High levels of the MDM2 oncogene in paediatric rhabdomyosarcoma cell lines may confer multidrug resistance.

The MDM2 protein is known to be overexpressed in some sarcomas including rhabdomyosarcoma. However, the extent to which the MDM2 protein influences sensitivity to chemotherapeutic drugs is unclear. We have analysed this further using stable transfection of the mdm2 gene into 4 well-characterised human paediatric rhabdomyosarcoma cell lines. Transfection with the mdm2 gene resulted in increased levels of the MDM2 protein in all the cell lines. In 2 of the lines, SCMC and RD, the mdm2 gene caused between 2-fold and 61-fold increase in resistance to vincristine, etoposide and doxorubicin but not to cisplatin. In these lines there was an increase in expression of the mdr-1 gene which encodes P-glycoprotein, but not the mrp1 gene which encodes the multidrug resistance protein (MRP). The resistance was reversible using the MDR modulator PSC833, confirming the presence of P-glycoprotein. We conclude that MDM2 overexpression may be a mechanism by which multidrug resistance is regulated in some rhabdomyosarcomas.

Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy.

A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically accessible and suitable for further pharmaceutical development. NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease.

Effect of p53 status on sensitivity to platinum complexes in a human ovarian cancer cell line.

Wild-type p53 is frequently mutated in late-stage ovarian cancer and has been proposed as a determinant of cisplatin chemosensitivity. We have therefore established a human ovarian cancer cell line differing only in p53 status and characterized its response after treatment with different platinum complexes. The wild-type p53-expressing cell line A2780 was stably transfected with HPV-16 E6 (E6) or an empty vector (VC) as control. Parental A2780 and VC had similar cisplatin sensitivities, whereas E6 was 3- to 4-fold more sensitive as measured by sulforhodamine B and clonogenic assay. E6 was 2- to 3-fold more sensitive to transplatin and the novel cisplatin analog ZD0473 than VC, whereas the trans-platinum analog JM335 was approximately equitoxic. Platinum uptake was similar for all of the cell lines after cisplatin. The removal of platinum-DNA adducts, as measured by atomic absorption spectroscopy, was reduced in E6 compared with VC after cisplatin but similar after JM335. After 10 microM cisplatin, the G(1) population (0-96 h) was reduced in E6 cells compared with VC, whereas the S phase (8-48 h) and G(2) phase (48-96 h) were increased. Similar proportions of VC and E6 cells died by apoptosis, as detected by annexin V binding, but more E6 cells died by necrosis relative to VC. Our results suggest that the loss of functional p53 can increase cisplatin cytotoxicity in A2780, with loss of G(1)/S checkpoint control and decreased cisplatin-DNA adduct repair, but these effects can be circumvented by the use of JM335, which forms different DNA-platinum adducts.

Crystal structure of human DT-diaphorase: a model for interaction with the cytotoxic prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954).

The crystal structure of human DT-diaphorase (NAD(P)H oxidoreductase (quinone); EC 1.6.99.2) has been determined to 2.3 A resolution. There are only minor differences in shape and volume between the active sites of the rat and human enzymes and in the hydrophobic environment in the vicinity of the substrate. The isoalloxazine ring of the bound FAD is more buried in the human structure. Molecular modeling was used to examine optimal positions for the antitumor prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) in both the human and rat enzyme active sites. This suggests that the position of CB1954 in the active site of the human enzyme is very similar to that in the rat, although there are detailed differences in the predicted patterns of hydrogen bonding between side chains and the drug. Some of the differences are a consequence of the shift in position for the FAD molecule and may contribute to the observed differences in rate of the two-electron reduction of CB1954.

Investigation of alternative prodrugs for use with E. coli nitroreductase in 'suicide gene' approaches to cancer therapy.

The most commonly employed 'suicide' gene/prodrug system used in cancer gene therapy is the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir system. We have examined the efficacy of an alternative approach utilising the E. coli nitroreductase B enzyme with CB1954 and a variety of other prodrugs. V79 cells transfected with a nitroreductase expression vector were up to 770-fold more sensitive to CB1954 than control non-expressing cells. In general other prodrugs which were found by HPLC to act as substrates for purified E. coli nitroreductase also exhibited increased cytotoxicity against the nitroreductase-expressing cells, although this correlation was not absolute. In particular nitrofurazone (97-fold) and additional aromatic nitro-compounds (nine- to 50-fold) showed a large differential whereas the quinones and the antimetabolite, B-FU, were less effective (< three-fold). The results support the possibility of using nitroreductase and CB1954 for 'suicide gene' therapy and in addition suggest that alternative prodrugs, such as nitrofurazone, warrant further investigation in this novel approach.

Interaction of aglycosyl immunoglobulins with the IgG Fc transport receptor from neonatal rat gut: comparison of deglycosylation by tunicamycin treatment and genetic engineering.

The role of carbohydrate in the structure and function of immuno-globulin Fc regions has been studied using the interaction of a monoclonal mouse IgG2b anti-NIP antibody with the IgG Fc transport receptor from neonatal rat gut. An aglycosyl variant of this immunoglobulin, in which site-directed mutagenesis had been used to eliminate the carbohydrate attachment site in the CH2 domain by changing Asn297 to Ala, was compared in this system to aglycosyl immunoglobulin prepared from immunoglobulin-secreting cells treated with tunicamycin to inhibit N-linked glycosylation. Loss of carbohydrate from the heavy chain was confirmed for both methods by Western blotting of the separated chains with Concanavalin A, and no significant differences in circular dichroism spectra were found between glycosylated and non-glycosylated mutants. Removal of carbohydrate by site-directed mutagenesis had no effect on binding of the immunoglobulin to the Fc transport receptor (FcTR) in vitro or transport from the gut to blood in vivo. Short-term clearance from circulation and degradation by gut contents in vitro were similarly unaffected. Mutation of Glu235 to Leu, an alteration that allows binding to human monocyte Fc gamma RI, did not alter the interaction with FcTR. However, treatment of wild-type or aglycosyl mutant cells with tunicamycin resulted in immunoglobulin which was less stable, cleared more rapidly and was transported slightly less efficiently. These findings indicate that the binding site for the FcTR may be unique among Fc-binding ligands, and that tunicamycin treatment may cause alterations in the immunoglobulin molecule in addition to loss of N-linked carbohydrate.

IgA antibodies in the bile of rats. V. Primacy of the GALT as a source of IgA.

In each of a series of rats the common bile duct and the thoracic duct (cisterna chyli) were cannulated so that both bile and thoracic duct lymph could be collected quantitatively for several hours. The concentrations of IgA in samples of lymph and bile were measured by radioimmunoassay so that the output of IgA per unit time could be calculated. Although the output of IgA in the lymph did not decline significantly, the output in the bile fell so that by 2 hr it had been reduced to less than 20% of the peak value. Similar experiments in rats which had been immunized actively by injecting antigens into the GALT showed a corresponding rapid decline in titres of specific biliary antibodies after fistulation of the thoracic duct. The low levels of IgA in the bile of rats that had been drained of thoracic duct lymph were restored quickly to normal values by the intravenous infusion of a volume of thoracic duct lymph equal to that which had been lost; this restoration was transient, and the concentration of IgA in the bile soon declined again after the infusion ceased.

Low affinity antibodies against collagen type II are associated with pathology in collagen-induced arthritis in mice.

The relationship between the functional affinity of antibodies against type II collagen (CII) and the development of arthritis was studied in mice with collagen-induced arthritis. The responses of DBA/1 strain mice were compared with those of mice selectively bred to produce antibodies of high functional affinity (HA mice) and low functional affinity (LA mice). HA and LA mice did not develop arthritis in response to immunization with CII whereas 86% of DBA/1 mice did, with 33% showing severe and 53% mild disease. Anti-CII antibodies of the highest titre, the lowest functional affinity, and the greatest affinity heterogeneity were associated with the development of the severest arthritis in DBA/1 mice: even in DBA/1 mice with moderate or no disease the amount of antibody and heterogeneity were higher and functional affinity lower than in either HA or LA mice. Antibodies of the G1, 2a, 2b and 3 subclasses were produced in all mice, and none of these alone accounted for the overall difference in IgG antibody titres or affinity in the groups of mice. Antibodies of the IgG2a subclass showed the closest association with the development of arthritis in the different groups. It is concluded that anti-CII antibodies of low functional affinity, and presumably also of the IgG2a subclass, influence the disease process in collagen arthritis.

Some properties of dendritic macrophages from peripheral lymph.

Peripheral lymph was collected from the skin and liver of sheep, and from the intestine of rats. The dendritic macrophages contained in it were isolated by centrifuging the lymph over a layer of 'Nycodenz'. Similar cells were produced by culturing mononuclear cells from venous blood, but the yields were very small. The numbers of dendritic cells in the lymph from the legs of sheep increased five-fold after xylene had been applied to the skin. Dendritic macrophages displayed abundant class II histocompatibility antigens on their surfaces, as well as immunoglobulins. Although the latter were probably acquired passively, they remained present for several days on cells cultured in vitro. When in vitro, dendritic cells could be shown to phagocytose marker particles, such as latex beads, but their performance was unimpressive compared to macrophages from the peritoneal cavities of rats. In contrast, their ability to phagocytose rapidly T4 phage or influenza viruses unequivocal and striking.

Role of carbohydrate in binding of IgG to the Fc receptor of neonatal rat enterocytes.

Monoclonal IgG1, IgG2a, IgG2b and IgG2c were prepared from rat hybridoma cells treated with tunicamycin in order to inhibit N-linked glycosylation. The IgG produced by these cells was about 70% lower in carbohydrate content compared to IgG from equivalent untreated cells, but was similar to the corresponding normal IgG in terms of antigen binding. However, the ability of carbohydrate deficient (CHO-) IgG to bind in vitro to Fc receptor extracted from jejunum of neonatal rats was impaired in most cases and, in all but one case, the amount of CHO- IgG transported from gut lumen to blood in vivo was markedly reduced. No reduction in binding of normal IgG to extracted receptor was observed in the presence of various sugars. It is postulated that N-linked carbohydrate acts to stabilize the structure within the IgG molecule which is responsible for binding to this Fc receptor, possibly in the CH2 domain.

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens. II. Effect of host age and immune status.

In the preceding paper it was suggested that the tumour localisation of 125I-labelled syngeneic rat monoclonal antibodies (mAbs) may be limited in immunocompetent hosts by the presence of competing endogenous serum antibodies. In syngeneic congenitally athymic (nu/nu) and cyclosporin-A-treated rats (both of which fail to mount immune responses to tumour antigens) increased uptake of mAbs in tumour tissue was obtained compared with that in immunocompetent animals. However, in the case of IgG2b and IgG1 mAbs, this appeared to be due primarily to enhanced "non-specific" localisation mediated by Fc binding, since it was abolished by the use of F(ab')2 fragments with two out of three mAbs tested. Normal tissue distribution was also influenced by host immune status: in nu/nu rats the uptake of IgG2b mAbs in the spleen was up to fivefold higher than that previously found in normal animals and the levels in liver were also increased. This effect was not seen in cyclosporin-A-treated hosts, suggesting that the reticuloendothelial system of congenitally athymic animals contains cells with enhanced IgG2b-FcR activity. This hypothesis was strengthened by the observation that splenic uptake was reduced by either the use of F(ab')2 fragments, or prior "blockade" of Fc receptors by "cold competition" with excess unlabelled IgG2b mAbs. This blockade could not be effected by mAbs of any other isotype or by IgG2b F(ab')2 fragments. The former manoeuvre resulted in higher tumour specificity ratios but usually at the expense of reduced levels of tumour associated radiolabelled mAb. The latter was found to increase "absolute" tumour localisation by up to 35%. In an attempt to characterise further and compare the Fc receptor activity of intratumour and intrasplenic host cells. The distribution of IgG2b mAbs was assayed in 3-week, 8-week and 12-week-old rats. We were able operationally to distinguish the activity of these two categories of cells, suggesting that they represent either different lineages or differentially activated subpopulations: the splenic IgG2b binding was fully expressed in weanling nu/nu rats whereas the FcR activity of cells infiltrating MC24 sarcoma was limited in 3-week-old compared with 8-12-week-old hosts. A further difference was apparent in the subclass "preference" of FcR binding: in immunodeprived rats both IgG1 and IgG2b mAbs were able to bind to tumour-infiltrating host cells, but uptake of IgG1 mAbs in the spleen was always low and not reduced further by the use of F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens. I. Comparison of IgG subclasses.

The object of our current investigations is to explore the potential of antibodies for localisation and treatment of disseminated disease, using as a model rat monoclonal antibodies (mAbs) raised against syngeneic tumour-specific antigens. As part of this study, antibodies of differing isotypes with specificity for either HSN or MC24 sarcoma were labelled with 125iodine and injected intravenously into normal rats or those bearing paired tumours in contralateral flanks. The blood clearance rates of the radiolabelled antibodies were found to be influenced by immunoglobulin subclass (IgG2b greater than IgG2a greater than IgG1) and to be increased non-specifically by the presence of growing tumours. The tumour and normal tissue distributions of the antibodies tested were also found to vary according to their apparent degree of interaction with host Fc-receptor-bearing cells, to the extent that tumour specificity in vitro was not necessarily reflected in selectivity of localisation in vivo. Three IgG2b monoclonal antibodies showed preferential uptake in the spleens of syngeneic rats and non-specific accumulation in tumours. This effect was not observed with antibodies of IgG2a or IgG1 subclass, and was abolished by the use of IgG2b F(ab')2 preparations. In spite of the use of immunoglobulin fragments, varying the assay time and testing tumours of different sizes, specific tumour localisation was low with all seven monoclonal antibodies tested. The maximum uptake achieved was less than 1% of the injected dose of antibody per gram of tumour. Much higher levels of antibody localisation have been reported for human tumour xenografts growing in nude mice, but these are rarely achieved in other systems. We propose that the use of autologous monoclonal antibodies recognising tumour-associated antigens of relatively low epitope density in syngeneic hosts provides a valid alternative model in which to investigate the factors limiting more effective, specific immunolocalisation of malignant disease.

Tolerogenic activity of polymerized type II collagen in preventing collagen-induced arthritis in rats.

Rats were exposed parenterally or pergastrically to polymerized type II collagen (POLCII) and became resistant to the subsequent induction of disease with arthritogenic type II collagen (CII) administered intradermally in Freund's incomplete adjuvant (FIA). POLCII was prepared by cross-linking native soluble arthritogenic CII, from bovine nasal septal cartilage, with glutaraldehyde. POLCII injected intradermally in FIA did not induce arthritis. Animals treated in this manner were resistant for a period of at least 100 days to induced disease. The change in the properties of the CII from an arthritogen to a tolerogen was related to the amount of glutaraldehyde (used to polymerize the CII) which was assumed to control the extent of cross-linking of the CII. Highly cross-linked POLCII administered pergastrically, like soluble CII, was not arthritogenic but was tolerogenic, inducing a state of unresponsiveness to a challenge with arthritogenic CII. In general serum anti-CII antibody levels were higher in arthritic than in tolerized non-arthritic rats. It is concluded that the breaking of self-tolerance to CII depends upon its physical state. When polymerized and insoluble, a form analogous to that in which it exists naturally, it is tolerogenic.

Binding of subclasses of rat immunoglobulin G to detergent-isolated Fc receptor from neonatal rat intestine.

Brush borders of cells lining the proximal small intestine of neonatal rats express a receptor specific for the Fc portion of IgG that mediates transport of IgG from gut lumen to blood. We have investigated the interaction of subclasses of rat IgG with this receptor, extracted in Triton X-114 solution, using phase separation to separate receptor-immunoglobulin complexes from free immunoglobulin. Binding of immunoglobulin showed the same pH dependence as is found in vivo, being active at pH 6 and reversibly inhibited at pH 8. The numbers of binding sites for each IgG subclass were similar, but polyclonal IgG2a was bound with higher affinity (1.2 X 10(8) M-1) than monoclonal IgG1 or IgG2b (2-3 X 10(7) M-1). Radiolabeled monoclonal IgG2c did not show specific binding, apparently as a result of the iodination process. Competition studies showed cross-inhibition between all IgG subclasses. IgG2a being approximately 10-fold more effective at competing for receptor than other isotypes, in the order IgG2a much greater than IgG1 greater than IgG2b greater than or equal to IgG2c. These data suggest that a single receptor capable of binding all subclasses of IgG is active in the detergent extract. However, investigation of radiolabeled immunoglobulins that were bound to isolated gut cells before detergent extraction showed evidence for other types of interaction in vivo.

Lymphotoxic activity of methyl prednisolone in vitro--I. Comparative toxicity of methyl prednisolone in human cell lines of B and T origin.

The cytotoxic activity of methyl prednisolone was compared in EB-3(B), NALM-6(B), CCRF-CEM(T) and RPMI-8226 (plasma cell) cell lines derived from human lymphoid malignancies. Whereas EB-3 cells were steroid-sensitive, NALM-6 cells were partially sensitive and CCRF-CEM and RPMI-8226 were steroid resistant at concentrations of methyl prednisolone up to 10(-4) M. A high concentration of methyl prednisolone, 2.5 X 10(-3) M was toxic to all cell lines. Steroid-sensitivity did not correlate with the incorporation of [3H] dexamethasone and could not be mimicked by flurbiprofen, a non-steroidal anti-inflammatory agent. Both theophylline and di-butyryl cAMP were toxic towards NALM-6, EB-3 and CCRF-CEM cells; however, this toxicity was reversible and did not reflect the cells' sensitivities towards methyl prednisolone. Furthermore, elevated levels of cAMP in theophylline-treated cells, were not demonstrable in cells treated with methyl prednisolone at toxic or non-toxic concentrations of the steroid. Steroid-sensitive EB-3 cells exposed to 10(-5) M methyl prednisolone, produced a soluble factor which was toxic CCRF-CEM cells.

Biochemical characterization of chicken secretory component.

Human dimeric IgA was injected i.v. into chickens whose bile was then collected. The human IgA which had been transported across the hepatocytes of the chicken was subsequently purified from the bile and shown to be associated with a protein of 80 kDa and pI 4.6, which reacted with rabbit antisera to chicken bile proteins but not with an antiserum to human alpha chain. The chicken bile protein thus has functional and biochemical properties similar to those of mammalian secretory component.

The production of hybridomas from the gut associated lymphoid tissue of tumour bearing rats. I. Mesenteric nodes as a source of IgG producing cells.

Rat X rat hybridomas secreting antibodies with tumour specificity have been prepared using cells from the mesenteric nodes of rats bearing syngeneic sarcomata in their Peyer's patches. The antibodies obtained, which embraced all of the major immunoglobulin classes, varied in cellular reactivity from the individually tumour specific to cross-reactive with both normal and tumour cells. Several IgA producing hybridomas were prepared using this protocol but IgG secretors were obtained more frequently and they accounted for about half of the specific hybridomas. Specific IgG producers were found to predominate also when hybridomas were prepared from the mesenteric nodes of a rat immunized by injection of horseradish peroxidase into the Peyer's patches. Comparison of these data with our earlier results using spleen cells taken from rats that were either hyperimmunized with, or were bearing the same tumours in the leg, show that mesenteric nodes draining a tumour growing in the Peyer's patches are a much better source specific IgG producing B cells.

The production of hybridomas from the gut associated lymphoid tissue of tumour bearing rats. II. Peripheral intestinal lymph as a source of IgA producing cells.

Peripheral intestinal lymph afferent to the mesenteric nodes has been collected from rats bearing syngeneic sarcomata in their Peyer's patches and the B cells used to produce rat X rat hybridomas. Analysis of the hybridoma supernatants by radioimmunoassay for the presence of immunoglobulins, showed that hybridomas secreting IgA predominated. Eleven out of the 15 hybridomas selected for antibody binding to cells of the immunising tumour secreted IgA antibodies, and six of these were tumour specific. Efferent mesenteric lymph (i.e. normal thoracic duct lymph), on the other hand, was found to be a poor source of B cells for hybridoma production and no specific IgA secreting hybridomas were obtained. The high yield of IgA secreting hybridomas obtained shows that peripheral intestinal lymph is a better source of IgA committed B cells than are the mesenteric nodes or thoracic duct lymph. We conclude that the IgA producing cells in the latter tissues are too far along the differentiation pathway to plasma cells to undergo successful somatic cell fusion.