The perfusion characteristics of the P22 carcinosarcoma were investigated in tissue-isolated tumour preparations in the ovarian and inguinal fat pads of BD9 rats. Tumours were perfused with a physiological buffer of known viscosity and changes in perfusion pressure were recorded at different perfusion rates in an ex vivo system. At perfusion pressures exceeding 30-40 mmHg tumour flow rate was directly proportional to the perfusion pressure in all tumours, indicating a constant resistance to flow. An apparent positive pressure difference across the tumour vasculature of 20-30 mmHg occurred under conditions of zero flow in either site. At low perfusion pressures, the flow resistance increased sharply due to increases in the geometric resistance of the tumours. These findings are in accord with previously published data. Geometric resistance increased with tumour volume in both sites and was approximately five times greater in the inguinal tumours than it was in the ovarian tumours, on a weight to weight basis. The dependence of tumour geometric resistance on perfusion pressure differs from the situation in normal tissues and may provide a means of manipulating the tumour microcirculation to the exclusion of the systemic blood supply. The dependence of geometric resistance on tumour site may partly explain why tumours located in different sites respond differently to various forms of therapy.


We have recently succeeded in producing transgenic mice carrying a hybrid gene consisting of mouse metallothionein promoter-enhancer and the ret oncogene (MT/ret). . A retroperitoneal tumour developed in one of 17 MT/ret transgenic founder mice. Histological analysis revealed that the tumour consisted of undifferentiated neuroblasts and differentiated ganglion cells, the latter of which were strongly positive for neuron specific enolase. Expression of the ret transgene was observed at high levels in RNA from the tumour, but not in those of other normal tissues. In addition, a 100kDa ret protein was detected in the cell lysate of the tumour. Taken together with our previous data, these results suggest a possible role for the ret oncogene in the proliferation of neural crest cells.


The fluorescent stain Hoechst 33342 (H33342) has been employed extensively as an in vivo marker of functional tumour vasculature. We have found that H33342 causes a transient, dose-dependent decrease in tumour red blood cell (RBC) flow in SCCVII tumours as measured using laser Doppler flowmetry. After intravenous bolus injection of 15 mg kg-1 to anaesthetised mice, blood flow in subcutaneous back tumours declined to 19 +/- 11% of pretreatment values, returning to normal in less than 7 min. The effect was less pronounced in mice bearing foot tumours in which flow decreased to 52 +/- 14% of pretreatment values in unanaesthetised mice and to 50 +/- 15% in anaesthetised animals. RBC flow in foot tumours remained significantly depressed for only 2-3 min. A dose of 5 mg kg-1 was not significantly vasoactive in back tumours. H33342 also caused a transient 20 +/- 6 mmHg decline in mouse arterial blood pressure. Blood pH and haematocrit, and tumour cell oxygen consumption were unchanged by H33342. H33342-induced flow changes did not affect results obtained using an in vivo double staining protocol provided that the interval between stain injections was greater than 5 min. Due to its transient effects on tumour perfusion, the stain caused radiobiological tumour hypoxia if injected immediately prior to X-irradiation. Injection 20 min before irradiation had no influence on tumour radiation response. We conclude that the transient nature of H33342-induced perturbations in mouse cardiovascular physiology and tumour blood flow must always be considered but do not preclude the use of the stain as a vascular marker to detect spontaneous tumour blood flow fluctuations or acute hypoxia.


A lipopolysaccharide (BP-LPS) isolated from killed Bordetella pertussis (Tohama strain) was determined to have low toxicity based on the mortality and decrease in body weight of BP-LPS-injected mice. BP-LPS, administered intradermally or intraperitoneally, clearly inhibited the growth of an MM46 murine mammary carcinoma. When compared with a toxic Escherichia coli-derived LPS, BP-LPS displayed excellent anti-tumour activity against MH134 hepatoma and Meth A fibrosarcoma. As part of a combined chemotherapy/immunotherapy regimen, BP-LPS also seemed to prolong the lifespan of mice inoculated with Lewis lung carcinoma. BP-LPS thus appears to have valuable characteristics as an anti-tumour agent.


Ifosfamide is clinically used in combination chemotherapy regimens for the treatment of patients with high-grade lymphomas, sarcomas and metastatic germ cell tumours. In order to reduce the oxazophosphorine-related urothelial toxicity, sodium mercaptoethane sulphonate (mesna) is used in different schedules following the administration of ifosfamide. The proposed mechanism of mesna activity is the binding of toxic oxazaphosphorine metabolites such as acrolein in the urine of the patients. Since an influence of mesna on ifosfamide anti-tumour activity is controversial, the current study has used xenografts from two human testicular cancer cell lines heterotransplanted into nude mice to study the anti-tumour activity of ifosfamide in combination with different dosages and schedules of mesna. In both human testicular cancer cell lines, H 12.1 and 2102 EP, ifosfamide demonstrated anti-tumour activity as a single agent. No reduction in ifosfamide activity was observed with the application of mesna at a dose range from 50% to 200% of the ifosfamide dose. Furthermore, the application of mesna before and 3 h after ifosfamide, a schedule used in many clinical protocols because of the short half life of mesna, not only maintained high ifosfamide anti-tumour activity but also seemed to be associated with the lower systemic and urothelial toxicity of ifosfamide therapy compared with ifosfamide given alone. In conclusion, the experimental in vivo system using human heterotransplanted testicular cancer cell lines confirms the significant anti-tumour activity of ifosfamide in malignant germ cell tumours and demonstrates that mesna does not impair ifosfamide anti-tumour activity in this model. These results are most likely transferable to the use of mesna in patients with metastatic testicular cancer.


The changes in the phosphorus metabolism of a xenografted hypopharynx carcinoma (Hyp 1), sensitive to cisplatin (CDDP), were compared to those occurring in two sublines of the tumour, characterised by moderate or high resistance to CDDP (Hyp 1/H and Hyp 1/R) following, i.p. administration of 4, 8 or 12 mg CDDP/kg-1. The investigations were performed by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy. Parallel to the NMR experiments, the cytokinetic and histological alterations in the tumours were studied under the same experimental conditions. No mentionable differences in the levels of the main phosphorus-containing metabolites could be detected between the three tumour lines before treatment. Following application of CDDP, the alterations in the NMR spectra were clearly related to the degree of tumour response. The most sensitive and earliest marker of tumour regression was a decrease in the phosphomonoester/phosphodiester ratio, parallelled by a gradual increase in the phosphocreatine/inorganic phosphorus quotient. In the resistant tumour lines Hyp 1/H and Hyp 1/R non-responding tumours showed alterations in the 31P NMR spectrum which were similar to those observed during uninfluenced tumour growth. Marked changes in the 31P NMR spectrum were always associated with severe cytotoxic lesions following therapy. The results suggest that the changes detected by 31P NMR spectroscopy following chemotherapy with CDDP are response-specific.


Twenty-one identical tumour specimens were cultured both in the Plasma-Clot Diffusion Chamber (PCDC) Technique and the Human Tumour Colony-forming Assay (HTCA). The culture results achieved in the PCDC-technique were clearly superior to the HTCA: in the PCDC the mean and median plating efficiency (PE) was 0.156 and 0.147, in the HTCA 0.103 and 0.028%; adequate growth rate in the PCDC-technique was 67% and in the HTCA 38%. Fewer cells were required for plating in the PCDC-technique: 6.4 X 10(4) vs. 2.6 X 10(5) in the HTCA. The mean and median coefficient of variation of the colony numbers in the PCDC-technique appeared much higher: 27.3 and 37.3 vs. 11.2 and 11.1% in the HTCA. The relation between the PEs obtained for the same specimen in the two techniques was compared. No positive correlation was found, which can possibly be ascribed to technical shortcomings in both techniques.


The effects of 131-labelled antiferritin polyclonal antibody for the treatment of established hepatocellular carcinoma (HC-04) in athymic nude mice were evaluated. 131I-labelled antiferritin antibody localised specifically to a subcutaneous tumour with a mean of 8.1% of the infused dose per gram of tumour at 24 h after infusion when the experiment was started 15 days after inoculation and with a mean of about 6.5% of the infused dose per gram of tumour when the experiment was started 30 days after tumour transplantation. The concentrations of 131I-antiferritin antibody in tumour delivered a mean of 1994 cGy to tumour following infusion of 500 microCi of radiolabelled antiferritin antibody in the early group and a mean of 1600 cGy in the late group. Treatment with 500 microCi led to regression of the tumour in 55% of animals in the early group and 44% in the late group. In contrast, unlabelled antiferritin and 131I-labelled IgG failed to exert any significant effect on tumour growth. The transplanted tumours in the early groups of animals had relatively higher concentration of ferritin than those in the late group. There was accelerated inhibition of tumour growth and prolonged survival in animals in the early group compared with those in the late group.


Age-related trends of gastritis and intestinal metaplasia (IM) were studied in 476 endoscopically examined and bioptically proved cases of gastric carcinoma (GC), 263 of which were of intestinal (IGC) and 213 of diffuse (DGC) types. Endoscopic biopsy specimens from the area around the tumour were available in all cases, and from the antrum and/or body distant from the tumour area in 238 cases. A representative sample of an endoscopically and bioptically examined Finnish population consisting of 431 subjects was used as control material. In patients with IGC the prevalence of atrophic gastritis in the gastric area affected by the tumour was higher and that of superficial gastritis lower than expected, and the age-group scores of gastritis and IM were situated above the age-dependent line of gastritis scores of controls in all age groups studied. This was seen to indicate a more rapid progression of gastritis in IGC patients than in the population at large. In the opposite area of the stomach, i.e. in the tumour-free area, the progression of gastritis and IM was virtually similar to that in controls. No such differences were seen with regard to DGC. It is concluded that IGC is dynamically closely linked to gastritis and IM, while in DGC no such relationship is demonstrable.


The previously reported concept of using mixtures of monodentate ligands in a combinatorial manner in order to influence enantio- or regioselectivity of transition metal catalyzed processes has been extended to include diastereoselectivity. Accordingly, 1,2- and 1,3-asymmetric induction in the Rh-catalyzed hydrogenation of a chiral allylic alcohol and a chiral homo-allylic alcohol has been studied by using mixtures of monodentate P-ligands. It was found that appropriate 1:1 mixtures of two different P-ligands enhance the degree of diastereoselectivity relative to the use of the respective pure ligands themselves. Here, as in the previous cases regarding enantio- or regioselectivity, this type of combinatorial catalysis leads to improved catalytic profiles without the need to prepare new ligands. The application of combinatorial chemical methods in asymmetric catalysis has emerged as a promising area of research, and indeed several reviews covering the subject have appeared.–8 It is a and Lb in metal catalysts of the type ML2, leading to two so-called homo-combinations and one hetero-combination which are in equilibrium with one another , 95% regioselectivity in favor of C-C bond formation at the higher substituted C-atom was achieved, whereas the use of the pure ligands as traditional homo-combinations led to low degrees of regioselectivity, but in the opposite sense.[We have recently extended this combinatorial principle beyond lectivity–26 to inectivity, specificte sense.diastereoselectivity, specifically in the Rh-catalyzed hydrogenation of chiral olefins. The literature contains several reports of 1,2- and 1,3-asymmetric induction in the hydrogenation of chiral olefins,[1 with formation of diastereomers 2 and 3, ketone 4 forming as a side product due to undesired isomerization.[anti-product 2 (32:1). In our study we also chose this transformation as the model reaction. All reactions were performed with the racemate of 1, although only one enantiomeric form is shown here for simplicity.We now show for the first time that mixtures of monodentate P-ligands can also be used to influence  olefins,–30 the u olefins, within trization. From a s5 with formation of diastereomers 6 and 7 was considered. Here again one enantiomeric form is shown arbitrarily, although a racemate was used.As a second model reaction the analogous Rh-catalyzed hydrogenation of the homo-allylic alcohol S)-form. In the case of the menthyl-derivative P15, a single stereoisomer was employed, although the configuration at phosphorus is unknown.[S)-configuration at phosphorus was described earlier.[1 and 5 were used as racemates, kinetic resolution may be involved when the chiral ligands are employed, i.e., diastereoselectivity may change with conversion. This aspect was not a subject of the present study. In all hydrogenation reactions the standard precursor Rh(nbd)2BF4 (nbd = norbornadiene) was treated with the ligands to form pre-catalysts of the type RhL2(nbd)BF4 and free nbd. Dichloromethane served as the solvent.Monodentate P-ligands P1 – P23 were employed in the hydrogenation reactions, the Rh:ligand ratio being 1:2 in all cases. These ligands were not chosen on the basis of structural or mechanistic considerations; rather, they happened to be available in our laboratory, and indeed many of them are commercially available . Most of the ligands are achiral, but some contain one or even two stereogenic centers. Ligands P18, P20 and P21 were use unknown. The ment earlier. Since th1, 21 of the 23 ligands were employed, which means a total of 210 different hetero-combinations in addition to the 21 conventional reactions (homo-combinations). Not all of them were actually tested. Rather, the combinatorial search was terminated after approximately 150 reactions had been performed in an arbitrary order. This random walk through a two-fold field of combinations led to the identification of 15–20 hits, i.e., those in which the hetero-combination results in a higher diastereoselectivity than either of the two homo-combinations themselves alone. anti-product 2 (entry 33). This contrasts with the results obtained using the homo-combination P11/P11 which shows totally sterorandom behavior (entry 11), and the other homo-combination comprising the phosphinine P17/P17[2:3 ratio of only 2:1, while P18/P18 and P22/P22 are both more selective .In the case of the hydrogenation of the allylic alcohol e P17/P17 which re5 involves 1,3-asymmetric induction, which is usually considered to be more difficult.[anti-product 6 is moderate . Since in the present study the racemic form of 5 was employed, the degree of "reagent control" in the case of the chiral ligands P16, P18 and P20 was not ascertained. The highest syn-selectivity favoring 7 amounts to only 1:1.8 when using ligand P4 (entry 4). From the list of hetero-combinations showing a stereochemical influence relative to the use of the respective pure ligands, three lead to substantial syn-selectivity (6:7 = 1:5), namely P4/P23, P9/P23 and P10/P23 . In all three cases the chiral phosphinite 23 is involved, which as a homo-combination actually favors the opposite diastereomer . With regard to maximizing anti-selectivity, the hetero-combination P7/P21 results in a diastereoselectivity of 18:1 in favor of 6 (entry 26) which is clearly better than P21 alone or any other homo-combination listed in Diastereoselectivity in the hydrogenation of the homo-allylic alcohol ifficult.–31 In th1 and the homo-allylic alcohol 5. Since the goal was proof-of-principle, only 23 randomly chosen monodentate P-ligands that happened to be available in our laboratory were employed, and not even all combinations were actually tested. We do not claim that the hits reported here constitute the optimal catalyst systems. If in the present reactions (or in other processes) higher diastereoselectivities are strived for, the combinatorial search needs to be systematized and the library of ligands extended. Indeed, numerous analogs of the present ligands are known, many of which are commercially available. Moreover, important classes of P-ligands not considered here can also be included in future studies of this kind.The purpose of the present study was to demonstrate that mixtures of two monodentate P-ligands can influence the diastereoselectivity of transition metal catalyzed reactions of chiral substrates. This was accomplished by studying the diastereoselective Rh-catalyzed hydrogenation of the chiral allylic alcohol a : Lb ratio may affect diastereoselectivity and can thus be used as a tool in future studies. It also remains to be seen if mixtures of chiral ligands affect diastereoselectivity in reactions of chiral substrates when they are used in enantiomerically pure form.At this time we refrain from proposing any hypotheses regarding the structural and mechanistic reasons for enhanced diastereoselectivity when using certain combinations of monodentate P-ligands. The same applies to the observed differences in rate. The combinatorial search is purely empirical. However, in addition to the inherent different degrees of diastereoselectivity arising from the homo- and hetero-combinations, their relative amounts present in catalytically active form and the relative reaction rates dictate the stereochemical outcome. Since the relative amount of catalytically active forms is thermodynamically controlled, changing the LIn conclusion, the previous concept of using mixtures of monodentate ligands in order to influence enantio-–26 and r


The expression of the EGF receptor, c-erbB-2 and PDGF receptor proteins has been studied in a series of human brain tumour biopsies and cell lines. Western blotting was used to determine the amount of protein present and their intrinsic and ligand promoted enzyme activities were studied by immunoprecipitation followed by autophosphorylation. EGF receptors were found to be expressed at very high levels in 40% of primary tumour biopsies, but at uniformly low levels in tumour derived cell lines. The c-erbB-2 protein was not detected in tumour biopsies, but was present at variable, but low levels in extracts of tumour cell lines. PDGF receptors were also found at moderate to low levels in both primary tumours and cell lines. The EGF receptor gene was amplified in four out of 14 primary tumours and this generally correlated with high levels of protein expression. The c-erbB-2 gene was not amplified. Employing the polymerase chain reaction and sequence specific oligonucleotides as probes there was no evidence of mutations in the c-erbB-2 gene transmembrane region. These results suggest that alterations of expression of the EGF receptor may play a role in human brain tumours. There was however no evidence for aberrant expression of the c-erbB-2 protein. Additional experiments are required to assess the influence of PDGF receptor expression in brain tumour cells.


Pyrazinamide, the pyrazine analogue of nicotinamide, has been evaluated for its ability to modify the radiation response of hypoxic cells both in vivo and in vitro. Results obtained with three different murine tumour systems EMT6, LLC and SCCVII showed that pyrazinamide at a dose of 0.5 mg g-1 i.p. resulted in enhanced radiation response. Dose modification factors of between 1.3 and 1.6 were observed using in vivo/in vitro clonogenic assays. This enhancement was greater than that obtained in mouse intestine using crypt cell survival as an endpoint (DMF 1.1). In contrast to the tumour data in vivo, the in vitro results indicate that pyrazinamide displays little radiosensitising or toxic properties towards hypoxic CHO cells in culture. These results suggest that pyrazinamide exerts its effects in vivo either by directly perturbing tumour physiology or by being converted to an active metabolite. Blood flow studies performed using laser Doppler flowmetry indicate that pyrazinamide produces a small (32%) increase in overall tumour blood flow in the SCCVII tumour. Based on this finding, additional studies on tumour perfusion at the microregional level were performed in the SCCVII tumour using a histological technique involving injection of fluorescent stains which demarcate functional vasculature. The data show that when compared to saline injected controls, pyrazinamide reduced the number of vessels opening and closing over a 20 min period from 10.2% to 3.8%. This finding suggests that pyrazinamide may exert its effects at least in part by reducing the occurrence of acute hypoxia resulting from dynamic changes in microregional perfusion.


The 11q13 chromosomal region encodes oncogenes relevant to a variety of human cancers as well as a tumour suppressor gene implicated in multiple endocrine neoplasia type 1. In addition, high affinity folate receptor (FOLR1), which maps to 11q13.3-13.5, is expressed at an elevated level on the surface of over 80% of nonmucinous epithelial ovarian cancers. Further telomeric, 11q breakpoints are found in many cancers. We studied the involvement of 11q markers in ovarian cancer by looking for tumour-specific loss of heterozygosity (LOH), as well as amplification or rearrangements that might explain the overexpression of FOLR1. Twenty eight epithelial ovarian cancers, along with lymphocyte DNA from the same individual were used for Southern blotting with polymorphic probes from 11q. PCR primers from 11q23.3 were also used. The 11q13 band was amplified in four out of 28 cancers. The amplicon included the probe D11S146 as well as FGF3 (formerly INT2) and FOLR1 in one out of these four cases, thus crossing the bcl1 translocation breakpoint. LOH was seen in three out of 16 cases with FGF3 (11q13). A much higher frequency of LOH (8/12) was found at 11q23.3-qter, implying the presence of a tumour suppressor gene in this region.


Three types of natural immune responses against malignant cells were studied in vitro: Cytotoxicity mediated by splenic NK cells; cytostasis mediated by splenocytes and binding of naturally occurring antibodies to various tumour targets. These responses were studied in untreated 3 and 12 month old mice and in mice of both age groups inoculated with B16 melanoma cells. The results showed that in normal mice NK activity decreases with age, cytostatic activity remains unchanged and the titre of natural antibodies increases. Twelve-month old mice were shown to be appreciably more resistant than 3 month old mice to the development of tumours from subthreshold numbers of B16 tumour cells. In mice injected with threshold amounts of the B16 tumour, there was no change in any of the responses in the tumour-free period, but there was a decrease in NK activity and an increase in cytostatic activity when a large tumour mass developed. An increase in the titre of natural antibodies in young mice injected with the tumour was also seen. The correlation between these changes and tumour appearance and development is discussed.


Tumour tissue oxygenation has been measured in man during carbogen breathing  using a commercially available polarographic electrode system (Eppendorf p02 histograph). At least 200 tumour measurements in each of 17 patients with accessible tumours were taken before, and subsequently continuously after the commencement of carbogen breathing for periods of 10 to 30 min. In 12 out of 17 patients studied there was a significant increase in median tumour p02 during the first 10 min of carbogen breathing (range 9 to 1800%). There was an initial rapid increase in tumour p02 which was maintained until 8 to 12 min, but then decreased throughout the subsequent treatment period. Although there was a reduction in the proportion of point measurements < or = 10 mmHg in 11 out of 13 patients, during carbogen breathing, measured points of < or = 2.5 mmHg were only eliminated in three out of 11 tumours. The time course has implications for the planning of clinical trials utilising radiotherapy with carbogen breathing.


Systemically administered radiolabelled anti-tumour antibody is ineffective in treating the majority of patients with liver metastasis from colorectal carcinoma. We have assessed whether agents which increase capillary permeability can increase tumour uptake of antibody isotope conjugate. We developed a xenograft model of colorectal carcinoma using an antibody directed against carcinoembryonic antigen (CEA). Tumours were grown subcutaneously in the hind limbs of athymic rats to derive their circulation from the femoral artery. Cannulae were placed in the common iliac artery and iliolumbar vein. Antibody was delivered systemically, regionally and regionally with histamine, leukotriene C4 and interleukin-2. Regionally administered anti-CEA antibody resulted in a significantly greater (P = 0.004) tumour to normal tissue ratio  compared to systematically administered antibody . The addition of vasoactive drugs produced an approximately 3-fold increase with an increase to a mean tumour:liver ratio of 3.24 (s.d. = 1.39) for histamine (P less than 0.001 compared to systemic delivery), 3.21  for leukotriene C4 and 3.80  for interleukin-2. The addition of histamine significantly (P = 0.004) increased the mean tumour to liver ratio  of non-specific antibody uptake compared with either systemic  or regional delivery  of non-specific antibody alone. Increasing tumour capillary permeability can produce a significant clinically useful increase in tumour uptake of antibody-isotope conjugate.


Intrahepatic tumour is associated with alterations in splanchnic haemodynamics. To investigate the hypothesis that these are the result of a circulating vasoactive agent, rat small bowel segments were cross-perfused with arterial blood from groups (n = 12) of paired tumour-bearing (intrahepatic HSN sarcoma) and control rats. The vascular resistance of the segment was significantly greater during perfusion by tumour-bearing animals , suggesting that intrahepatic tumour may be associated with a circulating vasoactive agent. A similar mechanism may underlie changes in the hepatic perfusion index in patients with liver metastases.


In vitro drug sensitivity testing, both by optical colony counting and by a [3H]-TdR incorporation assay, was performed on human tumour cells proliferating in soft agar cultures. Cells from two different human tumour cell lines, 5 different human tumour xenografts, and 94 different primary human tumour specimens of various histologic types were studied. Regression analysis comparing the results of the colony counting assay and the [3H]-TdR assay revealed good to excellent correlations between the two assay endpoints for quantitating the effect of in vitro anticancer drug exposure for a large number of different agents. The presence of pre-existing tumour cell aggregates complicates the performance of the optical colony counting assay. The [3H]-TdR incorporation assay is more sensitive and reproducible than the colony counting assay when performed on samples containing a large number of initially seeded tumour cell aggregates.


The response of 14 mouse tumour sub-lines to the radiosensitizing action of a large single dose of misonidazole (MISO) has been assessed by regrowth delay. In 13 of these, significant enhancement of radiation effect occurred under ambient conditions, indicating sensitization of naturally hypoxic cells. The enhancement observed (SER') varied with the radiation dose, as would be predicted for a mixed oxic/hypoxic cell population. The maximum SER' in these 13 tumours did not depend on histology or regrowth rate. The 14th tumour, a slow-growing sarcoma, was not sensitized under ambient conditions, but showed marked sensitization when clamped to produce acutely hypoxic cells. This is consistent with no hypoxic cells occurring naturally in a sarcoma with a slow rate of growth. Faster-growing variants of this tumour showed radiosensitization under ambient conditions. The slow-growing carcinoma, RH, however, appears to contain hypoxic cells and did show sensitization. The cytotoxic action of MISO was compared with the radiosensitization by administering it after irradiation in 8 of the tumour lines. In 2 tumours no cytotoxicity was observed. In the rest cytotoxicity was significant, but much smaller than the sensitization observed when MISO was administered before irradiation. These regrowth-delay data have been used to calculate hypoxic fractions in 3 ways. Estimates of hypoxic fraction ranged from less than 0.1% in the slow sarcoma to greater than or equal to 30% in several tumours. There is considerable variation in the estimate, according to the technique used.


The influence of photodynamic therapy (PDT) on vascular perfusion and the development of hypoxia was investigated in the murine RIF-1 tumour. Image analysis was used to quantify changes in perfusion and hypoxia at 5 min after interstitial Photofrin-mediated PDT. The fluorescent stain Hoechst 33342 was used as an in vivo marker of functional vascular perfusion and the antibody anti-collagen type IV as a marker of the tumour vasculature. The percentage of total tumour vasculature that was perfused decreased to less than 30% of control values after PDT. For the lower light doses this decrease was more pronounced in the centre of the tumour. The observed reduction in vascular perfusion showed a good linear correlation (r = 0.98) with previously published tumour perfusion data obtained with the 86Rb extraction technique. The image analysis technique provides extra information concerning the localisation of (non)-perfused vessels. To detect hypoxic tumour areas in vivo, an immunohistochemical method was used employing NITP [7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline]. A large increase in hypoxic areas was found for PDT-treated tumours. More than half the total tumour area was hypoxic after PDT, compared with < 4% for control tumours. Our studies illustrate the potential of image analysis systems for monitoring the functional consequences of PDT-mediated vascular damage early after treatment. This provides direct confirmation that the perfusion changes lead to tissue hypoxia, which has implications for the combined treatment of PDT with bioreductive drugs.


Similar protocols evaluated by visual scoring of tumour load at 40 days after inoculation supported these findings, although no quantitative assessment of treatment-induced growth delay could be made by this method. This study shows that in vivo imaging of luciferase-transfected tumour cells is a useful tool to investigate the dynamics of disseminated tumour growth and efficacy of anticancer treatment in orthotopic models of peritoneal cancer in rats. It offers an attractive alternative to invasive methods, and requires fewer animals for measuring tumour response to therapy.Animal tumour models using orthotopic tumours for the evaluation of cancer therapies are of greater clinical relevance than subcutaneous models, but they also pose greater difficulties for measuring tumour size and quantifying response to treatment. In this study, we used noninvasive bioluminescence imaging to monitor the intraperitoneal growth of luciferase-transfected CC531 colorectal cells in adult WAG/RIJ rats. The bioluminescence signal correlated well with post-mortem assessment of tumour load by visual inspection of the peritoneal cavity at specific follow-up times. Using bioluminescence imaging, we were able to monitor peritoneal tumour growth sequentially in time and to calculate a tumour growth rate for each animal; this is not possible with invasive methods of evaluating tumour load. Bioluminescence imaging of rats treated with a single dose of cisplatin (4 mg kg Hyperthermic Intraoperative Peritoneal Chemotherapy (HIPEC), followed by systemic chemotherapy. The aim with HIPEC is to achieve high local drug concentrations in direct contact with small peritoneal tumour deposits, with mild hyperthermia used to increase drug penetration. This technique was pioneered by Locally disseminated peritoneal surface malignancy is a major problem in the management of abdominal cancers. In all, 10% of patients with primary colorectal cancer initially present with peritoneal seeding and 25–35% of patients who underwent seemingly curative surgery will later develop peritoneal metastases . Once peUntil now, the most commonly used method to determine tumour load in the abdominal cavity of animals is to kill the animals and score the size and number of tumours at various times after treatment  camera, minutes after the administration of the substrate. Bioluminescence imaging has been used successfully for monitoring tumour growth in mice  for quantitative assessment of anticancer therapies directed against disseminated peritoneal colorectal cancer. We report on the noninvasive, −1) and streptomycin (100 U ml−1). These cells give rise to disseminated peritoneal tumour nodules after i.p. injection in syngeneic WAG/RIJ rats , were used in this study. All protocols were approved by the local Animal Experimentation Committee and carried out in compliance with national guidelines for the care and use of research animals.6) and treated at 10 days after tumour cell inoculation, by which time small (1–2 mm) peritoneal tumours were established at 1 or more peritoneal sites. Treatment groups consisted of a single i.p. bolus injection of cisplatin  or a 90 min intraperitoneal perfusion at 40 or 37°C with or without cisplatin (200 ml at 8 μg ml−1). Previous studies in rats had demonstrated that approximately 30–35% of the cisplatin administered during such a 90 min peritoneal perfusion was absorbed systemically (−1), but higher perfusate concentrations were associated with renal toxicity and were poorly tolerated. Animals were killed and the intraperitoneal tumour load was determined at 40 days after inoculation. Tumour load was scored according to a point scale indicating large (>5 mm: 3 points), moderate (1–5 mm: 2 points), small (<1 mm: 1 point) or no tumour (0 points) in seven abdominal regions . These scores were added to give the total peritoneal tumour load (maximum score 21 arbitrary units).Rats were inoculated i.p. with CC531 cells  and Dormicum . Prior to perfusion, animals were injected i.v. with 42 mg kg−1 gelofusin  to increase the resistance of the veins to the pressure of the peritoneal perfusion fluid, as described previously (μg ml−1) at the start of the timed perfusion. At the end of the 90 min perfusion, the perfusate was aspirated and the wound was closed.Rats were anaesthetised by i.m. injection of a mixture of Hypnorm (fentanyl: 0.15 mg kgCC531 cells were cotransfected with the firefly luciferase gene (pGL3) in combination with a vector for puromycin resistance (pHA262pur) or for green fluorescent protein (GFP) and neomycin resistance, using lipofectin reagent  or electroporation, respectively. Molar ratios of 10 : 1 were used for luciferase : selection plasmids.5 per six-well plate. A total of 1 μg vector DNA was then added to 100 μl Optimem medium  and incubated for 30 min at room temperature before mixing with 100 μl lipofectin solution (2–20 μl in 100 ml Optimem) and incubating for an additional 15 min. Plated cells were washed and 0.2 ml of the DNA/lipofectin mixture and 1.8 ml serum-free DMEM were added prior to overnight incubation at 37°C. Transfection medium was then replaced by fresh DMEM, containing 10% FCS, antibiotics and puromycin (5 μg ml−1). The resulting colonies were harvested and cultured independently to select for the highest luciferase activity.At 24 h before transfection with lipofectin, cells were seeded at a density of 2 × 10μl of HEPES-buffered mannitol buffer. Luciferase and GFP plasmid (10 μg)  were added to the cell suspension and incubated for 5 min at room temperature prior to electroporation at 350 V. Cells were then incubated at 37°C for 48 h before selection by incubation with 800 μg ml−1 neomycin .Electroporation transfections were carried out using a Genepulser equipped with an RF module (Bio-rad). A total of 4 million cells were suspended in 950 5 luciferase-transfected cells in 100 μl lysis buffer. Protein concentration was measured using pyrogallol total protein reagent  according to the manufacturer's instructions. Luciferin substrate (50 μl) was added to 5 μl aliquots of cell lysates to determine the luciferase enzyme activity, using a Top Count Luminescence Counter . Each lysate was measured twice and luciferase activity, corrected for protein content, was expressed as relative light units (RLUs).Luciferase activity was measured using the Dual Luciferase Assay kit . Cell suspensions were made with approximately 106 CC531-luc cells to monitor the dynamics of peritoneal tumour growth.All animals were imaged using a cooled CCD camera , coupled to the LivingImage acquisition and analysis software (Xenogen Corp.). To determine the detection limits of CC531-luc cells in rats, an increasing number of tumour cells was initially inoculated within the abdominal wall, immediately followed by the injection of luciferin and imaging of photon emission after 10 min. On the basis of this result, images were subsequently made at various time points after i.p. inoculation of 2 × 10D-luciferin (Xenogen) was dissolved at 15 mg ml−1 in sterile PBS and stored at −20°C. Animals were anaesthetised with hypnorm  and Dormicum (midazolam: 2.5 mg kg−1) prior to injection with luciferin . Peritoneal light emission was measured over an integration time of 1 min, and images were acquired using Ivis and LivingImage software. Signal intensity was quantified as the total counts measured over the region of interest.Before imaging, the abdomen of the rats was shaved to minimise photon scattering and signal quenching. n=5) was followed sequentially at weekly intervals from 1 to 5 weeks after inoculation and then killed for visual inspection of peritoneal tumour load. Separate groups of rats (n=5 or 6 per time point) were imaged at 1, 2, 3 or 4 weeks immediately prior to post-mortem evaluation by visual inspection. Peritoneal luciferase activity was correlated with the distribution and size of tumours determined using the scoring system described.One group of rats , saline only  or no treatment (n=5–7 per group). Luciferase activity was imaged weekly (for a maximum of 11 weeks) to determine the effect of the treatment on tumour growth. Animals were killed when the peritoneal RLU count was >2000 × 103.The time taken for peritoneal RLU signal to increase by 1000 counts above the starting value (on days 8–10 after inoculation) was calculated for each rat; this was termed ‘tumour regrowth time’.Rats were inoculated with 2 × 10CC531 cells grew intraperitoneally in WAG/RIJ rats and induced superficial tumours at several sites in the abdominal cavity, including omentum, mesentery, pelvis and subhepatic and subdiaphragmatic area. However, there was a large variation in tumour load within each group of animals killed at different times after inoculation  or intra-abdominal perfusion at 37 or 40°C , 10 days after inoculation with CC531 cells. The tumour load was assessed post-mortem at 40 days after inoculation . There was no reduction in tumour load after normothermic or hyperthermic perfusion with cisplatin, relative to perfuate-only controls (P>0.5). The i.p. bolus cisplatin treatment protocol was subsequently selected for evaluating the usefulness of bioluminescence imaging to detect tumour response to therapy.Separate groups of rats were treated with cisplatin given either as an i.p. bolus injection (4 mg kgin vitro luciferase activity detected in CC351 transfected cells was 4.2 × 105 counts mg−1 protein after lipofectin transfection (CC531-luc1) and 2.9 × 103 counts mg−1 after electoporation (CC531-luc2). There was no difference in the in vitro growth of CC531 parental cells and CC531-luc1 or -luc2 cells, indicating that the transfection procedure had no effect on the in vitro growth ability (data not shown). These two cell lines were used for further in vivo studies.The highest 1 and CC531-luc2 produced tumours in immune suppressed nude mice when injected subcutaneously or i.p. However, only the electroporation- transfected CC531-luc2 cells grew in immune competent WAG/RIJ rats. The detection limit for CC531-luc2 cells, imaged immediately after injection in the rat abdominal wall, was 105 cells.Both the transfected cell lines CC531-luc2 cells; that is, earlier than after i.p. inoculation of rats with the parental CC531 cells. This reflects the change in protocol to inject 2 × 106 transfected cells compared with 1 × 106 parental cells. However, there was no significant difference in the in vivo growth rates of CC531-luc2 cells compared with the parental cells; the mean tumour loads increased from about 5 to 12 arbitrary units over a 4-week period  between bioluminescence imaging and tumour load for individual rats , or for untreated rats . There was no significant difference between the untreated control group and the group given i.p. saline . Previous studies demonstrated that peritoneal perfusion with 15 μg ml−1 cisplatin (at 37 or 40°C) was required to give the same tumour concentration as an i.p. bolus injection of 4 mg kg−1  compared to lipofectin transfection. These CC531-luc2 cells did grow in the peritoneal cavity of the WAG/RIJ rats and no significant differences between growth rates of parental and transfected cells were seen. The high level of transfected gene expression in CC531-luc1 cells could have been responsible for their inability to form tumours in immunocompetent rats. It has been previously reported that expression of high levels of a transfected gene, for example, GFP, in mouse BM185 leukaemia cells resulted in a drastic reduction in disease development in immunocompetent mice, whereas in immunodeficient Nu/Nu mice the disease developed rapidly  with regard to both the extent and distribution of tumour, despite the multiple tumour foci at variable depth in the peritoneal cavity. Small tumours within the liver mass could not be easily detected by bioluminescence.Using in vivo bioluminescence, has already been reported in mice  was followed by subsequent regrowth of tumours. This could be detected well before the appearance of the common end points of weight loss, palpable tumours or death. A significant effect of cisplatin treatment on tumour growth, determined by Despite the advantages and convenience of bioluminescence imaging for monitoring tumour growth, there are some disadvantages. Firstly, light transmission is attenuated by tissue; therefore the deeper the tumours lie within the body, the greater the signal attenuation. This means that tumour load from deeper parts of the body will be relatively under-represented in the integrated images. It also means that small tumours that are shielded by bulky, pigmented organs may well be missed. Light–coloured organs, like prostate and mammary tissue or lungs, will tend to scatter rather than absorb the light, so the bioluminescence signal from tumour cells within such organs should be much less attenuated.1-transformed cells to grow in immunocompetent rats. Ideally, one would like to use clones expressing very high levels of the reporter gene, since this will enhance detection sensitivity. However, high expression levels of the transfected gene are also the most likely to introduce unwanted phenotypic changes, for example, immunogenicity and altered growth rates. In reality, a compromise between high luciferase expression and no, or minimal, changes in tumour cell behaviour will probably need to be sought. In any case, each transfected cell line should be checked for altered tumour growth before use in evaluating treatment responses.A second disadvantage of bioluminescence detection is that this procedure involves transfection of the parental cell line. The resulting clones do not always behave in exactly the same way as the parental cells, as was demonstrated by the inability of CC531-lucAlternative noninvasive techniques, such as PET, might overcome some of the limitations of bioluminescence detection of orthotropic tumour growth. However, this is a very expensive technique and it is not widely available for preclinical animal studies. PET may also be associated with its own problems, since the radioisotopes used tend to accumulate in excretion organs , which could interfere with the detection of small tumours in the peritoneal cavity. A direct comparison of these techniques would be interesting, but is outside the scope of the present study.in vivo bioluminescence imaging of tumour cells enables a rapid, noninvasive measurement of tumour load before, during and after treatment of rats with disseminated peritoneal disease. Using this technique, our understanding of in vivo tumour development and response to current and novel treatments can be increased.In summary, we showed that


Phagocytosis was estimated by the in vitro uptake of 14C-labelled Pseudomonas aeruginosa and glucose oxidation was evaluated by 14CO2 production from 1-14C-glucose. AM were harvested by lung lavage from rats prior to and at 7 and 21 days following i.v. tumour-cell challenge. Macroscopic lung tumour nodules were not observed by 7 days after tumour challenge. However, 3 weeks after tumour challenge, tumour nodules were clearly identifiable on the surfaces of the lungs. One week after the i.v. tumour challenge a marked increase in the number of AM was evident. The in vitro phagocytosis of 14C-labelled Pseudomonas aeruginosa was unaltered at that time, but became progressively depressed thereafter. Three weeks after tumour challenge, this decrease in phagocytic activity was evident when cells were incubated in normal serum, and was furtheri ntensified by serum obtained from tumour-bearing animals. Glucose oxidation by AM in either the resting condition or during bacterial phagocytosis was clearly decreased at both 1 and 3 weeks following i.v. tumour challenge. These findings indicate that the growth of pulmonary metastases is associated with a depression of alveolar macrophage bacterial phagocytic capacity, perturbations in serum opsonic activity and distinct alterations in macrophage energy metabolism. The metabolic dysfunction may impair pulmonary macrophage host defences against lung tumour growth.Alveolar macrophage (AM) phagocytic activity and glucose metabolism were evaluated during lung tumour growth in adult rats challenged i.v. with 10


The 62 kDa protein product of the c-myc oncogene (p62 c-myc) is thought to be involved in the control of normal cellular proliferation and differentiation. We have measured oncoprotein levels using a flow cytometric assay in 141 operable breast cancers and have correlated levels with prognostic variables, patient survival and disease free intervals. High levels of p62 c-myc were associated with well differentiated tumours. There was no correlation with tumour DNA index, lymph node or oestrogen receptor status. C-myc oncoprotein levels were not predictive of patient survival or disease free interval. This relationship of oncoprotein levels with tumour histological grade is in keeping with the suggestion that the c-myc oncogene is important in the control of cellular differentiation. The other findings imply that measurement of c-myc oncoprotein levels does not yield useful prognostic information.


Gradient recalled echo (GRE) MRI, sensitive to blood oxygenation changes, and spin echo (SE) MRI, sensitive to perfusion/flow, showed large signal intensity increases with carbogen breathing. Nicotinamide, thought to act by suppressing the transient closure of small blood vessels that cause intermittent tumour hypoxia, induced a small increase in blood oxygenation but no detectable change in perfusion/flow. Carbogen combined with nicotinamide was no more effective than carbogen alone. Both carbogen and nicotinamide caused significant increases in the nucleoside triphosphate/inorganic phosphate (βNTP/P i) ratio, implying that the tumour cells normally receive sub-optimal substrate supply, and is consistent with either increased glycolysis and/or a switch to more oxidative metabolism. The most striking observation was the marked increase in blood glucose (twofold) induced by both nicotinamide and carbogen. Whether this may play a role in tumour radiosensitivity has yet to be determined. Copyright 2000 Cancer Research Campaign© 2000 Cancer Research CampaignBoth host carbogen (95% oxygen/5% carbon dioxide) breathing and nicotinamide administration enhance tumour radiotherapeutic response and are being re-evaluated in the clinic. Non-invasive magnetic resonance imaging (MRI) and


Objective: To determine the prognostic value of patient and treatment parameters in osteosarcoma, and whether these areequally important across international boundaries.            Design: Retrospective, cross-sectional study of 428 patients diagnosed with around-knee osteosarcoma, between 1990 and1997 in Birmingham, UK, and Bologna, Italy. Disease-free survival (DFS) and overall survival (OS) assessed by Kaplan–Meier,Fisher's PLSD and Cox proportional hazard regression.            Results: Five-year DFS and OS were 56 and 73% at Centre 1, compared to 43 and 60% at Centre 2 . The most important bad prognostic factors for DFS and OS respectively were raised alkalinephosphatase at diagnosis (P=0.002 and P=0.003), tumour necrosis < 90% following chemotherapy (P=0.001 andP = 0.004) and volume > 150 cm3 at diagnosis (P=0.04 and P=0.006). The most significant combination of bad prognosticfactors was alkaline phosphatase and tumour necrosis. A total of 73% of patients at Centre 1 had greater than 90% necrosisof the tumour following neoadjuvant chemotherapy compared with 29% at Centre 2.            Conclusions: Tumour-based prognostic factors have similar significance across international boundaries. Chemotherapyeffectiveness appears to be a major factor in explaining the survival difference between the two centres.


For successful photodynamic diagnosis (PDD) and effective photodynamic therapy (PDT) with the clinically used 'photosensitiser' 5-aminolaevulinic acid (ALA), knowledge of the maximal fluorescence intensity and of the maximal tumour-host tissue fluorescence ratio following systemic or local application is required. Therefore, time course and type of porphyrin accumulation were investigated in neoplastic and surrounding host tissue by measuring the kinetics and spectra of ALA-induced fluorescence in vivo. Experiments were performed in the amelanotic melanoma A-Mel-3 grown in the dorsal skinfold chamber preparation of Syrian golden hamsters. The kinetics of fluorescent porphyrins was quantified up to 24 h after i.v. injection of 100 mg kg-1, 500 mg kg-1 or 1,000 mg kg-1 body weight ALA by intravital fluorescence microscopy and digital image analysis (n = 18). In separate experiments fluorescence spectra were obtained for each dose by a simultaneous optical multichannel analysing device (n = 3). A three-compartment model was developed to simulate fluorescence kinetics in tumours. Maximal fluorescence intensity  in the tumour arose 150 min post injection (p.i.)  and 120 min p.i. . The fluorescence in the surrounding host tissue was far less and reached its maximum at 240 min  and 360 min p.i.  and . Maximal tumour-host tissue ratio (90:1) was encountered at 90 min after injection of 500 mg kg-1. The spectra of tissue fluorescence showed maxima at 637 nm and 704 nm respectively. After 300 min (host tissue) and 360 min (tumour tissue) additional emission bands at 618 nm and 678 nm were detected. These bands indicate the presence of protoporphyrin IX (PPIX) and of another porphyrin species in the tumour not identified yet. Tumour selectivity of ALA-induced PPIX accumulation occurs only during a distinct interval depending on the administered dose. Based on the presented data the optimal time for PDD and PDT in this model following intravenous administration of 500 mg kg-1 ALA would be around 90 min and 150 min respectively. The transient selectivity is probably caused by an earlier and higher uptake of ALA in the neoplastic tissue most likely as a result of increased vascular permeability of tumours as supported by the mathematical model.


Congenital oral granular cell tumour of the newborn is an uncommon benign tumour of uncertain origin. The typical clinical appearance is of a single nodule occurring on the anterior maxillary ridge. In 10% of cases there are multiple lesions. The occurrence of congenital epulis in non-Caucasians is rare.Two firm pedunculated nodular lesions were noticed in the mouth of a 3-day-old black female: one on the anterior maxillary ridge and the other further posteriorly in the midline of the palate. Both lesions were excised when the baby was nine days old. Microscopic examination of the lesions showed densely packed round to oval cells with abundant granular eosinophilic cytoplasm and uniform nuclei. The diagnosis was congenital granular cell tumour.Congenital oral granular cell tumour occurs almost exclusively in Caucasian newborns but also rarely in black infants. The parents should be assured of the benign nature and the simple treatment of the condition. Congenital epulis is an uncommon benign gingival tumour of the newborn. Although the condition has been well reported in the literature, to our knowledge this is the first case to be reported in a black South African infant.Epulis is a non-specific term connoting a tumour-like mass of the gingiva and the term is used in association with several different gingival lesions, regardless of their aetiology or histogenesis . Since aThree days after the birth of a black female, two lumps were noticed in her mouth. No other oral abnormalities were noted. The lumps did not interfere with feeding or breathing. The mother was HIV-seropositive and had been receiving Neveripine since before the pregnancy. The mother would not allow the HIV-serostatus of the baby to be investigated.On examination there were two firm pedunculated nodular lesions Fig , one on The differential diagnosis included congenital granular cell tumour (CGCT), odontogenic tumour, teratoma, neuroectodermal tumour, haemangioma and fibroma with a provisional diagnosis of CGCT. Both lesions were excised under general anaesthesia when the baby was 9 days old. Post-operative healing was uneventful.Microscopic examination of the lesions showed round or ovoid cells with granular eosinophilic cytoplasm and centrally located nuclei Fig . The masCGCT is uncommon and occurs almost exclusively in Caucasian newborns . It is aCGCT occurs only in the newborn and is a distinct pathological entity . It may Local excision is curative. If left untreated malignant transformation never occurs. Spontaneous regression of untreated lesions has been reported ,3,5,7. CThe histogenesis of CGCT is obscure. Its possible origin may be odontogenic, fibroblastic, histiocytic, myogenic or neurogenic cells -5,7, or Microscopically, CGCT typically consists of large closely packed polygonal cells with clear granular eosinophilic cytoplasm and centrally positioned nuclei, within a vascular fibrous connective tissue stroma, covered with squamous cell epithelium -10. The CGCT is relatively common in Caucasia newborns but appears to be much less common in black newborns, and the occurrence of more than one lesion in a black infant is rare. The family of an infant with CGCT should be assured of the benign nature and the simple treatment of the condition.Written informed consent was obtained from the parent of the patient for the publication of this case report and for the use of images. A copy of the written consent is available for the Editor-in-Chief of this journal.The authors declare that they have no competing interests.LF, NHW, EJR, RM and JL provided the study concept, ASS and NHW performed the clinical work and case management, EJR performed histopathological studies, LF, NHW and ASS acquired data and performed the data analysis, LF, RM and EJR were responsible for manuscript editing, LF, NHW, ASS, RM and JL reviewed the manuscript. All authors read and approved the final manuscript.


Nitric oxide (NO), an endogenous free radical, has been implicated in a wide range of biological functions. NO is generated enzymatically from the terminal guanidinonitrogen of L-arginine by nitric oxide synthase (NOS). Despite intensive investigations, the role of NO--either as the primary product of the L-arginine/NOS pathway or provided from the NO donor sodium nitroprusside (SNP)--in carcinogenesis and tumour cell growth remains unclear and controversial. The objective of this study was to examine the growth effects of NO on a ductal pancreatic adenocarcinoma in the rat and on a human pancreatic tumour cell line (HA-hpc2). In vivo, both SNP and endogenous induction of NO by endotoxins [lipopolysaccharide (LPS)] plus L-arginine significantly reduced the tumour growth. To investigate the mechanisms of NO anti-tumour growth action, the effects of either the SNP or L-arginine/NOS pathway were analysed on the HA-hpc2 cell line. Nitrite/nitrate production, NOS activity and iNOS expression [assessed by reverse transcription-polymerase chain reaction (RT-PCR)] were tested and related to growth (assessed by [3H]thymidine incorporation assay) and apoptosis . SNP exerted a dual effect on tumour cells: stimulation of the proliferation up to 1 mM and inhibition at higher concentrations. These effects were related to NO production. Both proliferative and cytostatic responses were inhibited by NO scavenger 2-phenyl-4,4,5,5-tetramethyl-hemidazoline-1-oxyl3-oxide (carboxy-PTIO). The marked apoptotic DNA fragmentation induced by SNP was also abolished by PTIO association. Unlike macrophages, the human pancreatic tumour cells did not seem to express intrinsically the L-arginine/NOS pathway. Macrophages were activated by HA-hpc2 cells as well as by LPS plus cytokines [interleukin (IL)-1beta plus tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma]. In HA-hpc2/macrophage co-cultures, NOS activity and inducible NOS (iNOS) transcription were stimulated, whereas an antiproliferative response was observed. These effects were related to both macrophage amount and NO production. Addition of LPS plus cytokines to co-cultures doubled iNOS activity, nitrite/nitrate production and tumoricidal effect. These data suggest the involvement of NO in pancreatic tumour growth and support the fact that generation of high levels of NO with potential production of endogenous reactive nitrogen intermediates may contribute to induction of apoptosis and tumour growth inhibition.


Two mouse monoclonal antibodies against the human osteogenic sarcoma 791T were examined for their capacity to exert complement-dependent cytotoxicity against a panel of human tumour cell lines. Cytotoxicity was most evident against the immunizing tumour 791T although significant reactivity was directed against other osteogenic sarcomas. In admixture, the 2 antibodies displayed synergism in their cytotoxicity although this was only demonstrable with defined ranges of antibody concentration. The cytotoxicity of these antibodies was dependent upon the use of rabbit serum as complement and no tumour-cell lysis was produced using human, guinea-pig or mouse serum complement. The more potent cytotoxic antibody failed to modify the outgrowth of 791T tumour xenografts in immunodeprived mice even though localization of antibody at the tumour site has been demonstrated .


A technique for growing colonies from single-cell suspensions of human tumour xenografts using agar in diffusion chambers is described. Modified Millipore diffusion chambers containing tumour cells in semi-solid agar-medium were implanted into the peritoneal cavity of pre-irradiated mice and provided standard culture conditions for the study of colony-forming cells. All 11 xenograft tumours so far studied produced colonies. The incubation period for colony growth ranged from 12 to 28 days and the plating efficiency ranged from 0-3% to 16% for different tumours, but both parameters were constant for each individual tumour. The reproducibility of the system provides a colony-forming assay which can be used to study the effects of irradiation and cytotoxic drugs on human tumour clonogenic cells and may therefore have some advantages over similar assays based on experimental animal tumours.


Tumour antigen-induced inhibition of leucocyte adherence was modified for use in glass test tubes (Tube LAI assay) for the study of cell-mediated anti-tumour immunity to human malignant melanoma. Peripheral blood leucocytes (PBL) of 20 out of 25 patients (80%) with active malignant melanoma responded to an extract of malignant melanoma with LAI, whereas only 4-5% of 475 control subjects showed a response. The malignant melanoma patients reacted to both allogeneic and autologous extracts of malignant melanoma which indicates a common cross-reacting antigen. Malignant melanoma patients did not respond to unrelated tumour extracts. The LAI was mediated by PBL (monocytes) "armed" with cytophilic anti-tumour antibody specific for the sensitizing tumour antigen. The anti-tumour response of the malignant melanoma patients was dependent on the stage of the cancer, and 11 out of 13 Stage I patients had a positive NAI, whereas patients with disseminated cancer had decreased response. The diminished LAI in patients with large tumour burdens appeared to be the result of release of tumour antigen systemically. Also, surgery and chemotherapy depressed LAI. Although LAI was depressed after surgical excision of the cutaneous melanoma, most patients showed LAI 1-3 months later. Tumour-free melanoma patients monitored for one year by the Tube LAI assay showed a decline in their anti-tumour immunity 5-6 months after surgery. The NAI was low or negative after the 8th post-surgical month in tumour-free patients. Patients with residual malignant melanoma showed persistent or recurrent LAI after the 8th post-surgical month. LAI reactivity monitored after "curative" surgery for malignant melanoma may assist in determining whether the patient is tumour-free or has a recurrence.


The expression and prognostic significance of the c-erbB-2 oncogene product was studied in 55 cases of childhood medulloblastoma. Forty-six of the 55 tumours (83.6%) expressed the c-erbB-2 product. The percentage of tumour cells expressing the c-erbB-2 product proved to be a significant indicator of patient outcome when analysed as both a categorical and a continuous variable. As a categorical variable, patients with more than 50% positive tumour cells had a significantly worse survival, with only 10% alive at 10 years vs 48% for those with less than 50% positive tumour cells (log rank P = 0.0049). To demonstrate that this observed prognostic significance was both independent and not a result of 'data-driven' categorisation, it was also entered into the Cox model as a continuous variable. Prognostic significance was retained in P = 0.038.


For validation of this approach, we compared the results obtained in vivo with data provided by iodoantipyrine autoradiography as a reference technique for the measurement of tumour blood flow at a high resolution in an experimental tumour model. The MRI protocol allowed quantitative mapping of tumour blood flow at spatial resolution of 250 × 250 μm2. Correlation of data from the MRI method with the iodantipyrine autoradiography revealed Spearman's correlation coefficients of Rs = 0.851  and Rs = 0.821  for local and global tumour blood flow, respectively. The presented approach enables noninvasive, repeated and quantitative assessment of microvascular perfusion at high spatial resolution encompassing the entire tumour. Knowledge about the specific vascular microenvironment of tumours will form the basis for selective antivascular cancer treatment in the future. © 2001 Cancer Research Campaign http://www.bjcancer.comTumour blood flow plays a key role in tumour growth, formation of metastasis, and detection and treatment of malignant tumours. Recent investigations provided increasing evidence that quantitative analysis of tumour blood flow is an indispensable prerequisite for developing novel treatment strategies and individualizing cancer therapy. Currently, however, methods for noninvasive, quantitative and high spatial resolution imaging of tumour blood flow are rare. We apply here a novel approach combining a recently established ultrafast MRI technique, that is T


The goal of this work was to determine the molecular basis for the induction of tumour vascularization and progression by injury. Magnetic resonance imaging (MRI) studies demonstrated that administration of wound fluid derived from cutaneous injuries in pigs reduced the lag for vascularization and initiation of growth of C6 glioma spheroids, implanted in nude mice, and accelerated tumour doubling time. The former effect can be attributed to the angiogenic capacity of wound fluid as detected in vivo by MRI, and in vitro in promoting endothelial cell proliferation. The latter effect, namely the induced rate of tumour growth, is consistent with the angiogenic activity of wound fluid as well as with the finding that wound fluid was directly mitogenic to the tumour cells, and accelerated growth of C6 glioma in spheroid culture. Of the multiple growth factors present in wound fluid, two key factors, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and platelet-derived growth factor (PDGF), were identified as the dominant mitogens for C6 glioma, and inhibition of their activity using specific neutralizing antibodies suppressed the mitogenic effect of wound fluid on DNA synthesis in C6 glioma. This study suggests that the stimulatory effect of injury on tumour progression can possibly be attenuated by therapeutic targeting directed against a limited number of specific growth factors. © 1999 Cancer Research Campaign


Side effects of drug treatment include both fluid retention and a lowering of blood pressure. Data from animal and human studies suggest that these effects arise, at least in part, from drug-induced changes in the kidney. In order to capitalize on the positive aspect (lowering of blood pressure) and exclude the negative one (fluid retention), it is necessary to understand the mechanisms of action underlying each of the effects. When interpreted with known physiological principles, current hypotheses regarding potential mechanisms produce enigmas that are difficult to resolve. This paper is a summary of the current understanding of PPARγ agonist effects on both blood pressure and fluid retention from a renal perspective and concludes with the newest studies that suggest alternative pathways within the kidney that could contribute to the observed drug-induced effects.Peroxisome proliferator activated receptor  One unanticipated, and poorly understood, side effect of the TZDs is a lowering of blood pressure. In the vast majority of patients treated with these drugs, the change in blood pressure per se can be viewed as a positive consequence of drug therapy. Statistically the classic type 2 diabetic patient has increased blood pressure that is treated by pharmaceutical intervention. Indeed, this beneficial property as well as positive effects on lipid profiles and inflammatory responses has prompted the suggestion that this class of drugs might be useful in treating metabolic syndrome, a prediabetic state that is reaching epidemic proportions in industrialized societies [PPARocieties , 2. γ agonists is not without negative side effects. Particularly noteworthy when considering using TZDs to treat overweight or obese patients is the propensity for these drugs to cause weight gain. The weight gain is multifactorial and includes a positive effect on adipogenesis [ However, treatment with PPARogenesis  and a diogenesis , 5. As w This paper examines the positive (lowering of blood pressure) and the negative (fluid retention) side effects of TZD treatment from a renal perspective. An important consideration for the development of drugs to treat metabolic syndrome is the question of whether the beneficial side effect can be dissociated from the negative action. Unfortunately, at this point one can only outline the issues—the answers await further experimentation to elucidate the mechanisms of action involved in these side effects.γ agonist-induced fluid retention results in plasma volume expansion (often measured as a decrease in hematocrit) and peripheral edema [The PPARal edema . The excal edema , 5. In aal edema .  The propensity to cause fluid retention is serious enough to raise questions about the continued use of these agonists—particularly in a marginal patient population. Rat studies have indicated that the increased plasma volume can cause relatively rapid cardiac remodeling, even in healthy animals . A recenγ agonists are a consistent finding in studies conducted in normal, diabetic, and hypertension-prone rodents and humans . The efans . These γ in the renal collecting duct of mice [γ-mediated fluid retention. Unfortunately, continuous monitoring of blood pressure was not reported in these studies. Two separate knockout technologies were exploited to specifically ablate the PPAR of mice , 19. In γ agonists cause fluid retention. The principal cells lining the distal tubule and collecting duct are the site of hormonally regulated Na+ transport. These hormones regulate whole body salt and water homeostasis and, therefore, blood pressure. Principal cells respond to steroid  and peptide  hormones with an increase in Na+ reabsorption leading to increased plasma volume. All three hormones exert their effects via an insertion of the epithelial Na+ channel (ENaC) into the luminal plasma membrane of the principal cells thereby initiating the Na+ resorptive cascade [γ is expressed in the distal tubule and collecting duct. The collecting duct knockout animal studies were the “icing on the cake” for an emerging hypothesis to explain how PPAR cascade –24. Thusγ collecting duct-specific knockout mice demonstrated a remarkable degree of Na+ and fluid retention and biochemical changes consistent with ENaC activation. Song et al. found that normal Sprague-Dawley rats fed a high dose of rosiglitazone (94 mg/kg body weight) exhibited a 22% decrease in urine volume and a 44% decrease in Na+ excretion [γ agonist, changed electrolyte and water reabsorption in the distal nephron of Sprague-Dawley rats [γ activation increased the cell surface expression of the α subunit of ENaC by upregulating serum glucocorticoid kinase (SGK), an enzyme previously shown to be a convergence point for hormone activation of ENaC [+ channel. Further corroboration was found in studies demonstrating that amiloride (a specific inhibitor of ENaC) prevented the TZD-induced increase in body weight gain in mice [γ agonist-induced fluid retention [γ agonist-induced salt and water retention.  Studies prior to the creation of the PPARxcretion . Chen etley rats . Hong etley rats  demonstr of ENaC , 28. All in mice  and the etention . From alγ agonists in any of three well-characterized principal cell lines that endogenously express the receptor [γ agonist treatment resulted in a decreased aldosterone level [ In the myriad of studies that followed the initial findings, some discrepancies began to appear. For example, not all studies were able to demonstrate inhibition of fluid retention by amiloride. There was a lack of consistency between studies as to which, if any, of the three ENaC subunits was regulated by the agonists and whether SGK actually changed in response to treatment. Nofziger et al. were unable to reproduce the stimulation of ENaC by PPARreceptor . In humareceptor , 30, andne level , 18, 25.While the ENaC hypothesis is appealing in its simplicity, is this the whole story? Do all of the findings correlate with known physiological principles? First and foremost, does the correlation between increased fluid retention and decreased blood pressure make sense, particularly when evoking regulation via an aldosterone/ENaC mechanism? decreasing Na+ and fluid retention. In the treatment of hypertension, an increase in Na+ reabsorption via ENaC is equated with an increase in blood pressure not a decrease as seen during PPARγ agonist therapy.  Amiloride and aldosterone antagonists are clinically used diuretics that inhibit ENaC activity directly or secondarily, and lower blood pressure by γ agonist stimulation of ENaC will result in a decreased blood pressure. Thus, physiological principles as we understand them seem inadequate to explain how ENaC stimulation and increased fluid retention correspond to a decrease in blood pressure.  There are some interesting experiments of nature that can inform this issue as well. There are human mutations in ENaC subunits that give rise to both gain-of-function and loss-of-function in channel plasma membrane expression or activity. The gain-of-function mutations known as Liddle's syndrome result in severe, diuretic-resistant hypertension . In lossα subunit of ENaC in the collecting duct of mice. This technique functionally inactivates the Na+  channel in the same area of the kidney as the previous PPARγ depletion experiments. The mice in which collecting duct ENaC was inactivated still demonstrated the PPARγ agonist fluid retention as observed in the control mice. Blood pressure measurements were not reported in this study. The authors concluded that activation of ENaC is not the primary mechanism in the TZD-induced fluid retention.  In a recent study, Vallon and colleagues  conditioγ knockout mice [+ retention are secondary consequences in response to changes in other ion fluxes. Such secondary effects would explain the diuretic efficacy and also the variability seen in hormonal responses and changes in ENaC subunit abundance. If the ENaC hypothesis does not fit well with known physiological principles, are there alternative possibilities? Putting all the data together, it does appear that one of the sites of action for both fluid retention and blood pressure effects is indeed the kidney, specifically the collecting duct. The strongest support for this contention results from the collecting duct-specific PPARout mice , 19. If −. Diets contain approximately equal concentrations of Na+ and Cl− but the movement of Na+ is considered to be of primary importance because it is hormonally controlled. However, there are emerging studies that suggest that control of renal Cl− transport can modulate blood pressure as well.  When examining other potential ion transporters, one intriguing possibility is a TZD-mediated change in Cl− channels found in the collecting duct is the cystic fibrosis transmembrane regulator (CFTR) [− channels in other portions of the nephron have been linked to changes in blood pressure, a role for the CFTR Cl− channel has yet to be established. Tantalizingly, like ENaC, this channel is activated by ADH and could, theoretically, be involved in hormonal control of salt and water balance in this portion of the nephron [ A number of different chloride channels have been found in various segments of the renal nephron , 36. Mutr (CFTR) . CFTR is nephron , 39, 40.γ agonists inhibit ADH-stimulated Cl− secretion in a cell culture model of the principal cells of the cortical collecting duct. The potency of the agonists to inhibit hormone-stimulated Cl− transport mirrors receptor transactivation profiles except that the IC50 for the effect on Cl− transport is an order of magnitude more sensitive. Analyses of the components of the ADH-stimulated intracellular signaling pathway indicate no PPARγ agonist-induced changes in any of the known steps in the transepithelial signaling pathway. The PPARγ  agonist-induced decrease in anion secretion is the result of a decrease in the mRNA encoding the final effector in the pathway, the apically located CFTR Cl− channel [γ agonists provide new theoretical possibilities for PPARγ agonist-induced fluid retention. The data, if substantiated by in vivo experiments, indicate that CFTR plays a role in fluid balance.  We have recently found that a variety of PPAR channel . These dγ agonist-mediated effect on Cl− flux has been demonstrated in mouse intestinal epithelia. Mice fed with rosiglitazone for 8 days had reduced intestinal forskolin-stimulated anion secretion and substantially inhibited cholera toxin-induced intestinal fluid accumulation. Both of these processes occur via CFTR mechanisms. In the HT29 intestinal cell line, 5-day treatment with rosiglitazone inhibits cAMP-dependent Cl− secretion. This decreased secretion was accompanied by decreases in the protein levels of CFTR, the Na+/K+/2Cl− cotransporter that allows basolateral influx of Cl− and in KCNQ1, which serves as a basolateral K+ recycling channel [ Recently, a similar PPAR channel . γ agonists on ion transporters in polarized epithelial cells is due to a decrease in the expression of the CFTR channel and, perhaps, in other transporters that mediate Cl− secretion. Exactly how this translates into simultaneous effects on fluid retention and decreased blood pressure is unknown. The theoretical possibilities range from changing electrochemical driving forces for transepithelial transport to altering ion and fluid partitioning between the interstitium and the vascular fluid compartment. Thus, there are data to suggest that the primary effect of PPARγ agonists in lowering blood pressure and causing fluid retention remain unknown, several important concepts are beginning to emerge. The most recent data suggest that the TZDs do not directly regulate the ENaC although changes in ENaC-mediated Na+ transport may occur secondarily. Until the physiological mechanisms of action leading to the TZD side effects are fully understood, drugs such as mineralocorticoid antagonists and ENaC channel blockers may be the most effective diuretics in the treatment of PPARγ-mediated edema. However, for development of new drug therapies devoid of the negative side effect of fluid retention, the biochemical mechanism of action of the TZDs on renal and vascular ion and water channels must be more thoroughly investigated.While the mechanisms of action of the PPAR


WT1 mutation with 11p LOH, LOH at 11p, LOH at 16q, and loss of imprinting of IGF2. Analysis of three tumour-associated lesions from 7p LOH cases revealed a cystic nephroma-like area also having 7p LOH. However, a nephrogenic rest and a contralateral WT from the two other cases showed no 7p LOH. No particular clinical phenotype was associated with the WTs which showed 7p LOH. The frequency and pattern of 7p LOH demonstrated in our studies indicate the presence of a tumour suppressor gene at 7p involved in the development of Wilms' tumour. © 2000 Cancer Research CampaignChromosome 7p alterations have been implicated in the development of Wilms' tumour (WT) by previous studies of tumour cytogenetics, and by our analysis of a constitutional translocation ) in a child with WT and radial aplasia. We therefore used polymorphic microsatellite markers on 7p for a loss of heterozygosity (LOH) study, and found LOH in seven out of 77 informative WTs (9%). The common region of LOH was 7p15–7p22, which contains the region disrupted by the t breakpoint. Four WTs with 7p LOH had other genetic changes; a germline


The investigational anti-tumour agent, 5,6-dimethylxanthenone-4-acetic acid , an analogue of flavone acetic acid (FAA), has been scheduled for clinical evaluation. Like FAA, 5,6-MeXAA exhibits excellent experimental anti-tumour activity and is an efficient inducer of cytokines in mice. We have examined the effect of pharmacological suppression of tumour necrosis factor (TNF) production on the anti-tumour activity of 5,6-MeXAA, taking advantage of previous observations that TNF production in response to endotoxin in vitro is inhibited by thalidomide. Thalidomide at doses of between 8 and 250 mg kg-1 efficiently suppressed serum TNF activity in response to 5,6-MeXAA at its optimal TNF inducing dose of 55 mg kg-1. Suppression was achieved when thalidomide was administered at the same time as, or up to 4 h before, 5,6-MeXAA. Under conditions in which TNF activity was suppressed, the degree of tumour haemorrhagic necrosis and the proportion of cures in the subcutaneous Colon 38 tumour were increased. In mice administered thalidomide (100 mg kg-1) together with 5,6-MeXAA (30 mg kg-1), complete tumour regression was obtained in 100% of mice, as compared with 67% in mice receiving 5,6-MeXAA alone. The results suggest a possible new application for thalidomide and pose new questions about the action of 5,6-MeXAA and related compounds.


Successive sampling of ovarian cancers during cytostatic treatment showed several cases of notable changes in their ploidy distribution and one change in model chromosome number, indicating selection of a resistant tumour-cell population. 2. Studies of cell suspensions from human tumour specimens incubated with [3H]-TdR after exposure in vitro to various cytostatic agents have shown variation in labelling between different parts of the same tumour, as well as between the primary tumour and its metastases or ascitic tumour-cell population, which may be accounted for by variation in sensitivity of the tumour-cell population. 3. Studies of nuclear morphology in 20 endometrial cancers before and after progesterone therapy demonstrate considerable variation in the proportion of cells undergoing secretory conversion within the same tumour, indicating primary heterogeneity of the tumour-cell population in response to progesterone.


Unsaturated fatty acids, including n-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid  and eicosapentaenoic acid , and a series of n-6 PUFAs were investigated for their anti-tumour and antimetastatic effects in a subcutaneous (s.c.) implanted highly metastatic colon carcinoma 26 (Co 26Lu) model. EPA and DHA exerted significant inhibitory effects on tumour growth at the implantation site and significantly decreased the numbers of lung metastatic nodules. Oleic acid also significantly inhibited lung metastatic nodules. Treatment with arachidonic acid showed a tendency for reduction in colonization. However, treatment with high doses of fatty acids, especially linoleic acid, increased the numbers of lung metastatic nodules. DHA and EPA only inhibited lung colonizations when administered together with the tumour cells, suggesting that their incorporation is necessary for an influence to be exerted. Chromatography confirmed that contents of fatty acids in both tumour tissues and plasma were indeed affected by the treatments. Tumour cells pretreated with fatty acids in vivo, in particular DHA, also showed a low potential for lung colony formation when transferred to new hosts. Thus, DHA treatment exerted marked antimetastatic activity associated with pronounced change in the fatty acid component of tumour cells. The results indicate that uptake of DHA into tumour cells results in altered tumour cell membrane characteristics and a decreased ability to metastasize.


The influence on stroma of heat alone, X-rays alone or the combined treatment, has been studied using the tumour bed effect (TBE) as an assay. Ca NT cells have been implanted into previously treated subcutaneous sites as an angiogenic stimulus. The vascular damage is then assessed by the reduced tumour growth rate, which results from inadequate vascular proliferation. A range of X-ray doses was used and large alterations in latent period for growth to 2 mm diameter were followed by smaller alterations in the growth rate of established tumours. A dose response relationship was seen for latency (0-20 Gy) and for growth rate (0-16 Gy). A range of subcutaneous temperatures was obtained by immersion in a water bath for 60 minutes at 40 degrees, 41.5 degrees, 43 degrees or 44.5 degrees C. A slight retardation of tumour growth was seen after 41.5 degrees C, but an unexpected acceleration resulted from the highest heat treatment. Combined heat and X-ray treatments showed thermal sensitization of the X-ray induced TBE at 41.5 degrees C, with a reversal at higher temperatures. At 43 degrees C and 44.5 degrees C a mild thermal burn was induced and this appeared to elicit neovascularisation that could be utilized by the implanted tumour cells. Delayed implantation of tumour cells (at 4 weeks instead of 1 day) abolished this effect.


Anti-tumour effectiveness was correlated with platinum accumulation in tumours and the amount of platinum bound to DNA, as determined by atomic absorption spectrometry. In tumours treated with electrochemotherapy, cell kill was increased by a factor of 20 compared with treatment with cisplatin only, as determined from tumour growth curves. The amount of platinum bound to DNA and platinum content in the tumours treated by electrochemotherapy was approximately two times higher than in cisplatin-treated tumours. Based on our results, we conclude that in vivo application of electric pulses potentiates anti-tumour effectiveness of cisplatin by electroporation that consequently results in cisplatin increased delivery into the cells. In addition, besides electroporation, immune system and tumour blood flow changes could be involved in the observed anti-tumour effectiveness of electrochemotherapy. © 1999 Cancer Research CampaignElectrochemotherapy is an anti-tumour treatment that utilizes locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. The aim of our study was to determine whether anti-tumour effectiveness of electrochemotherapy with cisplatin is a consequence of increased plasma membrane permeability caused by electroporation that enables cisplatin binding to DNA. For this purpose, anti-tumour effectiveness of electrochemotherapy was evaluated on SA-1 tumours treated with electric pulses 3 min after intravenous injection of cisplatin (4 mg kg