Flow cytometric double labeling technique for screening of multidrug resistance.

We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16. Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR-content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR-content were not related to differences in DNA-content. In experimental cell lines immunofluorescence data were inversely related with those of DNR-content: MDR cells had high levels of P-gp expression and low levels of DNR-content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.     

Current issues in the utility of 19F nuclear magnetic resonance methodologies for the assessment of tumour hypoxia

It is now well established that uncontrolled proliferation of tumour cells together with the chaotic and poorly regulated blood supply of solid tumours result in tissue hypoxia, and that hypoxic regions of tumours are resistant to radiotherapy and chemotherapy. The development and application of non-invasive methods to rapidly determine the degree and extent of tumour hypoxia in an individual tumour would clearly enhance cancer treatment strategies. This review describes the current status of two (19)F nuclear magnetic resonance (NMR) methodologies that have been exploited to investigate tumour hypoxia, namely: (i) (19)F NMR oximetry following administration of perfluorocarbons, from which tumour p(O)(2) measurements can be made; and (ii) (19)F NMR measurements of the tumour retention of fluorinated 2-nitroimidazoles.     

Amphotericin B susceptibility testing of Candida species by flow cytometry.

We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.     

The physiological role of biogenic amines redox reactions in mitochondria. New perspectives in cancer therapy

Summary. In tumours, polyamines and amine oxidases increase as compared to normal tissues. Cytotoxicity induced by bovine serum amine oxidase (BSAO) and spermine is attributed to H₂O₂ and aldehydes produced by the reaction. Increasing the incubation temperature from 37 to 42 °C enhances cytotoxicity in cells exposed to spermine metabolites. The combination BSAO/spermine prevents tumour growth, particularly well if the enzyme has been conjugated with a biocompatible hydrogel polymer. Since the tumour cells release endogenous substrates of BSAO, the administration of spermine is not required. Combination with hyperthermia improves the cytocidal effect of polyamines oxidation products. Our findings show that multidrug resistant (MDR) cells are more sensitive to spermine metabolites than their wild-type counterparts, due to an increased mitochondrial activity which induces the generation of intracellular ROS prior to the onset of mitochondrial permeability transition (MPT). It makes this new approach attractive, since the development of MDR is one of the major problems of conventional cancer therapy.     

Downregulation of taurine uptake in multidrug resistant Ehrlich ascites tumor cells

Summary.  In daunorubicin resistant Ehrlich ascites tumor cells (DNR), the initial taurine uptake was reduced by 56% as compared to the parental, drug sensitive Ehrlich cells. Kinetic experiments indicated that taurine uptake in Ehrlich cells occurs via both high- and low-affinity transporters. The maximal rate constant for the initial taurine uptake was reduced by 45% (high-affinity system) and 49% (low affinity system) in the resistant subline whereas the affinity of the transporters to taurine was unchanged. By immunoblotting we identified 3 TauT protein bands in the 50–70 kDa region. A visible reduction in the intensity of the band with the lowest molecular weight was observed in resistant cells. Quantitative RT-PCR indicated a significant reduction in the amount of taurine transporter mRNA in the resistant cells. Drug resistance in DNR Ehrlich cells is associated with overexpression of the mdr1 gene product P-glycoprotein (P-gp). Using 5 progressively DNR resistant Ehrlich cell sublines with different P-gp expression pattern no correlation between taurine uptake and P-gp expression was found. Taurine uptake in MDR1 transfected NIH/3T3 mouse fibroblasts was in contrast to the findings in Ehrlich cells increased compared to the parental fibroblasts. It is concluded that the reduced taurine uptake in resistant Ehrlich cells reflects a down regulation of the taurine transporter at the mRNA and protein level and it is most probably not related to P-gp overexpression. Received October 22, 2001 Accepted November 26, 2001     

Protein kinase CK1α regulates erythrocyte survival

Protein kinase CK1 (casein kinase 1) isoforms are involved in the regulation of various physiological functions including apoptosis. The specific CK1 inhibitor D4476 may either inhibit or foster apoptosis. Similar to apoptosis of nucleated cells, eryptosis, the suicidal death of erythrocytes, is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity following energy depletion (removal of glucose) or oxidative stress (exposure to the oxidant tert-butyl hydroperoxide [TBOOH]). Western blotting was utilized to verify that erythrocytes express the protein kinase CK1α, and FACS analysis to determine whether the CK1 inhibitor D4476 and CK1α activator pyrvinium pamoate modify forward scatter (reflecting cell volume), annexin V binding (reflecting phosphatidylserine exposure), and Fluo3 fluorescence (reflecting cytosolic Ca(2+) activity). As a result, both, human and murine erythrocytes express CK1 isoform α. Glucose depletion (48 hours) and exposure to 0.3 mM TBOOH (30 minutes) both decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence. CK1 inhibitor D4476 (10 μM) significantly blunted the decrease in forward scatter, the increase in annexin V binding and the increase in Fluo 3 fluorescence. (R)-DRF053, another CK1 inhibitor, similarly blunted the increase in annexin V binding upon glucose depletion. The CK1α specific activator pyrvinium pamoate (10 μM) significantly enhanced the increase in annexin V binding and Fluo3 fluorescence upon glucose depletion and TBOOH exposure. In the presence of glucose, pyrvinium pamoate slightly but significantly increased Fluo3 fluorescence. In conclusion, CK1 isoform α participates in the regulation of erythrocyte programmed cell death by modulating cytosolic Ca(2+) activity.     

p38 MAPK activation and function following osmotic shock of erythrocytes

p38 protein kinase is activated by hyperosmotic shock, participates in the regulation of cell volume sensitive transport and metabolism and is involved in the regulation of various physiological functions including cell proliferation and apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include hyperosmotic shock, which increases cytosolic Ca(2+) activity and ceramide formation. The present study explored whether p38 kinase is expressed in human erythrocytes, is activated by hyperosmotic shock and participates in the regulation of eryptosis. Western blotting was utilized to determine phosphorylation of p38 kinase, forward scatter to estimate cell volume, annexin V binding to depict phosphatidylserine exposure and Fluo3 fluorescence to estimate cytosolic Ca(2+) activity. As a result, erythrocytes express p38 kinase, which is phosphorylated upon osmotic shock (+550 mM sucrose). Osmotic shock decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence, all effects significantly blunted by the p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM). In conclusion, p38 kinase is expressed in erythrocytes and participates in the machinery triggering eryptosis following hyperosmotic shock.     

Mechanisms of "cytostasis" of tumours in vitro by syngeneic lymphoid cells of tumour bearers.

The cytostasis assay is an in vivo-in vitro radioactive technique which detects antitumour responses of the syngeneic tumour-bearing hosts. Examination and characterization of effector mechanisms at the cellular and humoral levels revealed that the cytostasis assay using Meth A (a 3-methylcholanthrene-induced) tumour was T cell independent. Furthermore, both B cells and macrophages were required. It was concluded that the mechanism involved complement-dependent antibody-mediated lysis of the tumour cells, with B cells producing antibody and macrophages producing the complement components during incubation. However, antibody-dependent cell-mediated cytotoxicity with or without complement could not be completely excluded. Although antibody was detected in vivo, specific antibody against Meth A tumour was produced in vitro by cultured lymphoid cells from the tumour-bearers. Antibody-coated Meth A cells caused regression of some tumours when inoculated into BALB/c mice. When these regressor mice were rechallenged with tumour, they were found to be permanently immune to the tumour. In the light of these findings, the role of antibody in the protection of tumours and its implications are discussed.     

Dietary red palm oil protects the heart against the cytotoxic effects of anthracycline

Strong anti-neoplastic anthracyclines like daunorubicin (DNR) and doxorubicin (DOX) have high efficacy against systemic neoplasm and solid tumours. However, clinically, they cause chronic cardiomyopathy and congestive heart failure. Red palm oil (RPO) supplementation can protect the heart against ischemic injury. We therefore hypothesize that supplementation with RPO during chemotherapy may protect the heart. Control rats received a standard diet, and the experimental group received RPO in addition for 4 weeks. Each group was subsequently injected with either saline or DNR over a 12-day period towards the end of 4 weeks. Hearts were excised and perfused on a working heart system. Functional parameters were measured. Tissue samples were collected for analysis of mRNA and protein levels. DNR + RPO increased aortic output by 25% (p < 0.05) compared with DNR only. Furthermore, DNR treatment significantly reduced tissue mRNA levels of superoxide dismutase 1 (SOD1) and nitric oxide synthase 1 (NOS1) compared with untreated controls. Protein expression of SOD1 followed the same pattern as mRNA levels. NOS1 protein levels were significantly increased in DNR treated rats when compared with untreated controls. In addition, DNR increased phosphorylation of p38 and Jun N-terminal kinase compared with untreated controls, whereas DNR + RPO completely counteracted this activation. DNR + RPO significantly up regulated the protein extracellular signal-regulated kinase 1 level compared with DNR only. In this model of DNR treatment, RPO is associated with stabilization of important antioxidant enzymes such NOS and SOD, and inhibition of the 'stress' induced mitogen-activated protein kinase pathways. Dietary RPO also maintained function, similar to control, in DNR treated hearts.     

Low and high concentrations of the topo II inhibitor daunorubicin in NIH3T3 cells: reversible G2/M versus irreversible G1 and S arrest

Daunorubicin (DNR) blocks the cell cycle by interfering with synthesis and repair of DMA. In both drug-sensitive 3T3 cells and drug-resistant 3T3 cells (NIH-MDR-6185, created by transfection with a human MDR1 cDNA), low concentrations of DNR (up to 80 ng/ml in sensitive cells, 1600 ng/ml in resistant cells) initially slowed S-phase progression for 2 to 3 hours, but the treated cells then continued in progression at a steady rate, close to that of untreated cells, and accumulated in G(2)/M. The 2 to 3 h lag period represents the time taken for fully establishing the G(2)/M block. The time required to bring about cessation of proliferation is the sum of this lag period and the time taken to travel through the cell cycle. This low concentration effect is cytostatic, and fully reversible on washing out the daunorubicin. At higher drug concentrations (above 160 ng/ml in sensitive cells, 3200 ng/ml in resistant cells) the cells became blocked in both G] and S, and did not reach G(2)/M. The high concentration effect was cytotoxic and irreversible, and was followed by cell death. Only cells that were in S phase were subject to this block in S, since cells that had accumulated in G(2)/M by using a low concentration (60 ng/ml DNR for 20 h) were not blocked in S, and did not die, when subsequently treated with high drug concentrations (320 ng/ml, 30 h). The low concentration effect occurred at the same maximal rate (4 %/h) in sensitive or resistant cells, but the external drug concentration required to produce half the maximal rate was, appropriately, twenty-fold higher in the resistant cells (20 ng/ml and 400 ng/ml, respectively).     

The effect of insulin on the growth of transplanted tumors in mice

The growth of four murine transplantable tumours, Ehrlich ascites or solid tumour, an aplastic carcinoma, B-16 melanoma, and a thymoma, were suppressed in mice treated daily with insulin (2 IU). Since insulin increased the number of plaque-forming cells and the phagocytic activity of the liver and spleen cells, the retardation of the tumour growth was ascribed to immunological mechanisms.     

Low and High Concentrations of the Topo II Inhibitor Daunorubicin in NIH3T3 Cells: Reversible G2/M Versus Irreversible G1 and S Arrest

Daunorubicin (DNR) blocks the cell cycle by interfering with synthesis and repair of DNA. In both drug-sensitive 3T3 cells, and drug-resistant 3T3 cells, NIH-MDR-6815, (created by transfection with a human MDR1 cDNA), low concentrations of DNR (up to 80 ng/ml in sensitive cells, 1600 ng/ml in resistant cells), cells initially slowed S-phase progression for 2 to 3 hours, but the treated cells then continued in progression at a steady rate, close to that of untreated cells and accumulated in G2/M. The 2 to 3 h lag period represents the time taken for fully establishing the G2/M block. The time required to bring about cessation of proliferation is the sum of this lag period and the time taken to travel through the cell cycle. This low concentration effect is cytostatic, and fully reversible on washing out the daunorubicin. At higher drug concentrations (above 160 ng/ml in sensitive cells, 3200 ng/ml in resistant cells) the cells became blocked in both G1 and S, and did not reach G2/M. The high concentration effect was cytotoxic and irreversible, and was followed by cell death. Only cells that were in S phase were subject to this block in S, since cells that had accumulated in G2/M by using a low concentration (60 ng/ml DNR for 20 h) were not blocked in S, and did not die, when subsequently treated with high drug concentrations (320 ng/ml, 30 h). The low concentration effect occurred at the same maximal rate (4 %/h) in sensitive or resistant cells, but the external drug concentration required to produce half the maximal rate was, appropriately, twenty-fold higher in the resistant cells (20 ng/ml and 400 ng/ml, respectively).     

CELL LOSS FROM SEVERAL TYPES OF SOLID MURTNE TUMOUR: COMPARISON OF 125I-IODODEOXYURIDINE AND TRITIATED THYMIDINE METHODS

The method of measuring tumour cell loss rates in situ following radioactivity loss after a single injection of ¹²⁵I-iododeoxyurudine (¹²⁵I-UdR) was tested for its accuracy in five different types of murine tumour. To achieve this the method was compared with two others: (1) using ¹²⁵I-UdR, but excising tumours before the radioactivity determinations, with or without extracting DNA; (2) using tritiated thymidine and autoradiography. A third method was used on three of the tumours, in which ¹²⁵I-UdR-labelled tumours were grown in unlabelled hosts, followed by whole body counting of the tumour-bearing mice. In two of the tumours an increase was observed in total tumour radioactivity with time after ¹²⁵I-UdR injection. This prevented the estimation of cell loss parameters in these tumours. Approximately half the increase was due to reutilization of ¹²⁵I-UdR supplied from tissues within the mouse; approximately a third to an influx of labelled inflammatory cells (probably in response to infection accompanying ulceration of overlying skin); and the remainder to an increase in non-DNA radioactivity. In these tumours cell loss rates could be obtained from the whole body counting technique in which influxes of labelled cells and reutilizable radioactivity were eliminated. A comparison of either ¹²⁵I-UdR technique with the ³H-TdR technique showed good agreement of the cell loss factors for the low cell loss tumours. However, for tumours with high cell loss factors the ¹²⁵I-UdR technique gave lower values for cell loss. This implied that reutilization of ¹²⁵I-UdR within the tumour (i.e. from internal, not external sources) occurred in the high cell loss tumours. It is concluded that equating radioactivity loss with cell loss after an injection of ¹²⁵I-UdR is reasonable for some tumours, but will result in significant underestimates in others. For high cell loss tumours the ³H-TdR technique will give the     

Nitric oxide and P‐glycoprotein modulate the phagocytosis of colon cancer cells

The anticancer drug doxorubicin induces the synthesis of nitric oxide, a small molecule that enhances the drug cytotoxicity and reduces the drug efflux through the membrane pump P‐glycoprotein (Pgp). Doxorubicin also induces the translocation on the plasma membrane of the protein calreticulin (CRT), which allows tumour cells to be phagocytized by dendritic cells. We have shown that doxorubicin elicits nitric oxide synthesis and CRT exposure only in drug‐sensitive cells, not in drug‐resistant ones, which are indeed chemo‐immunoresistant. In this work, we investigate the mechanisms by which nitric oxide induces the translocation of CRT and the molecular basis of this chemo‐immunoresistance. In the drug‐sensitive colon cancer HT29 cells doxorubicin increased nitric oxide synthesis, CRT exposure and cells phagocytosis. Nitric oxide promoted the translocation of CRT in a guanosine monophosphate (cGMP) and actin cytoskeleton‐dependent way. CRT translocation did not occur in drug‐resistant HT29‐dx cells, where the doxorubicin‐induced nitric oxide synthesis was absent. By increasing nitric oxide with stimuli other than doxorubicin, the CRT exposure was obtained also in HT29‐dx cells. Although in sensitive cells the CRT translocation was followed by the phagocytosis, in drug‐resistant cells the phagocytosis did not occur despite the CRT exposure. In HT29‐dx cells CRT was bound to Pgp and only by silencing the latter the CRT‐operated phagocytosis was restored, suggesting that Pgp impairs the functional activity of CRT and the tumour cells phagocytosis. Our work suggests that the levels of nitric oxide and Pgp critically modulate the recognition of the tumour cells by dendritic cells, and proposes a new potential therapeutic approach against chemo‐immunoresistant tumours.     

Cytotoxic susceptibility and defective MHC class I expression do not correlate with mutation of p53 in human carcinomas

Twenty-nine samples of ex vivo ovarian and lung carcinomas were investigated for the relationship between the presence of mutated protein 53 (mp53) and cytotoxic susceptibility. Unaltered expression of MHC class I alleles was required for the cytotoxic susceptibility of tumour cells to the autologous ex vivo blood lymphocytes, i.e. all 4 sensitive tumours belonged to the group of 11 tumours without defect in MHC class I expression. In contrast, the susceptibility did not correlate with the presence of mp53, i.e. cases with mp53 were randomly distributed between the sensitive and resistant tumours (2/4 and 10/17 respectively). There was no correlation either between the p53 mutation and down-regulation of MHC class I alleles. The results suggest that in these tumours the mutated p53 is not the source of immunogenic peptides and that the lack of recognition of the tumours with mp53 is not caused by a defect in the expression of MHC class I molecules.     

Diversity of penetration of anti-cancer agents into solid tumours

Failure of anti-cancer agents to reach all clonogenic cells at cytotoxic concentrations is recognized as an important form of resistance in solid tumours. Subcutaneously implanted mammary adenocarcinoma 16/C was used to evaluate the intratumour distribution of five alkylating, bioreductive alkylating and intercalating agents and two radiation sensitizers. The agents were classified according to their in vivo distribution in well- and poorly-perfused tumour regions, as delineated by lissamine green. The classifications were: (1) distribution in direct proportion to the vascular supply; (2) uniform distribution to well- and poorly-perfused tumour regions; and (3) preferential retention in the poorly-perfused tumour regions. Our current state of knowledge did not allow reliable prediction of the classification based on chemical structure or mechanism of action.     

Sulfated glycosaminoglycan and collagen patterns in parietal yolk sac carcinoma (PYSC)

Electrophoretic analyses of collagenous material have shown that the parietal yolk sac carcinoma (PYSC) ascitic tumour synthesizes polypeptide chains that migrate as type IV procollagen. Having molecular weights of 185,000 and 160,000, these polypeptides are sensitive to collagenase. When the PYSC cells are injected subcutaneously, they form a solid tumour, and type I collagen predominates. The electrophoretic analyses of sulfated glycosaminoglycans and enzymatic degradation have shown a predominance of heparan sulfate in the ascitic tumour, and of chondroitin sulfate B in the solid tumour. Cells cultured from ascitic tumours have maintained the same collagen and sulfated glycosaminoglycan patterns as the original cells, whereas in the solid tumour culture only chondroitin sulfate AC has been detected.     

The effect of tumour geometry on the quantification accuracy of planar 123I phantom images

IntroductionAccurate activity quantification is applied in radiation dosimetry. Planar images are important for quantification of whole-body images, enabling assessment of biodistribution from radionuclide administrations. We evaluated the effect of tumour geometry on quantification accuracy of ¹²³I planar phantom studies, including various tumour sizes, tumour-liver distances and two tumour-background ratios.Methods and materialsAn in-house manufactured abdominal phantom was equipped with a liver, different size cylindrical tumours, and a rod for tumour-liver distance variation. The geometric mean method with scatter and attenuation corrections was used for image processing. Scatter and attenuation corrections were made using the triple energy window scatter correction technique and a printed transmission sheet source, respectively. Region definitions for tumour activity distribution compensated for the partial volume effect (PVE). Activity measured in the dose calibrator served as reference for determining quantification accuracy.ResultsThe smallest tumour had the largest percentage deviation with an average activity underestimation of 34.6 ± 1.2%. Activity values for the largest tumour were overestimated by 3.1 ± 3.0%. PVE compensation improved quantification accuracy for all tumour sizes yielding accuracies of <12.4%. Scatter contribution to the tumours from the liver had minimal effect on quantification accuracy at tumour-liver distances >3 cm. With PVE compensation, increased tumour-background ratio resulted in a percentage increase of up to 26.3%.ConclusionWhen applying relevant corrections for scatter, attenuation and PVE without background activity, quantification accuracy of <13% was obtained. We demonstrated the successful implementation of a practical technique to obtain quantitative information from ¹²³I planar images.     

Inhibition of suicidal erythrocyte death by blebbistatin

Blebbistatin, a myosin II inhibitor, interferes with myosin-actin interaction and microtubule assembly. By influencing cytoskeletal dynamics blebbistatin counteracts apoptosis of several types of nucleated cells. Even though lacking nuclei and mitochondria, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include energy depletion and osmotic shock, which enhance cytosolic Ca(2+) activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. The present study explored the effect of blebbistatin on eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in fluorescence-activated cell sorting analysis and cytosolic Ca(2+) concentration from Fluo3 fluorescence. Exposure to blebbistatin on its own (1-50 μM) did not significantly modify cytosolic Ca(2+) concentration, forward scatter, or annexin V binding. Glucose depletion (48 h) was followed by a significant increase of Fluo3 fluorescence and annexin V binding, effects significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 10 μM). Osmotic shock (addition of 550 mM sucrose) again significantly increased Fluo3 fluorescence and annexin binding, effects again significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 25 μM). The present observations disclose a novel effect of blebbistatin, i.e., an influence on Ca(2+) entry and suicidal erythrocyte death following energy depletion and osmotic shock.     

Vascular endothelial growth factor (VEGF), a survival factor for tumour cells: implications for anti-angiogenic therapy

Angiogenesis is central to both the growth and metastasis of solid tumours. Anti-angiogenic strategies result in blood vessel regression accompanied by tumour cell apoptosis. Radiotherapy and many chemotherapeutic agents kill tumours by inducing apoptotic cell death. We propose that, in addition to its role as an angiogenic factor, vascular endothelial growth factor (VEGF) can act as a survival factor for tumour cells protecting them from apoptosis. Thus anti-angiogenics, in particular those directed against VEGF, have multiple anti-tumour effects. We suggest that anti-VEGF strategies prevent vessel growth and block a tumour cell survival factor, VEGF, rendering tumour cells more sensitive to chemotherapy and radiotherapy. In addition, as chemotherapy and radiotherapy have been shown to increase VEGF expression, anti-VEGF strategies may overcome therapy- induced tumour cell resistance.