeloid/monocytoid lines had semicircular distribution and B-cell lines cytoplasmic distribution of activity. Acid phosphatase activity appeared to decline with maturation along both myeloid and T-cell lineage. High activity of alpha-naphthyl acetate esterase and alpha-naphthyl butyrate esterase both at pH 5.8 and 8.0 and of beta-glucuronidase was found in myeloid/monocytoid lines although both B- and T-cell lines in contrast to peripheral blood B-cells also had significant esterase activity. alpha-Naphthyl butyrate esterase activity declined with increasing cell maturation along myeloid lineage. Except for weak activity in two B-cell lines alkaline phosphatase was not detected in any cell lines. Monocyte esterase activity was inhibited by sodium fluoride whereas acid phosphatase, only from hairy cell leukaemia line, was resistant to L-tartarate. Although periodic acid Schiff's staining could not distinguish myeloid, T-, B-, or non-T/non-B cell lines it gave characteristic reaction (large number of coarse granules against a clear background forming a ring around the nucleus) with erythroblastic leukaemia cell line and along myeloid series its intensity increased in more mature cells. Deoxyribonuclease II and chymotrypsin-like protease staining were not discriminatory. The results of this study show that cytochemical staining characteristics of various leukaemia/lymphoma cell lines are comparable to those of corresponding cells from patients and that the intensity and pattern of expression of these activities are related to cell type and degree of cell maturation. These studies give further credence to the use of these cell lines in cell differentiation, differential drug cytotoxicity, and many other studies.


Four cell lines of human (CCRF CEM and U266BL) or murine (L1210 and P388D1) origin, resistant to the anthracycline antibiotic Adriamycin (doxorubicin) were selected in vitro from cultured cells by serial passage in the presence of Adriamycin. The resistant sublines were also cross-resistant to Mitoxantrone, 4'-epi Adriamycin and a number of novel anthracyclines including 4'-deoxy and 4'-methoxy analogues. However, a series of 9-alkyl substituted 4-demethoxyanthracyclines retained full activity against all the resistant sublines as did Aclacinomycin A. These results suggest that 9-alkyl substitution of 4-demethoxy-anthracyclines is an important determinant of activity against Adriamycin-resistant cell lines in vitro.


Resistance to chemotherapy is a major problem in the treatment of patients with head and neck squamous cell carcinoma (HNSCC). Important factors involved are drug detoxification by glutathione (GSH) and reduced drug accumulation due to active transport out of the cell by so-called 'multidrug resistance-related proteins'. We have studied a panel of eight HNSCC cell lines showing differences in sensitivity to the anti-cancer drug cisplatin. Our previous studies indicated that the IC50 values were inversely correlated with the intracellular accumulation of platinum (Pt). In the present study, cellular GSH levels were found not to be related to the IC50 values. The expression levels of the enzymes glutathione S-transferase (GST) alpha, mu, and pi, the multidrug resistance-related proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and the lung resistance protein (LRP) were determined semiquantitatively by means of immunocytochemistry. The levels of the GSTs, P-gp and LRP were not found to be correlated with the IC50 values of the HNSCC cell lines. Surprisingly, however, an inverse correlation was found between MRP levels and IC50 values. The MRP expression levels were in agreement with the results of the MRP functional assay, based on the transport of calcein across the cell membrane as performed for two of the cell lines. Further studies should prove whether other pump mechanisms or DNA repair are involved in the cisplatin accumulation and the subsequent HNSCC cell growth inhibition.


The present studies were initiated to quantitate the oxygen dependence of bioreductive metabolism-induced binding of EF5, a pentafluorinated derivative of the 2-nitroimidazole, etanidazole. Two different assays were compared: first, radioactive drug incorporation into cell lysates, which provides a direct measure of drug metabolism or uptake; second, monoclonal antibody detection of cellular macromolecular adducts of EF5 after whole cell permeabilisation and fixing. The antibodies (a single clone designated ELK3-51) were conjugated with the fluorescent dye Cy3, with fluorescence determined by fluorescence microscopy and flow cytometry. For the two cell lines tested (V79 Chinese hamster fibroblasts and 9L rat glioma), the oxygen dependence of binding was found to be the same for the two techniques. Using the antibody binding technique, the fluorescence signal was highly reproducible between experiments, resistant to light or chemical bleaching and stable over time following cell or tissue staining. Flow cytometric analysis of cells from rat 9L tumours treated with EF5 in vivo or in vitro showed a distribution of fluorescent signal which was very compatible, on both a relative and absolute basis, with the in vitro results. Our results indicate that immunofluorescent techniques provide a quantitative assay for bioreductive drug adducts, and therefore may be able to measure the absolute oxygen concentration distribution in cell populations and tissues of interest.


TP53 and the T309G polymorphism of MDM2 were associated to the in vitro cytotoxicity of anticancer agents, extracted from the NCI database. For validation, the same study was performed independently on a second panel of tumour cell lines, JFCR-45.We investigated, in the panel of 60 human tumour cell lines of the National Cancer Institute (NCI-60), whether the R72P polymorphism of Both SNPs were identified in cell DNA using PCR-RFLP techniques confirmed by direct sequencing and by pyrosequencing. For the analysis of the results, the mutational status of p53 was taken into account.TP53 rare-allele frequency was 32% and the MDM2 rare-allele frequency 39%. The MDM2 alleles were distributed according to Hardy–Weinberg equilibrium whereas this was only found, for the TP53 alleles, in p53 non-mutated cell lines. Comparable results were obtained in the JFCR-45 validation set. The TP53 SNP had low impact on anticancer drug cytotoxicity in either panel. In contrast, the MDM2 gene polymorphism had a major impact on anticancer drug cytotoxicity, essentially in p53 non-mutated cell lines. Presence of the rare allele was associated to significantly higher MDM2 protein expression and to increased sensitivity to DNA-interfering drugs. In the JFCR-45 panel, a similar effect of the MDM2 gene polymorphism was observed, but was less dependent on the p53 mutational status.In the NCI-60 panel, the MDM2 G allele presented a lower availability of p53 for DNA repair, translating into higher sensitivity to DNA-damaging agents.We hypothesised that cell lines harbouring the  This polymorphism has been found with an allele frequency of about 25% in Caucasian populations, giving rise to about 6% variant homozygous subjects. It has been shown that the R common form of p53 is able to induce apoptosis markedly better than does the P variant form in cell lines containing inducible wild-type p53  . Sequencing was performed on randomly chosen PCR products from the various genotypes of the variations studied. Concordance with RFLP was obtained in 100% of the cases.Polymerase chain reactions (PCRs) were performed on genomic DNA using appropriate primers (see below). Polymorphisms were detected in the NCI-60 panel by restriction fragment length polymorphism (RFLP) analysis on PCR products, using appropriate restriction enzymes. Electrophoresis of the PCR products was performed before and after digestion on 10% polyacrylamide gels. The presence of a variation was translated into the occurrence or the disappearance of a restriction site on the PCR product, leading to two shorter products. This technique allowed the unambiguous discrimination between homozygous cell lines with the common-allele, homozygous cell lines with the rare-allele and heterozygous cell lines, for both polymorphisms studied  of MDM2, we used the following primers: sense, 5′-GAGTTCAGGGTAAAGGTCAC-3′; antisense, 5′-TCAAGAGGAAAAGCTGAGTC-3′. The PCR products were digested by MspAI, which specifically cleaves the variant G allele, and subjected to 10% polyacrylamide gel electrophoresis.For the T309G variation of TP53: sense, 5′ dR-Biotin-CACTGAAGACCCAGGTCCAGAT 3′; antisense, 5′-CCGGTGTAGGAGCTGCTGG-3′; MDM2: sense, 5′-CAGGGTAAAGGTCACGGG-3′; antisense, 5′ dR-Biotin-AGGCACCTGCGATCATCC 3′.To confirm these results by a second independent technique, we also identified both polymorphisms by pyrosequencing. The DNA extracts of the JFCR-45 collection were exclusively studied by pyrosequencing. Direct sequencing of the PCR fragments without any further purification was performed on the Pyrosequencer PyroMark ID system  according to the instructions of the manufacturer. In that case, the primers we used were as follows: for 50) of 136 core drugs vis-à-vis the 59 NCI-60 cell lines, expressed as –log10(GIC50), were extracted from the DTP database (http://dtp.nci.nih.gov). Similarly, the growth-inhibitory concentrations (GI50s) of 53 agents against the cell lines of the JFCR-45 collection were extracted from the original publication , which indeed presented good correlations with most other data sets . MDM2 expression was also obtained on Affymetrix microarrays in the JFCR-45 collection as described by MDM2 probe sets were available on these arrays; based on expression intensity, we chose the probe 217373_x_at for comparisons of MDM2 expression according to TP53 and MDM2 genotypes.The mutational status of χ2-test was used for comparing the distribution of the cell lines among genotypes. Pearson coefficients of correlation were computed for comparing continuous variables (GIC50 and gene expression data). To study the pharmacological parameters as a function of the genotype, we analysed the variances of drug GIC50s values or MDM2 expression for each genotype and calculated the significance of the differences in mean values, using a general linear model taking into account the unbalanced size of the groups. The Bonferroni correction was applied to take into account the number of tests performed. The two cell line collections were studied independently, the NCI-60 collection as a training test and the JFCR-45 collection as a validation test, to decrease the probability of generating falsely positive observations.The TP53 variation (P<0.001). When considering separately the cell lines with wild-type and mutated p53, it appeared that the frequency of the rare C allele was significantly higher (P=0.026) in cell lines with mutated p53 (38.4%) than in cell lines with wild-type p53 (15.6%). Especially, all the cell lines with the homozygous C/C genotype were found among the p53-mutated cell lines. In addition, the heterozygous cell lines harboured less frequently a p53 mutation than homozygous cell lines. Concerning the MDM2 variation . However, these differences were not observed when the mutational status of p53 in the cell line panel was taken into account. Indeed, and independent of the polymorphisms of TP53, one can notice a highly significant difference between cell lines with wild-type and mutated p53: cell lines having a p53 mutation had a significantly lower sensitivity to most drugs than cell lines having no p53 mutation. The mean GIC50 ratio reaches 2 for alkylating agents .We looked for associations between the presence of a given genotype of MDM2 genotype and cell sensitivity to the 136 core drugs could be evidenced when they were sought in the whole NCI-60 panel. When the drugs were pooled as a function of their mechanism of action, a significant difference could be evidenced for alkylating agents: the cell lines containing a G allele were slightly more sensitive to this class of agents than the common homozygous cell lines   and 2.4 for topoisomerase II inhibitors . Due to the fact that only two cell lines had a homozygous G/G genotype in the p53 wild-type subset, it was not possible to study them separately from heterozygous cell lines, but it should be noticed that these two cell lines presented the lowest GIC50 values for most DNA-interfering drugs.No consistent association between  and II) . When thMDM2 expression and polymorphism in the NCI-60 panel. As expected, there was a significantly lower MDM2 expression in cell lines having a mutated p53 than in cell lines having no p53 mutation . There was no association between MDM2 polymorphism and MDM2 expression in the whole cell line panel; however, when the cell lines with and without a p53 mutation were considered separately, it appeared that, among the cell lines with no p53 mutation, those harbouring at least one G allele at position 309 of MDM2 had a significantly increased MDM2 expression   , right.  × 10−5) , left. T50 values between wild-type and p53-mutated cell lines was 1.35 (P=6.0 × 10−3). However, the TP53 polymorphism was not associated to differences in the cytotoxicity of any anticancer drug or drug class. The polymorphism of the MDM2 gene was in contrast associated, as in the NCI-60 panel, to major differences in drug sensitivity, the cell lines harbouring the variant MDM2 G allele being more chemosensitive than those harbouring only the common T allele; this was detected mainly in cell lines with wild-type p53 but also in cell lines where a mutation of p53 was detected, although to a lesser extent , whereas it was only 2.4 in p53-mutated cell lines. As in the NCI-60 panel, the expression of MDM2 in the JFCR-45 panel was higher in p53 wild-type cell lines than in p53-mutated cell lines  and, in p53 wild-type cell lines, MDM2 expression was higher in cell lines harbouring at least one G allele (expression ratio=1.9) (P=0.11) because of the small number of cell lines with common homozygous MDM2 genotype.In the JFCR-45 panel, we observed, as in the NCI-60 panel, a significant relationship between the mutational status of p53 and the cytotoxicity of most anticancer drugs belonging to the classes of alkylating agents, topoisomerase inhibitors and antimetabolites. When the cytotoxic drug panel was considered as a whole, the mean ratio of GICr extent . In p53 tio=1.9)  but thisvs 11% in the p53 mutated subset) and would not be a characteristic of the genotype. Indeed, there was no significant difference in drug sensitivity as a function of the TP53 polymorphism in the JFCR-45 panel, which confirms that the difference seen in the NCI panel should be a bias due to the distribution of the genotypes. However, this last observation is interesting per se: the higher proportion of heterozygotes among wild-type p53 tumours in both panels has never been mentioned before. It could be simply related to the loss of heterozygosity at the TP53 locus during evolution of p53 mutated, genetically unstable, tumours. It can also be hypothesised that the TP53 heterozygous genotype may protect against the occurrence of p53 mutations. Researching both the p53 mutational status and the TP53 polymorphism in clinical samples would be warranted to confirm this hypothesis.The higher sensitivity of p53 wild-type cells, as compared to p53 mutated cells, to most anticancer agents (excluding spindle poisons), is a very general feature when cytotoxicity is evaluated by growth inhibition . It was MDM2 gene is one of the numerous transcriptional targets of p53 and the MDM2 protein induces the proteasomal degradation of p53. Its polymorphism present in the 5′ untranslated region has been consistently associated with an increased transcription rate and a subsequent attenuation of p53, leading to an activation of tumour formation and growth  , but also of inducing DNA repair following drug-induced damage. One can hypothesise that the second function of p53 would be preponderant in the culture conditions of the cell line panels. As a consequence, the higher


Homozygous nude and normal mice of 3 strains, BALB/c, CBA and C57BL, were used as sources of nucleated haemopoietic "natural killer" (NK) cells. These killer cells could lyse a wide range of syngeneic and allogeneic lymphoid tumour cell lines in vitro, and it was found that cell suspensions from nude mice were always significantly more active than those from normal mice, and that the most active effector population was a polymorph-enriched peritoneal-exudate cell suspension. Eosinophils did not appear to be involved in the phenomenon, and mononuclear peritoneal-exudate cell suspensions were actually highly inhibitory. Three non-lymphoid tumours, a carcinoma, a fibrosarcoma and a mastocytoma, were totally resistant to in vitro lysis. Although all susceptible tumour cell lines were C-type virus-associated, not all of these tumours were killed by all strain sources of spleen cells, indicating a specificity of killing.


Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation.


The anti-tumour alkaloid taxol shows strong cytotoxic and antiproliferative activity in two human malignant glioma cell lines, T98G and LN-229. CD95 (Fas/APO-1) ligand is a novel cytotoxic cytokine of the tumour necrosis factor (TNF) family that exerts prominent antiglioma activity. At clinically relevant taxol concentrations of 5-100 nM, taxol and CD95 ligand showed significant synergistic cytotoxicity and growth inhibition. High concentrations of taxol induced G/M cell cycle arrest in both cell lines. The synergy of taxol and CD95 ligand was independent of cell cycle effects of taxol as synergy was achieved at much lower taxol concentrations than G2/M arrest and as cell cycle effects of taxol were unaffected by co-exposure to CD95 ligand. Similarly, high concentrations of taxol were required to induce p53 activity in the p53 wild-type cell line LN-229. This effect was not modulated by CD95 ligand, suggesting that synergy is also independent of p53 activation. However, taxol induced a mobility shift of the bcl-2 protein on immunoblot analysis, indicative of bcl-2 phosphorylation. Bcl-2 phosphorylation on serine was confirmed by immunoprecipitation and phosphoserine immunoblot analysis. Considering (1) that phosphorylation of bcl-2 interferes with its heterodimerization with bax and (2) the inhibition of CD95-mediated apoptosis by bcl-2, we propose that taxol sensitizes malignant glioma cells to CD95 ligand by increasing the functional bax/bcl-2 rheostat in favour of bax and thus cell death.


Evidence is presented that the platinum–AG adduct, in which platinum is bound to guanine and an adjacent adenine, may be the cytotoxic lesion because a significant correlation was found between the platinum–AG levels and the sensitivities in our panel of HNSCC, in vitro as well as in vivo. This correlation with the platinum–AG levels was established at 1 h (in vitro) and 3 h (in vivo) after the start of the cisplatin treatment, which emphasizes the importance of early sampling. © 1999 Cancer Research CampaignTotal platinum contents and cisplatin–DNA adduct levels were determined in vivo in xenografted tumour tissues in mice and in vitro in cultured tumour cells of head and neck squamous cell carcinoma (HNSCC), and correlated with sensitivity to cisplatin. In vivo, a panel of five HNSCC tumour lines growing as xenografts in nude mice was used. In vitro, the panel consisted of five HNSCC cell lines, of which four had an in vivo equivalent. Sensitivity to cisplatin varied three- to sevenfold among cell lines and tumours respectively. However, the ranking of the sensitivities of the tumour lines (in vivo), also after reinjection of the cultured tumour cells, did not coincide with that of the corresponding cell lines, which showed that cell culture systems are not representative for the in vivo situation. Both in vitro and in vivo, however, significant correlations were found between total platinum levels, measured by atomic absorption spectrophotometry (AAS), and tumour response to cisplatin therapy at all time points tested. The levels of the two major cisplatin–DNA adduct types were determined by a recently developed and improved


Mapping of sub-networks in the EGFR-MAPK pathway in different breast cancer cell lines reveals that PAK1 may be a marker for sensitivity to MEK inhibitors. Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EgfR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes.We were interested in identifying subnetworks within the EgfR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EgfR-MAPK signaling. This model was composed of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype-specific subnetworks, including one that suggested Pak1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that Pak1 over-expressing cell lines would have increased sensitivity to Mek inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three Mek inhibitors. We found that Pak1 over-expressing luminal breast cancer cell lines are significantly more sensitive to Mek inhibition compared to those that express Pak1 at low levels. This indicates that Pak1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to Mek inhibitors.All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets. Cancer is a heterogeneous disease that results from the accumulation of multiple genetic and epigenetic defects -4. TheseAmong breast cancers, unique subsets can be defined at the genomic, transcriptional and proteomic levels. For many years, breast cancers were classified by whether or not they express various receptors, namely the estrogen receptor (ER/EsR1), the progesterone receptor (PR/PGR) and ErbB2 -25. ThisA critical question remaining is how to identify meaningful subsets of cancers that differ in their cell signaling pathways. One approach to this problem is to identify gene expression signatures that reflect the activation status of oncogenic pathways ,33. WhilOur goal was to identify signaling pathway modules that are deregulated in particular cancer subtypes. To that end, we populated a well-curated cell signaling model with molecular information from a panel of breast cancer cell lines. We used a combination of transcriptional, proteomic and mutational data to create a unique signaling network for each cell line. Specifically, we discretized transcript and protein data and used them to populate the network models; genes or proteins that are differentially expressed across the cell lines were evaluated as present in some cell lines and absent from others. The resultant network models can be viewed as a statistical formalism of the pathways activated in each of the cell lines.We created our network models with Pathway Logic -38, a syWe focused our modeling on the ErbB/MAPK pathway because deregulation along this pathway is both frequent in breast cancers and heterogeneous across them ,41. Furtet al., unpublished data). Overall, the genomic heterogeneity in the cell lines mirrors that observed in a large population of primary tumors, and as an ensemble constitutes a useful model of the molecular diversity of primary tumors  × 100, where T0 is the cell count at day 0, C is the vehicle control (for example 0.3% DMSO without drug) cell count at day 3, and T is the cell count at the test concentration. We calculate the GI50 and total growth inhibition (TGI) values after 72 h drug exposure. The GI50 is the drug concentration that results in 50% growth inhibition; the TGI is the drug concentration that yields 100% growth inhibition.Data calculations were made according to the method described by the NCI/NIH DTP Human Tumor Cell Line Screen Process  and as pPathway Logic  is a sysrl[793.ErbB2.on]:{CLm | clm [EgfR - act] ErbB2}= >{CLm | clm [EgfR - act] [ErbB2 - act]}.The first term on each line represents a cellular location. In this case, CLm indicates that EgfR and ErbB2 are located in the cell membrane. A reaction will occur ('fire') only if the components are located in the specified cellular compartment. Most rules in our database describe changes to the state of a protein, such as activation, exchange of GDP for GTP, or translocation to a different cellular compartment. In total, the relevant rule database contains 396 rules, all of which have been individually curated from primary literature sources.The initial state specifies the model components present in a cell, as well as their locations. We created the initial states for each network model from a set of 286 components. Models are generated by 'rewrites.' In a simple rewrite, the initial state is presented to the rules. Whenever the state meets the conditions required by a rule input, the state is adjusted in accordance with the rule. The new state is then presented to the rules and more adjustments are made. This iterative process continues until either no further alterations can be made, or a user-defined condition is reached. We visualize the result of these rewrites as a Petri net, a directed bipartite graph that contains places, transitions, and directed arcs that connect the places and transitions . In PetrWe discretized the protein and transcript data in order to determine which components were present in (or absent from) the initial state of each cell line network model. Conceptually, the idea was to analyze the expression data for each protein in the initial state in order to decide if it showed differential expression across the panel of cell lines. Proteins that showed a highly variable expression pattern across the panel of cell lines were considered present in some cell lines and absent from others. Our approach to discretization and creation of the initial states was quite conservative. That is, we did not omit a component from the initial state unless there was strong evidence that it is absent from a particular cell line. We chose a conservative approach because in discrete networks such as these, errant omission of a component from the initial state can lead to significant effects on the structure of the network, in the form of truncated signaling pathways Figure .We developed the following discretization method and applied it to both the protein and transcript data. First, for each gene or protein, we used PAM clustering and a mean split silhouette (MSS) statistic to determine whether the log-transformed expression values are best represented as 1, 2 or 3 groups of cell lines . We searNext, for genes that clustered into two or three groups, we compared the mean expression levels of the groups. If the expression levels between the highest and lowest group differed by less than a four-fold change, we collapsed the groups together. This ensured that expression differences between the groups were great enough to be meaningful. We assigned proteins to the initial states in the following way. If a single group best described the distribution of expression values, the protein was considered present in all the cell lines. For distributions that yielded more than one group, the protein was considered absent from the initial state of the cell lines with the lowest mean expression; the protein was present in the initial state of cell lines in the highest group(s). We considered the protein present in the two clusters with highest mean expression in order to avoid erroneous omissions from the initial state of cell lines in the middle expression group. Finally, if we had no data available from which to estimate the initial state, we considered the protein present in all cell lines.For model components that had both transcript and protein data available, we used the clustered protein (rather than transcript) data to populate the model. To ensure that we made the most robust initial state assignments possible, we used data from as many of the 51 cell lines for the discretization step, even if we ultimately did not create a network model for the cell line. We performed the analyses above in R with the hopach package, available as part of the BioConductor tools suite .We used the following method to compare the networks. First, we decomposed each network into a list of all the components and rules contained within it. This list describes all the state changes  and reactions in each network. We clustered the network features with PAM and an MSS, which searched for the optimal number of clusters, up to a maximum of 40. Each cluster can be considered a unique 'signaling module' that represents a small portion of the total network. We compared the presence or absence of these signaling modules across the panel of cell lines.The discretized data used to populate the initial states were hierarchically clustered using an average linkage algorithm and a Pearson correlation for the distance measure . We alsoCav1: Caveolin-1; DMSO: dimethyl sulfoxide; EgfR: epidermal growth factor receptor; ER: estrogen receptor/EsR1; MAPK: mitogen-activated protein kinase; MIP: molecular inversion probe; MSS: mean split silhouette statistic; PAM: partitioning around medoids; Pi3k: phospho-inositide-3-kinase.LMH participated in the design of the study, constructed the computational models and drafted the manuscript. NJW conducted the Pak1 molecular studies. CLT created the Pathway Logic software and provided guidance of the computational modeling. MK curated the rule library. MK and KRL provided biological guidance of the modeling work. YG, ZH, SZ and WLK conducted the growth inhibition assays. BLW, SL, JRJ and RFW provided the Mek inhibitor GSK1120212. JWG and PTS conceived of the study and participated in its design and coordination.


The action of methotrexate, actinomycin D, bleomycin, vincristine and hydroxyurea on the production of human chorionic gonadotrophin (hCG) by a choriocarcinoma cell line (BeWo) has been studied. hCG production per unit of cell protein was increased, and this was a continuing process only halted by cell death. The proportion of hCG-producing cells in the population increased from approximately 5% to a maximum of approximately 40% during incubation with vincristine. A possible explanation for these and related observations is that cytotoxic agents promote the differentiation of cytotrophoblast to syncytium. A similar mechanism could contribute to the unusual sensitivity of choriocarcinoma to cytotoxic agents.


The morphology and ultrastructure of 48 primary tumours established from 5 cell lines of adult mouse salivary gland epithelial cells transformed in vitro are described. Tumours from 4 of the cells lines were adenocarcinomas with a wide range of structural variation, and resembled human salivary gland carcinomas. The fifth cell line produced tumours with carcinomatous and sarcomatous elements.


Apoptosis induced by rhodium II amidate, rhodium II propionate, cisplatin and interactions withdexamethaxone were studied on some human leukemia cell lines Raji, Jurkat and U937. Apoptosis wasstudied by flow cytometry, agarose gel electrophoresis and morphological analysis. Rhodium II propionateinduced apoptosis in all the three cell lines, Rhodium II amidate, in the lymphoid cell lines Jurkat and Raji,and cisplatin, only in the Jurkat, a T lymphoid cell line. It has also been observed that the addition ofdexamethasone enhances the apoptosis index only in U937, a monocytic line with a glucocorticoid receptorbearing.


It has been suggested that sensitivity of ovarian carcinomas to cisplatin is in part related to an endogenous folate deficiency. In this work, we investigated whether overexpression of the folate-binding protein (FBP), a receptor involved in folate transport, might be associated with cisplatin sensitivity. The results obtained on a panel of ten ovarian carcinoma cell lines that overexpress different levels of the FBP showed a statistically significant relationship between FBP overexpression and cisplatin responsiveness, with the most sensitive cell lines expressing higher FBP levels on their membrane than the less sensitive ones. The relationship was observed both in cells growing in standard medium-containing high-folate concentrations (2.3 microM) and in cells adapted to growth in low-folate (20 nM) medium. Analysis of two cisplatin-resistant cell lines derived from the cisplatin-sensitive IGROV1 ovarian carcinoma cell line indicated that resistance was associated with a significant decrease in FBP expression. However, the receptor does not appear to be directly responsible for drug sensitivity per se as different cell lines transfected with FBP cDNA did not become more sensitive to the drug. Together, the data suggest the possible predictive value of FBP in ovarian carcinoma, as higher levels of expression can be indirectly but significantly associated with increased drug sensitivity.


The streptococcal preparation OK432 was studied for its effects on natural killer (NK) activity of peripheral blood lymphocytes (PBL) from normal donors and from ovarian cancer patients, and of tumour-associated lymphocytes (TAL) from peritoneal effusions. OK432 augmented NK activity against the susceptible K562 line and induced killing of the relatively resistant Raji line. Freshly isolated ovarian carcinoma cells were relatively resistant to killing by unstimulated PBL and TAL. OK432 induced significant, though low, levels of cytotoxicity against 51Cr-labelled ovarian carcinoma cells. Augmentation of killing of fresh tumour cells by OK432 was best observed in a 20 h assay and both autologous and allogeneic targets were lysed. PBL were separated on discontinuous Percoll gradients. Unstimulated and OK432-boosted activity were enriched in the lower density fractions where large granular lymphocytes (LGL) and activity against K562 were found. Thus, OK432 augments NK activity of PBL and TAL in human ovarian carcinomas and induces low, but significant, levels of killing of fresh tumour cells. Effector cells involved in killing of fresh ovarian tumours copurify with LGL on discontinuous gradients of Percoll.


Using a fibrin-agarose-overlay technique, high levels of plasminogen-dependent fibrinolytic activity have been demonstrated in cell lines derived from an ethylnitrosourea-induced glioma of the rat brain. Cell lines derived from normal adult rat brain showed only low levels of activity. The degree of lysis produced by a cell line was dependent on the average number of cells per colony, and a different pattern of response was observed for tumour and normal cell lines. A good positive correlation existed between the level of fibrinolytic activity, growth in agar and tumourigenicity of a cell line. Fibrinolytic activity was associated with cell lines derived at various times in the latent period, before the appearance of a visible tumour. Many cell lines derived from rat brains at 57-60 (E7), 90-91 (E8) and 111-112 (E6) days after transplacental exposure to ethylnitrosourea showed fibrinolytic activity, and in the latter group the close association with growth in agar and tumourigenicity was also demonstrated. Results from cell lines derived in the E7 and E8 experiments indicated that the possession of fibrinolytic activity preceded the ability of cells to form colonies in agar.


We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.


Intracellular protoporphyrin IX (PpIX) content was quantified by extraction and fluorescence spectroscopy. Cellular PpIX content did not always correlate with cell proliferation rate as measured by the doubling times of cell lines. Cellular efflux of PpIX was also investigated. In a bladder carcinoma cell line, the observed rapid efflux was not blocked by verapamil, an inhibitor of the P-glycoprotein efflux pump. These data support the view that cellular PpIX accumulation is a dynamic process that is determined by both the efflux of PpIX from the cells and enzyme activities in the haem biosynthesis pathway. Desferrioxamine , a modulator of PpIX biosynthesis, enhanced ALA-induced cellular PpIX content significantly in all carcinoma cell lines but not in non-malignant cell lines. The enhanced PpIX cellular accumulation is attributed to inhibition of ferrochelatase activity, the enzyme responsible for the conversion of PpIX to haem. PpIX-mediated cellular photodestruction following irradiation with an argon ion laser at 514.5 nm was determined by the 'MTT assay'. There appeared to be a 'threshold' effect of cellular PpIX content; cells that synthesised less than 140 ng/mg-1 protein exhibited very little phototoxic damage, while cell lines having greater than 140 ng PpIX/mg-1 protein [corrected] exhibited a consistent phototoxic response. Among the cell lines which did undergo phototoxic damage, there was not a strict correlation between PpIX cellular content and ALA-induced phototoxicity. Desferal enhanced the PpIX content and phototoxic effect in the responsive cells. Fluorescence microscopy of the ALA-treated cells revealed marked accumulation of PpIX in mitochondria (rhodamine 123 co-staining). That the primary site of phototoxic damage is also the mitochondria was confirmed by electron micrographs of cells photosensitised with ALA-induced PpIX, which showed swelling of mitochondria within minutes after irradiation while other suborganelles appeared to be unaffected. The repair or further destruction of the mitochondria was fluence and cell-type dependent. The data from this study suggest that the basis of increased ALA-induced PpIX accumulation in tumours is a combination of various aspects of the metabolic process and pharmacokinetics and that the efficacy of photodestruction of malignancy will be determined not only by the rate of PpIX synthesis but also by specific cellular and tissue characteristics.


HLA-DM region in humans, the β-chain locus is duplicated in mouse, with the H2-Mb1  and the H2-Ma (Ma) α-chain gene). Here, we show that murine MHC class II and H2-M genes are coordinately regulated in murine tumour cell lines by T helper cell 1 (IFN-γ) and T helper cell 2 (IL-4 or IL-10) cytokines in the presence of the MHC class II-specific transactivator CIITA as determined by mRNA expression and Western blot analysis. Furthermore, Mαβ1 and Mαβ2 heterodimers are differentially expressed in murine tumour cell lines of different histology. Both H2-M isoforms promote equally processing and presentation of native protein antigens to H2-Ad- and H2-Ed-restricted CD4+ T cells. Murine tumour cell lines could be divided into three groups: constitutive MHC class II and CIITA expression; inducible MHC class II and CIITA expression upon IFN-γ-treatment; and lack of constitutive and IFN-γ-inducible MHC class II and CIITA expression. These differences may impact on CD4+ T cell recognition of cancer cells in murine tumour models. © 2000 Cancer Research CampaignQualitative differences in the MHC class II antigen processing and presentation pathway may be instrumental in shaping the CD4+ T cell response directed against tumour cells. Efficient loading of many MHC class II alleles with peptides requires the assistance of H2-M, a heterodimeric MHC class II-like molecule. In contrast to the


We have immortalized thymicstromal elements using recombinant retroviral constructs containing a temperature-sensitivesimian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1aregion linked to the gene coding for resistance to G418. Cell lines containing thethermolabile large T antigen encoded by SV40 proliferate at the permissive temperatureof 33°C and arrest growth when transferred to the nonpermissive temperature of 39°C.At the nonpermissive temperature, ts-derived cell lines are shown to alter theirphenotype but remain metabolically active, as indicated by the inducible expression ofclass I and class II MHC antigens. Here we describe the generation of a total of 84thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, andfunctional properties consistent with fibroblastoid, epithelial, or monocytoid origins.Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelialcharacteristic of desmosome formation and, in addition, two of these lines (15.5 and15.18) form multicellular complexes (rosettes) when incubated with unfractionatedthymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecificesterase activity, suggesting it may represent a macrophagelike cell type. We describethe generation of stromal cell lines with different properties, which is consistent with theheterogeneity found in the thymic microenvironment. In addition to documenting thisdiversity, these cell lines may be useful tools for studying T-cell development


The human breast cancer cell line MCF-7 requires oestrogen to produce and promote growth of tumours in athymic mice. In vitro, however, MCF-7 cells proliferate rapidly without supply of oestrogen . Oestrogen stimulation of proliferation of MCF-7 cells can be achieved when the cells are grown at high concentration of newborn calf serum  or oestrogen deprived foetal calf serum (10%). The stimulation involves an abolishment of inhibitory activity present in the serum. The oestradiol stimulated cultures grow rapidly for a much longer time period and attain a much higher cell density than the unstimulated cultures. Oestrogen is specific for the promotion of cell proliferation and only oestrogen receptor positive cell lines with a functional oestrogen receptor mechanism can be stimulated. We assume that oestradiol acts directly on the cells and via the oestrogen receptor mechanism induces the synthesis of a substance which abolishes the inhibitory activity in serum. We call this mechanism of action an indirect stimulation of cell proliferation. A similar mechanism may exist in vivo since we find that serum from athymic mice contains a growth inhibitory activity towards MCF-7 cells and the inhibitory effect can be abolished by oestradiol.


CLDN11) is silenced in gastric cancer via hypermethylation of its promoter region.Claudins are membrane proteins that play critical roles in tight junction (TJ) formation and function. Members of the claudin gene family have been demonstrated to be aberrantly regulated, and to participate in the pathogenesis of various human cancers. In the present study, we report that claudin-11  and quantitative reverse transcriptase-PCR (qRT-PCR), respectively. Analyses of paired gastric cancers and adjacent normal gastric tissues revealed hypermethylation of the CLDN11 promoter region in gastric cancers, and this hypermethylation was significantly correlated with downregulation of CLDN11 expression vs. normal tissues. The CLDN11 promoter region was also hypermethylated in all gastric cancer cell lines tested relative to immortalized normal gastric epithelial cells. Moreover, CLDN11 mRNA expression was inversely correlated with its methylation level. Treatment of CLDN11-nonexpressing gastric cancer cells with 5-aza-2′-deoxycytidine restored CLDN11 expression. Moreover, siRNA-mediated knockdown of CLDN11 expression in normal gastric epithelial cells increased their motility and invasiveness.Levels of CLDN11, leading to downregulated expression, contributes to gastric carcinogenesis by increasing cellular motility and invasiveness. A further understanding of the mechanisms underlying the role of claudin proteins in gastric carcinogenesis will likely help in the identification of novel approaches for diagnosis and therapy of gastric cancer.These data suggest that hypermethylation of  Gastric cancer (GC) remains the second most-common cause of cancer deaths globally. It is one of the most lethal malignancies and a leading cause of cancer deaths in developing countries, with overall 5-year survival rates below 20% Studies of GCs and their preneoplastic precursor lesions have identified several genetic and epigenetic alterations, including microsatellite instability, point mutations, and loss of heterozygosity (LOH) affecting tumor suppressor genes (TSGs) Epigenetic alterations are extremely important in cancer development and progression Previously, studies have been published on promoter hypermethylation in GCs and their premalignant precursors CLDN11), a tight junction (TJ) protein, as a potential target of epigenetic inactivation in gastric cancers (unpublished data).A pilot microarray-based genome-wide search conducted by our group, performed to discover novel epigenetically silenced genes in gastric carcinogenesis, identified claudin-11  were obtained from Dr. Duane T. Smoot (Howard University) and GC cell lines AGS, SIIA, MKN28, KATOIII, and SNU-1 were obtained from ATCC. All cell lines were cultured and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution (Invitrogen).Paired primary gastric normal and tumor tissues were collected at the Johns Hopkins Hospital (JHH). Specimens were snap-frozen immediately after resection.Johns Hopkins University (JHU) Institutional Review Board (IRB) approval was obtained for all the cases included in the study. Cases were obtained from JHU surgical pathology under JHU IRB-approved exemption 02-07-19-05e under rule 45 CFR 46.101(b), which waives the requirements for obtaining patient consent.Genomic DNA was extracted from snap-frozen tissue samples or cell lines using a DNeasy Blood & Tissue Kit  according to the manufacturer's protocol. Total RNA was extracted with Trizol reagent (Invitrogen). Extracted DNA and RNA were quantified using a NanoDrop ND-1000 Spectrophotometer .CLDN11 gene. CLDN11-specific primers and probe sequences used were: forward primer 5′ CGCGATTGGTCGGCGCGTTTC 3′; reverse primer 5′ GACGAAAACAACAACGCTACT 3′; TaqMan probe 5′TCGGAGTCGCGGGGTTTAAAGAG 3′. CpGenome Universal Methylated DNA  served as a positive control, and serial dilutions of it were used to plot a standard curve. qMSP with TaqMan probe were performed on an iQ5 thermal cycler  using iQ Supermix (ibid.). Fifty cycles of PCR amplification starting with 50 ng of bisulfite-treated genomic DNA were performed in triplicate, according to the manufacturer's protocol. Duplex PCR with β-actin (ACTB) primer and probe sequences containing no CpGs were performed for normalization. The primer and probe sequences used were as published previously CLDN11-S/CLDN11-FM)/(ACTB-S/ACTB-FM) * 100, where CLDN11-S and CLDN11-FM represent CLDN11 methylation levels in the sample and fully methylated DNAs, respectively, while ACTB-S and ACTB-FM correspond to β-actin in the sample and fully methylated DNAs, respectively. Whole-genome amplified DNA (WGA) was used as an unmethylated negative control.Genomic DNAs obtained from 36 patient samples comprising 18 GC and 18 matched noncancerous stomach mucosal (NS) tissues, as well as from various gastric cell lines, were subjected to qMSP. qMSP was performed as described previously, with minor modifications CLDN11 RT-PCR amplicon was designed to overlap an intron-exon boundary in order to exclude genomic DNA (gDNA) amplification. Primer sequences were as published previously CLDN11expression was normalized to β -actin expression. Total RNA from HFE145 cells was used for the standard curve. (CLDN11-S/CLDN11-C)/(ACTB-S/ACTB-C), where CLDN11-S and CLDN11-C represent CLDN11 mRNA expression levels in the test sample and control mRNAs, respectively, while ACTB-S and ACTB-C correspond to β-actin expression levels in the test sample and control mRNAs, respectively.The CLDN11 promoter in conjunction with absent CLDN11 mRNA expression. 5-aza-2′-deoxycitidine (5-aza-dC) treatment of cells was performed as described previously 5 cells/ml in a T-75 flask. Twenty-four hours later, cells were treated with 1 µM 5-aza-dC for 72 hours. Media containing 5-aza-dC was replaced with freshly prepared media every 24 hours. Cells were then harvested for total RNA extraction. Total RNAs from AGS cells before and after 5-aza-dC treatment were subjected to quantitative real-time RT-PCR analysis.AGS is a GC cell line manifesting hypermethylation of the CLDN11-specific siRNA oligos were purchased from Ambion, Inc. . The HFE145 cell line, which is CLDN11 positive, was selected to study the effect of claudin-11 knockdown on migration and invasion properties of gastric cells. Experiments were conducted as described previously . Cells cultured in 6-well plates were transfected with siRNA duplexes using LipofectAMINE 2000 (Invitrogen), following the manufacturer's instructions. Mock-transfections and nonspecific siRNA duplexes were used as negative controls. Cells were treated for 48 to 72 hours to allow maximum knockdown, after which they were either harvested for Western blot analysis or used for migration and invasion assays.Confluent cell cultures were washed with HBSS (Invitrogen) and whole cell lysates were made using lysis buffer: 62.5 mmol/L Tris-HCl (pH 6.8), 10% glycerol, and 2% SDS. Protein concentration was determined using a bicinchoninic acid (BCA) assay kit . Twenty micrograms of total proteins were separated by 10% to 20% SDS-PAGE on Tris-Glycine gels (Invitrogen) and transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5% nonfat dry milk, washed in TBST buffer, and probed with an anti-claudin-11 antibody . Blots were then washed and incubated in horseradish peroxidase-conjugated secondary antibody . For detection, chemiluminescence was carried out using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).i.e., the well beneath the filter) to act as a chemoattractant. Assay plates were incubated at 37°C for up to 48 hrs. Cells that did not migrate or invade through the pores of the Transwell inserts were manually removed with a cotton swab. Cells present at the bottom of the membrane were fixed in cold methanol for 10 min and then stained with 0.01% crystal violet in 20% ethanol. After 10 min incubation, the filters were washed thoroughly in water and suspended in 200 µL of 5% acetic acid and 5% methanol. Colorimetric readings were taken at OD595. Experiments were repeated at least three times, with triplicates in each experiment. To assess cell migration, assays were carried out essentially as above, except that cells were plated on top of uncoated (Matrigel-free) inserts.Invasiveness of siRNA-transfected cells was determined using Matrigel-coated invasion chamber inserts  using a modified Boyden chamber assay p<0.05 considered statistically significant.Statistical analyses were performed using Student's t-test , with CLDN11 promoter is hypermethylated and that this hypermethylation correlates inversely with CLDN11 mRNA expression in primary GC tissues. Paired primary gastric normal and tumor tissues were collected at the Johns Hopkins Hospital (JHH). Specimens were obtained immediately after resection and snap-frozen until use. Only cases obtained with informed consent as approved by the Institutional Review Board (IRB) were included in this project. Genomic DNAs were obtained from 36 clinical specimens, comprising 18 GC and 18 matched noncancerous gastric mucosal (NS) tissues. CLDN11 promoter methylation levels in these samples were analyzed using quantitative real-time methylation-specific PCR (qMSP). i.e., the ratio of methylated value of the CLDN11 promoter region in each specimen to a fully methylated control DNA. CLDN11 NMVs were significantly higher in GC specimens than in their matching NS tissues (P<0.001). To assess whether CLDN11 promoter hypermethylation in GCs was associated with silencing of CLDN11 expression, CLDN11 mRNA levels were measured using quantitative real-time (qRT–PCR) in RNAs extracted from the 36 specimens used for methylation analysis. As demonstrated in CLDN11 in GC specimens were significantly lower than in paired NS samples (P<0.001). This data establishes that CLDN11 promoter hypermethylation correlates with diminished or silenced CLDN11 mRNA expression in primary GCs.We first tested our hypothesis that the CLDN11 promoter methylation and expression in gastric cell lines. Immortalized human normal gastric epithelial cells (HFE145) and GC cell lines AGS, SIIA, MKN28, KATOIII, and SNU-1 were studied. All GC cell lines tested  demonstrated promoter hypermethylation, whereas no methylation was observed in immortalized normal HFE145 cells . CLDN11 mRNA levels were assessed using qRT-PCR on RNAs purified from the cell lines, while claudin-11 protein expression was analyzed by Western blotting. As shown in CLDN11 mRNA or protein, while HFE145 cells manifested high CLDN11 expression levels. This finding establishes that CLDN11 is coordinately hypermethylated and downregulated in GC cell lines relative to normal gastric epithelial cells (NGECs).Next, we evaluated 45 cells . CLDN11 CLDN11 expression by hypermethylation, we treated the GC cell line AGS with the demethylating agent, 5-aza-2-deoxycytidine , for varying time intervals. Total RNAs extracted before vs. after treatment were subjected to qRT-PCR for CLDN11. As can be seen in CLDN11 mRNA became re-expressed upon treatment with 5-aza-dC, confirming that CLDN11 is silenced by promoter hypermethylation in AGS GC cells.To further validate silencing of CLDN11 influences cell motility or invasiveness. HFE145 cells, which express abundant CLDN11, were chosen to study effects of CLDN11 knockdown on the invasive and migratory properties of gastric epithelial cells. Transient siRNA transfections were carried out using CLDN11-specific siRNA duplexes. Transfection with CLDN11-specific siRNA duplexes efficiently repressed claudin-11 protein levels by>90%, whereas expression remained unchanged in mock- or control siRNA-treated cells . This scan identified putative binding sites for Sp1 and GATA-1 and GATA-2. Previous studies have shown that hypermethylation of promoter DNA contributes to silencing of CLDN3 and CLDN4 in ovarian cell lines, in part via methylation-induced disruption of binding of Sp1 to its cognate binding site . In addition, members of the GATA family have been shown to be positive regulators of CLDN11 transcription CLDN11 gene transcription.Promoter hypermethylation is known to be associated with transcriptional silencing of certain genes. DNA methylation can interfere with binding of transcription factors whose binding sites contain CpG dinucleotides. Using the TFSEARCH software program, we scanned the Tumor cells typically exhibit structural and functional deficiencies in their TJs Modulations in expression of TJ-associated proteins, particularly claudin proteins, have been shown in a number of cancers. Recent studies by us and others have shown alterations in claudin protein regulation in various epithelial cancers CLDN18 gene as downregulated in GCs possessing an intestinal phenotype CLDN23 gene is also downregulated in intestinal-type GCs et al. reported CLDN4 expression to be increased in intestinal metaplasia and gastric epithelial dysplasia, identifying this gene as a marker of GC precursor lesions CLDN11 expression to be downregulated or silenced in GC via hypermethylation of its promoter region. Moreover, we found that unlike CLDN18 and CLDN23, CLDN11 expression was downregulated in GC patient specimens as well as in cell lines, irrespective of diffuse vs. intestinal subtype.Studies have previously reported changes in expression profiles of members of the claudin family to be associated with GC. Serial analysis of gene expression (SAGE) identified the CLDN11 from the blood–testis barrier in Sertoli cells to be associated with testicular intraepithelial neoplasia in men CLDN11 is expressed in normal gastric tissues and immortalized NGECs. However, its complete functions in normal stomach as well as in gastric carcinogenesis remain unclear.Claudin-11, also known as oligodendrocyte-specific protein, was first identified to be specifically expressed in the tight junction strands of oligodendrocytes in brain and in Sertoli cells of rats and mice CLDN11 in GC, we studied phenotypic changes associated with silencing of CLDN11 expression in CLDN11-expressing gastric epithelial cells (HFE145). Very interestingly, siRNA-mediated knockdown of CLDN11 resulted in increased cell motility and invasion.In an attempt to explore the possible involvement of Previous studies have reported cancer-specific phenotypic changes to be associated with modulations in claudin expression in various cancer types. Overexpression of claudin-3 and claudin-4 proteins in ovarian cancer cell lines results in increased invasion by these cells Although in recent years it has become clear that TJs and TJ-associated proteins have extensive and diverse functional and physiological activities in normal and cancerous conditions, molecular mechanisms underlying these activities remain unclear. TJs are sophisticated intercellular apparatuses capable of recruiting signaling proteins, thereby regulating various cellular processes including cell growth, differentiation, and tumorigenesis CLDN11 as a target of epigenetic modification, as well as a promising biomarker for gastric cancer early detection, diagnosis, and therapy. These findings also suggest involvement of CLDN11 downregulation in carcinogenesis via promotion of cell invasion and motility. The mechanisms for this phenomenon are subject to further investigation.In summary, data from the current study identify


The results of cytotoxicity trials against a panel of seven human cell lines for a series oftriorganophosphinegold(I) 3- and 4-mercaptobenzoates are reported. While the new compounds showmoderate to high toxicity, their potencies are inferior to those reported previously for their isomeric 2-mercaptobenzoate derivatives. The results therefore suggest a structure-activity relationship in that the 2-isomeric species are more active, particularly against the non-small cell lung cancer and renal cancer celllines, results that may indicate some selectivity in their cytotoxic profile.


The relationship between ageing and transformation has been investigated by a serial study of the changes in cell-surface morphology as normal and carcinogen-treated cells progressed in culture. A progressive increase in the density of cell surface microvilli occurred in association with the adoption of a more rounded profile and concomitant increase in the rate of cell detachment. These changes occurred earlier after carcinogen treatment, which appeared to indicate a carcinogen-induced acceleration of ageing. The alterations have also been described as characteristic of the transformed state. The observations suggest that the expression of in vitro transformation may be the result of continuous selection from a population with genetic instability and variable morphology.