20th-century advances in drug therapy in oncology--Part. II.

Ongoing research in cancer therapy has led to the development of antineoplastic agents which target specific components of the cell cycle. In Part II of this series, we discuss agents which target the mitotic mechanism by inhibiting microtubules. Although many of these agents are being shown to have multiple effects, the Vinca alkaloids and the taxanes are known as antimitotic drugs. They are among the most important anticancer agents currently available, and because of their unique mechanisms, can be combined with a wide variety of other antineoplastic agents in a spectrum of diseases. In addition, in part II, we are discussing agents that target DNA and prevent replication and thus cell growth by inhibiting the enzymes which protect DNA during replication, the topoisomerases. These drugs, too, have unique mechanisms of action and have become major components of combination regimens. The topoisomerase I inhibitors are new drugs derived from an older parent drug, and their full possibilities are still being explored.

Autologous bone marrow and peripheral blood stem cell transplantation in haematological malignancies: current status.

Autologous bone marrow and peripheral blood stem cell transplants permit the use of higher doses of chemoradiotherapy than would have been possible without "rescuing' bone marrow function. This may well allow cure of malignant disorders in cases where this was previously not feasible. This short review attempts to summarize the current status of such a treatment approach for the various haematological malignancies.

Radioimmunoassay for assessing exocrine pancreatic insufficiency, based on the differential enzymatic degradation of cobalamin-binding proteins.

Pancreatic proteases degrade the protein moiety of the R protein-cobalamin complex but not the intrinsic factor-cobalamin complex. Accordingly, we used these two proteins as substrates in an in vitro enzymatic assay to assess pancreatic function by incubating basal jejunal fluids with a mixture of intrinsic factor and cyano[57Co]cobalamin coupled to R-type protein and then using immunoprecipitation to determine the distribution of isotopically labeled cobalamin bound to the two proteins. With normal jejunal fluids, 91.2 (SD 6.1)% and 4.5 (SD 5.5)% of cyano[57Co]cobalamin was precipitated with antisera to intrinsic factor and anti-R protein, respectively. In the patients' jejunal fluids, the cyano[57Co]cobalamin precipitated with the respective antisera was 5.3 (SD 10.0)% and 96 (SD 6.2)%. In patients with other gastrointestinal problems, the sequestration of cobalamin was indistinguishable from that observed with the normal fluids. The clearcut discrimination this radioimmunoassay provided between abnormal and normal samples was confirmed by parallel comparative chromatographic analysis.

Isolation and characterization of two species of receptor which bind intrinsic factor.

The intrinsic factor receptor from guinea pig ilea has been characterized following purification by affinity chromatography. The purified receptor complexed to intrinsic factor-cobalamin (holo-receptor) had an anhydrous molecular weight of 680,000, a Stokes radius of 10.9 nm, and a sedimentation coefficient of 15.1 S. In contrast, unsaturated receptor (apo-receptor) resolved into distinct "large" and "small" molecular species having, respectively, (i) molecular weights of 700,000 and 350,000; (ii) Stokes radii of 11.1 and 7.06 nm; (iii) sedimentation coefficients of 15.5 S and 11.9 S; (iv) association constants for binding the intrinsic factor-cobalamin complex of 7.3 and 2.5 X 10(10) liters mol-1; and (v) two isoproteins for the larger species (pI's 4.05 and 4.80), and one isoprotein for the smaller species (pI 4.90). A rabbit anti-receptor serum gave only one precipitation line in immunodiffusion against either the large or the small receptor, and each one of these two lines fused completely with the one of two lines which formed with the purified preparation containing both receptor species. Autoradiography of the precipitin lines obtained when the receptor was coupled to intrinsic factor-cyano[57Co]cobalamin demonstrated that both species of receptor were functional. The reaction of complete immunologic identity, the similar electrical properties and similar kinetics for binding to intrinsic factor, and the observed molecular weight differences indicate that the small and large apo-receptors are chemically interrelated, and suggest that the large receptor may consist of two small functional proteins.

Measurement of cyanocobalamin in serum by a specific radioimmunoassay.

Antiserum to cobalamin was raised in rabbits by immunization with the monocarboxyl derivative of cyanocobalamin coupled to albumin. The antiserum was treated to remove transcobalamin II and transcobalamin I. The partially purified antibody bound free cyano[57Co]cobalamin, but not the vitamin precoupled to the transcobalamins. Cyano[57Co]cobalamin bound by the antiserum eluted from Sephadex G-200 as a single peak with a mol wt of 160,000 and was precipitated by goat anti-rabbit gamma globulin, indicating that the vitamin was bound to an IgG immunoglobulin. Unlabeled cyanocobalamin and hydroxocobalamin competitively inhibited the binding of cyano[57Co]cobalamin to this antibody. Neither adenosylcobalamin, in a similar concentration range, nor cyanocobinamide at a concentration 30-fold greater than the tracer cobalamin competed appreciably with the binding of cyano[57Co] cobalamin. The association constant for the interaction of the antibody with cyanocobalamin and cyanocobinamide was estimated to be 8.6 X 10(9) and 9.6 X 10(6) L/mol, respectively. The association constant for adenosylcobalamin, methylcobalamin, and hydroxocobalamin was indirectly determined, and values of 2.5 X 10(5), 1.7 X 10(9), and 2.3 X 10(9) L/mol, respectively, were obtained. Photolysis in the presence of potassium cyanide rendered each of the three cobalamins equivalent to cyanocobalamin in immunoreactivity. The mean concentration of cobalamin in normal human sera and cobalamin-deficient sera measured as cyanocobalamin by radioimmunoassay using this anticobalamin antibody was significantly lower than the concentration measured in the same extracts by competitive ligand-binding radioassays using intrinsic factor and transcobalamin I. These findings, although indirect, support the proposition that there may be factor(s) in normal and cobalamin-deficient sera that falsely elevate the concentration of true cobalamin if the radioassay uses R protein as the binder. However, the lower concentration of serum cobalamin measured by radioimmunoassay compared with the intrinsic factor radioassay also indicates that this "purported" factor(s) reacts to some extent with intrinsic factor but not with the cobalamin antibody.

Functional activity of intrinsic factor measured by using solubilized receptor protein.

The traditional radioimmunoassay for gastric intrinsic factor, in which this protein is measured on the basis of immunoreactivity rather than function, is of no value for identifying intrinsic factor that binds cobalamin but does not bind to the ileal receptor site, or for detecting animal intrinsic factor, which does not cross react with human intrinsic factor. Accordingly, we have applied a radioassay for the intrinsic factor receptor protein to measure the functional activity of intrinsic factor in gastric juice. The receptor protein reagent was partly purified from guniea pig ilea and its interaction with intrinsic factor--CN[57Co]-cobalamin was determined by precipitation with sodium sulfate at a final concentration of 150 g/L. Results of this assay were comparable with results obtained for intrinsic factor by radioimmunoassay. The receptor protein did not bind immunoreactive intrinsic factor that was functionally abnormal. This functional radioassay for intrinsic factor is not species specific and will be of value when specific antiserum to intrinsic factor is not available and when cobalamin malabsorption is to be evaluated in patients who are secreting normal amounts of immunoreactive intrinsic factor.

Intrinsic factor-mediated intestinal absorption of cobalamin in the dog.

The purpose of these studies was to determine whether gastric intrinsic factor and the ileal intrinsic factor receptor participate in the process of cobalamin absorption in the dog. Physicochemical analysis of gastrointestinal fluids and mucosal extracts obtained 3-5 h after cyano[57Co]-cobalamin was fed to dogs demonstrated that 1) all cyano-[57Co]cobalamin became bound to proteins during intraluminal transport; and 2) mucosal cyano[57Co]cobalamin in the extract of the ileal mucosa was bound to intrinsic factor, to intrinsic factor coupled to receptor protein, and to proteins with properties similar to R protein and transcobalamin II. A significant fraction of the cyano[57Co]cobalamin in the mucosal extract was membrane bound and, upon solubilization with Triton X-100, was found to contain immunoreactive intrinsic factor that, however, could no longer couple to the isolated receptor. The formation of the complex of cobalamin with intrinsic factor and the receptor protein and the selective accumulation of cobalamin in the ileum indicate that the intrinsic factor-mediated mechanism for absorption of this vitamin is active in the dog.

In vivo evidence that intrinsic factor-cobalamin complex traverses the human intestine intact.

Ingested cyano[57Co]cobalamin was recovered as coupled to intrinsic factor from the jejunum of healthy humans. This vitamin-protein complex and the analogous complex from patients having exocrine pancreatic insufficiency were indistinguishable from each other in terms of molecular radius (3.30 nm), ionic nature (eight well-defined isoproteins isoelectric at pH 4.71, 4.84, 4.90, 5.13, 5.23, 5.31, 5.40 and 5.69), and antigenic structure. These findings indicate that the pancreatic proteases do not alter the intrinsic factor cobalamin complex in vivo. Purified R type protein-cyano [57Co]cobalamin complex recovered from patients with exocrine pancreatic insufficiency was similar to the analogous gastric protein in terms of molecular radius (alpha = 4.78 nm) and types of isoproteins (seven well-defined isoproteins isoelectric at pH 2.70, 3.03, 3.38, 3.74, 3.87, 4.05 and 4.20). However, this R protein complex from patients and the intrinsic factor complex from both control subjects and from patients was comprised of more of the acidic type of isoproteins, thereby supporting the notion that carbohydrate-rich isoproteins are more resistant to digestion in the intestine.

A derivative of R-type cyanocobalamin binding proteins in the human intestine. A candidate antibacterial molecule.

Human jejunal fluid contains a protein which has a molecular radius of 3.37 nm, an Mr or 60600 and a mean pI of 3.35 and binds cobalamin with a K of 0.1 . 10(9) l/mol. This protein also couples cobalamin analogues lacking the nucleotide moiety, cross-reacts with R-type protein and is resistant to proteolysis in the intestine. These findings refute the hypothesis that cobalamin-analogue binders are present and effectively inhibit the bacterial uptake of analogues in the intestine.

Cobalamin malabsorption due to nondegradation of R proteins in the human intestine. Inhibited cobalamin absorption in exocrine pancreatic dysfunction.

In vivo studies demonstrate that the pancreatic enzymes and the ionic environment in the upper gastrointestinal tract are essential determining factors for transport and absorption of cobalamin in man. Jejunal fluid was aspirated from healthy human volunteers after administration of cyano[57Co]cobalamin preparations. Immunochemical analysis of the aspirates demonstrated that all isotopic vitamin was transferred to a protein that is identical to the gastric intrinsic factor in terms of molecular mass (57,500), ionic nature (mean pI, 5.09), and reactivity with anti-intrinsic factor sera. However, in the aspirates from patients with exocrine pancreatic dysfunction the vitamin was found to be coupled > 60% to a protein identical to R proteins in terms of molecular mass (125,000), ionic nature (mean pI, 3.51), and reactivity with anti-R protein and anti-intrinsic factor sera. The preferential transfer of cobalamin to R proteins in the patients and to intrinsic factor in healthy subjects was associated, respectively, with low and normal levels of pancreatic enzymes in the intestine and these in turn were paralleled respectively by impaired and normal ileal absorption of cobalamin. These findings confirm the suggestion that the formation of unabsorbable cobalamin complexes may be the reason of impaired vitamin absorption in exocrine pancreatic insufficiency. Observations made with other selected patients demonstrate: (a) that decreased enzyme activity and nondegradation of R proteins may also be due to nonactivation of pancreatic zymogens in an acidic pH of the intestinal juice the vitamin transported to the jejunum couples to intrinsic factor when pancreatic function is normal, and to intrinsic factor and R protein in exocrine pancreatic insufficiency. The observations made with these selected patients may explain why not all patients with exocrine pancreatic insufficiency develop imparied cobalamin absorption, and also why the malabsorption is corrected by the administration of bicarbonate in certain patients.

[Role of pancreatic enzymes in the intraluminal transport and ileal absorption of vitamin B 12 (Cb 1)]. / Rôle des enzymes pancréatiques dans le transport intraluminal et l'absorption iléale de la vitamine B12 (Cb1).

The present paper outlines the classical concepts of transport and absorption of vitamin B12 and discusses findings which provide new insight into the important role of pancreatic enzymes in the absorption of the vitamin B12. In vivo experiments with healthy subjects and patients with exocrine pancreatic insufficiency demonstrate that the pancreatic enzymes do not activate "the precursor" intrinsic factor molecule but solely dissociate vitamin from the inactive R type proteins with a consequent coupling to the biologically active intrinsic factor.

Blocking and binding type antibodies against all major vitamin B12-binders in a pernicious anaemia serum.

The simultaneous occurrence of blocking and binding antibodies to intrinsic factor (IF), transcobalamin (TC- II, TC I) and other R type vitamin B12 proteins in the serum of a patient with treated pernicious anaemia (PA) is reported here for the first time. The dialysed and purified immunoglobulin-G (IgG), but not the immunoglobulin-M (IgM), from a PA patient neutralized the total unsaturated vitamin B12 binding capacity (UB12BC) of human gastric juice, saliva and serum and also of rabbit serum, suggesting that the PA IgG contained blocking antibodies against, IF, TC II, TC I and other R-binders. In addition, the PA IgG and IgM preparations contained binding antibodies since they could form macromolecular complexes with 57Co-B12 bound to IF, TC I or TC II so that each one of the latter was totally excluded from Sephadex G-200. The presence of the binding antibodies was further confirmed by the formation of radioactive precipitation lines on agarose with each one of the vitamin B12-binders bound to 58Co-B12. The PA serum did not exhibit any measurable UB12BC after dialysis against 7.5 M guanidine-HCl followed by renaturation with phosphate buffer (pH 7.4). But it did form TC I and TC II complexes with 57Co-B12 when the latter was added during the renaturation step indicating that the serum contained circulating immunoglobulin-TC complexes. The blocking antibodies should be distinguished from the previously described binding antibodies. The blocking of the binding of vitamin B12 to TCs resulted in relatively lower serum vitamin B12 levels in the present case in contrast to the presence of binding antibodies where high serum vitamin B12 levels have been reported. In addition, the binding antibodies form immunocomplexes with TCs which can easily be detected because they can bind radioactive vitamin B12 while the corresponding immunocomplexes of blocking antibodies are hidden because they prevent the binding of the vitamin to TCs.

Comparative studies on intrinsic factor and cobalophilin in different parts of the gastrointestinal tract of the pig.

The vitamin B(12) binders in the pig pyloric mucosa gastric and intestinal juice from the upper gastrointestinal tract were fractionated into only two molecular forms, classified as intrinsic factor and cobalophilin. The unsaturated vitamin B(12)-binding power due to cobalophilin was lower in the intestinal than in the gastric juice. Electrofocusing revealed that intrinsic factor and cobalophilin in the intestinal juice contained more of the ;neutral'-type isoproteins, and the suggestion is made that this is due to enzyme activity. The gastric-juice intrinsic factor contained more acidic isoproteins, which supports the hypothesis that carbohydrate is added on to the polypeptide chain of this protein before it is secreted into gastric juice. The gastric- and intestinal-juice cobalophilins, studied also by electrofocusing, differed from that of pyloric mucosa and they appeared to be of salivary origin. With regard to molecular dimensions there was no significant difference between the intrinsic factors and cobalophilins from all sources studied. All cobalophilins had molecular weights by the formula of Svedberg of approx. 92500, Stokes radii of 4.62nm and sedimentation coefficients of 5.15S. The corresponding values for the intrinsic factors were 63600, 3.57nm and 4.38S. In addition, the intrinsic factors exhibited similar avidities for binding to the solubilized ileal intrinsic-factor receptor. Also the intrinsic factors and cobalophilins, irrespective of their source, bound to the analogous specific xenoantibodies with the same avidity. The present results demonstrate that intrinsic factor remains practically unaltered during its passage through the proximal intestine and render unlikely the speculations made about the presence of an endogenous binder for intrinsic factor as well as the existence of a ;pancreatic intrinsic factor'. In addition, they are compatible with the theory that the interference by undegraded cobalophilin may be the reason for the abnormal vitamin B(12) absorption observed in patients with pancreatic insufficiency.

Porcine serum cobalophilin and transcobalamin. Identification, isolation and properties including electrofocusing patterns.

Pooled porcine serum was found to contain cobalophilin (also called transcobalamin I) and transcobalamin (also called transcobalamin II). The two proteins were harvested by batchwise absorption with vitamin B-12 covalently coupled to Sepharose, and then separated from each other either by gel filtration or using an immunoadsorbent. Both proteins were finally isolated as single proteins using a second vitamin B-12-Sepharose chromatography step. Cobalophilin and transcobalamin complexed with vitamin B-12 had molecular weights by gel filtration of 135 000 and 38 000 and by the formula of Svedberg 104 000 and 44 000, Stokes radii 4.97 nm and 2.65 nm, and sedimentation coefficients 5.39 S and 3.75 S, respectively. Electrofocusing resolved the cobalophilin complex into three main isoproteins isoelectric at pH 3.23, 3.42 and 3.69, and transcobalamin into only the main component isoelectric at a value as low as pH 3.47. Neither protein was capable of binding to the ileal intrinsic factor receptor.

Sequential affinity chromatography for the purification of antigens extracted from Onchocerca volvulus adult worms.

Adult Onchocerca volvulus worms were extracted sequentially with buffers of various ionic strengths. The extract was incubated with purified human onchocerciasis immunoglobulin-G (IgG) convalently coupled to Sepharose. Antigens were then eluted with 8 M urea and 7.5 M guanidine-HCl. IgG contained in the eluates was removed by incubating the eluates with rabbit anti-human IgG covalently coupled to Sepharose. As demonstrated by double immunodiffusion and "double" crossed immunoelectrophoresis at least three antigens were isolated. Most of the antigens were totally excluded from Sephadex G-200 but entered the included volume of Sepharose 6B. In electrofocusing they focused in acid regions. Antibodies were generated in rabbits against the three antigens isolated together. The antibodies were covalently coupled to Sepharose, which was subsequently used as a new affinity medium for the isolation of antigens. Such isolated antigens appeared to be identical with those with human onchocerciasis IgG. The isolated antigens and their corresponding artificial antibodies generated in rabbits were successfully applied in the enzyme linked immunoadsorbent assay. Host material and onchocerciasis IgG left behind during the purification procedure interfered in the assay system.

Isolation of vitamin B-12-binding proteins by combined immuno and affinity chromatography. Comparative studies on the isolated and unisolated proteins.

Intrinsic factor or cobalophilin were removed by incubating human gastric juice and pig pyloric extract with purified anti-intrinsic factor and anti-cobalophilin immunoglobulin-G, respectively, covalently coupled to Sepharose. Cobalophilin (transcobalamin I) was also removed from pig serum either by using anti-cobalophilin immunoglobulin-G Sepharose or by Sephadex G-200 chromatography. The one remaining semipurified vitamin B-12-binding protein (intrinsic factor, cobalophilin or transcobalamin II) was then isolated by vitamin B-12-Sepharose affinity chromatography. Intrinsic factors, cobalophilins and transcobalamin II isolated by this two-step procedure were compared by double isotope techniques with the corresponding protein not subjected to affinity chromatography and found to be identical in reaction to antiserum, gel filtration and electrofocusing. The avidity of the isolated and unisolated intrinsic factors for the ileal intrinsic factor receptor was also the same.