Stimulation of growth in human and murine cells by adriamycin.

Adriamycin causes a variety of biological actions and is an effective cytotoxic agent against proliferating cells. In this paper we show that the drug is not limited in its action solely to cytotoxicity, but can also stimulate cell growth under the appropriate conditions. Using the survival assay of cloning in soft agar, we present data showing that the conditions for Adriamycin-induced growth stimulation are that the drug be in a subtoxic concentration range of 10(-10)-10(-9) M (greater than 10(-8) M causes cytotoxicity) and that the growth medium be suboptimal. This latter condition is satisfied by either growing cells for an extended period in order to exhaust the growth supporting capacity of the medium, or by growing the cells at low (less than 10%) serum concentrations. Several active anthracycline congeners also have the ability to stimulate growth. The results indicate that the cytotoxic anticancer agent Adriamycin can stimulate the proliferation of some cells.

Adriamycin: protection from cell death by removal of extracellular drug.

Adriamycin is a cytotoxic drug which has enjoyed considerable success in the treatment of cancer. This agent has a bewildering variety of biological effects both within and on the surface of cells exposed to drug, and it has proved difficult to unambiguously assign a single mechanism of action. In this report we are able to separate intracellular and extracellular actions by taking advantage of the complete lack of Adriamycin-induced cytotoxicity at low temperature. For example, cells exposed to 100 microM Adriamycin at 0 degree C are not killed by the drug, even though this concentration is orders of magnitude higher than the concentration needed to cause 100% cell death at 37 degrees C. If cells exposed to 100 microM Adriamycin at 0 degree C are shifted to fresh drug-free medium at 37 degrees C, there is a time-dependent decrease in survival. However, if the drug-free medium contains calf thymus DNA (1.5 mg/ml) to act as a reservoir for Adriamycin binding of effluxed drug, there is no ensuing cytotoxicity. Thus, the results show that no matter how much drug is present inside the cell, there must also be extracellular drug available for membrane interaction in order to initiate nuclear DNA damage and the cytotoxic cascade.

Protection from adriamycin cytotoxicity in L1210 cells by brefeldin A.

We present studies which suggest that the cytotoxic action of Adriamycin (ADR) may involve intracellular pathways of vesicular transport. The movement of proteins or lipids from the endoplasmic reticulum to the plasma membrane via the Golgi organelle and associated compartments exhibits several temperature-sensitive steps between 15 degrees C and 20 degrees C. In this same temperature range, ADR loses its cytotoxic capacity. Using the inhibitor brefeldin A (BFA), we have investigated the possible role of intravesicular trafficking in the loss of ADR activity and the induction of protection from cytotoxicity at low temperature in L1210 cells. We show here that cells pretreated at 37 degrees C for 2 h with BFA could be protected from a subsequent exposure to ADR. The concentration causing 50% inhibition, determined by cloning in soft agar, was increased approximately 3.5 fold. L1210 cells could also be protected from the topoisomerase II inhibitors etoposide and amsacrine, but to a lesser extent; the concentration causing 50% inhibition for the latter inhibitors were increased 2-fold. Spectrofluorometric analysis of intracellular ADR accumulation revealed that there was no significant difference in the level of ADR in cells with or without BFA pretreatment. In addition, examination of ADR-induced cleavable complex formation by alkaline elution showed no significant difference in the level of DNA strand breaks in cells which had been pretreated with BFA even though there was a large difference in survival. Further examination of the persistence of DNA damage after a period of up to 6 h of repair revealed that cells which were pretreated with BFA removed DNA strand breaks at rates equivalent to those of cells which had received ADR directly. These results suggest that the protective effect induced by brefeldin A does not involve uptake, DNA damage, or repair but instead implicates protein or lipid interactions which may be independent of DNA damage and which may influence critical events that take place after the topoisomerase II-DNA complex has been formed.

Cell surface effects of adriamycin and carminomycin immobilized on cross-linked polyvinyl alcohol.

Previous reports have claimed Adriamycin to be cytotoxic to cultured tumor cells when the drug is covalently immobilized on a solid support, thus suggesting a cell surface mechanism of action for the drug. Although these previous reports attempted to rule out released drug or endocytosis of drug-support particles as alternative explanations for the observed cytotoxicity, a more thorough analysis is necessary to substantiate fully the cell surface idea. In the present work, the stability of the drug-support linkage was increased by use of cross-linked polyvinyl alcohol as the support and cyanuric chloride or a diazonium salt for attachment of the drug. Different anthracycline orientations were tested by coupling Adriamycin at the amino sugar and carminomycin at the D-ring. The Adriamycin cross-linked polyvinyl alcohol and carminomycin cross-linked polyvinyl alcohol preparations had much lower drug release rates than did the earlier used carbamate-linked Adriamycin cross-linked agarose materials. All three immobilized drug preparations inhibited the growth of L1210 or S180 clones following 2- or 20-h incubation with cells at 37 degrees C. The results strongly support the concept that immobilized anthracyclines can be cytotoxic to cultured cells, for at least two different orientations of the drug on the support.

Protein kinase C in adriamycin action and resistance in mouse sarcoma 180 cells.

Adriamycin has a wide variety of biological actions on susceptible cells, several of which may be integrally involved in cytotoxicity. In this paper, we present evidence that one of the alterations in cell function that occurs in the presence of Adriamycin is an elevation in the production of diacylglycerol. The effect is rapid, reaches a peak within 10 min of exposure of Sarcoma 180 cells to Adriamycin, and can thus be classified among the earliest alterations that occur in cells damaged by Adriamycin. Concomitant with the rise in diacylglycerol is an increase in cytosolic protein kinase C activity. Although Adriamycin does not appear to modulate the activity of this enzyme by direct binding, drug-exposed Sarcoma 180 cells have a 56% increase in intrinsic cytosolic protein kinase C (PKC) activity, with no change in the activity of the membrane form. Experiments with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate suggest that the PKC effect is linked to Adriamycin action, since activation of the enzyme by short 12-O-tetradecanoylphorbol-13-acetate exposure enhances Adriamycin's cytotoxicity as well as its ability to provoke DNA damage (measured by alkaline elution). Likewise, down-regulation of PKC by extended 12-O-tetradecanoylphorbol-13-acetate exposure partially protects the cells from Adriamycin-induced cytotoxicity as well as from DNA damage. Thus, the ability of cells to be injured by Adriamycin appears to be correlated with the activity of PKC. Multidrug-resistant subline Sarcoma 180A10 cells have the same total quantity of membrane-recruitable PKC as the sensitive parent Sarcoma 180 cells, as determined by [3H]phorbol-12,13-dibutyrate binding. However, the resistant cells have a significantly higher intrinsic PKC activity and an altered ability to translocate the enzyme to the cell surface. Taken together, the results raise the possibility that cell signaling mechanisms, particularly those involving protein kinase C, may play an important role in mediating the biological action of the anticancer drug Adriamycin.

Temperature dependence of adriamycin-induced DNA damage in L1210 cells.

We report alkaline elution experiments that reveal the temperature dependence of DNA lesions, both single-strand breaks and DNA-protein cross-links, in L1210 cells exposed to Adriamycin. DNA damage, which at 37 degrees C is equivalent to several hundred rads of ionizing radiation exposure, diminishes as the temperature of drug exposure is lowered. At all temperatures below about 15 degrees C no DNA damage is detectable in L1210 cells exposed to Adriamycin, even at relatively high doses. The low temperature inactivity is not due to a redistribution of intracellular drug since at both 37 and 0 degrees C there is a high concentration of Adriamycin in both nuclear and cytoplasmic locations. The temperature profile for DNA damage parallels the profile for cytotoxicity, i.e., at low temperature, the drug is completely inactive as a cytotoxic agent (P. Lane, P. Vichi, D. L. Bain, and T. R. Tritton, Cancer Res., 47:4038-4042, 1987). Thus, DNA breaks and cell kill appear to be correlated with one another. However, when we examined DNA lesions in nuclei isolated from L1210 cells we found that the low temperature inability to sustain Adriamycin-induced single-strand breaks or DNA-protein cross-links was absent. In nuclei, then, the drug can provoke DNA damage at low temperature, while in whole cells it cannot. Topoisomerase II, an enzyme implicated in catalyzing DNA lesions in cells exposed to intercalating agents, retains its catalytic activity both to unknot P4 DNA at 0 degrees C, and to be induced by drug to alter the release of pBR322 supercoils, so a low temperature inactivation of this enzyme cannot explain the results. We propose that intact L1210 cells have a regulatory factor which controls DNA damage, possibly through topoisomerase II, but which is lost when nuclei are isolated.

Photoaffinity labeling of the Sarcoma 180 cell surface by daunomycin.

We have used photoaffinity labeling to investigate the distribution and function of daunomycin binding sites in Sarcoma 180 cells. When native daunomycin is irradiated at 366 or 488 nm in the presence of cells, the drug is irreversibly incorporated into cellular molecules. The cellular acceptor for the photoincorporation cannot be extracted by chloroform-methanol nor can it be degraded by DNase. However, the drug acceptor is susceptible to trypsin digestion. These results show that the photoincorporation site is composed of protein but not of lipid or DNA. Furthermore, the fact that photoincorporation proceeds equally well at 0 degrees (where drug does not accumulate inside the cells) as compared to 37 degrees (where free drug concentrates in the cells) suggests that the labeling reaction occurs principally at the cell surface. The photolabeling process is not highly specific since it is not saturable at high drug concentrations and cannot be competed for by unlabeled daunomycin. When 2 X 10(5) daunomycin molecules are incorporated per Sarcoma 180 cell, the cells can still accumulate free drug. This result suggests that the photolabeling reaction does not occur at the drug transport locus. Photoincorporation of daunomycin also does not affect the viability of Sarcoma 180 cells, as judged by a cloning assay. Thus, there is probably no surface receptor for the drug which mediates cytotoxicity when occupied. This result is as expected from previous work predicting that the mechanism of daunomycin involves disruption of some generalized membrane property like fluidity. However, in a series of Sarcoma 180 sublines selected for increasing resistance to daunomycin, the photoincorporation increases in direct proportion to drug sensitivity. Consequently, daunomycin appears to be capable of photoaffinity labeling a cell surface protein which, although not directly involved in the mechanism of cytotoxicity is implicated in the expression of drug resistance.

Cell surface-directed interaction of anthracyclines leads to cytotoxicity and nuclear factor kappaB activation but not apoptosis signaling.

Anthracyclines are, above all, DNA intercalators, which induce genetic damage leading to cell death. However, increasing evidence firmly suggests that the underlying mechanism for anthracycline cytotoxicity is the induction of apoptosis through intracellular-mediated signaling pathways. Whether drug/DNA interaction is necessary for such apoptosis signaling is unknown. We investigated the cellular effects of the anthracyclines daunorubicin (DNR) and doxorubicin (DOX) using the myeloid leukemia cell line U937. By comparing free drug against agarose bead-immobilized drug iDNR and iDOX (which cannot accumulate within the cell), we observed that whereas both free and immobilized anthracyclines were cytotoxic, only the former induced apoptosis; the latter induced necrosis. Indeed, we did not observe ceramide generation, neutral sphingomyelinase activation, poly (ADP-ribose) polymerase cleavage, or other apoptotic events with iDNR or iDOX. However, both free and immobilized drug were similarly capable of triggering nuclear factor kappaB activation. These observations demonstrate that whereas activation of certain cellular signaling pathways can be achieved solely through membrane interaction, apoptosis signaling requires anthracycline internalization. These results also show that the initiation of cell survival pathways (illustrated by nuclear factor kappaB activation) is independent of intracellular drug/target interaction.

Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor.

Asbestos fibers are human carcinogens with undefined mechanisms of action. In studies here, we examined signal transduction events induced by asbestos in target cells of mesothelioma and potential cell surface origins for these cascades. Asbestos fibers, but not their nonfibrous analogues, induced protracted phosphorylation of the mitogen-activated protein (MAP) kinases and extracellular signal-regulated kinases (ERK) 1 and 2, and increased kinase activity of ERK2. ERK1 and ERK2 phosphorylation and activity were initiated by addition of exogenous epidermal growth factor (EGF) and transforming growth factor-alpha, but not by isoforms of platelet-derived growth factor or insulin-like growth factor-1 in mesothelial cells. MAP kinase activation by asbestos was attenuated by suramin, which inhibits growth factor receptor interactions, or tyrphostin AG 1478, a specific inhibitor of EGF receptor tyrosine kinase activity (IC50 = 3 nM). Moreover, asbestos caused autophosphorylation of the EGF receptor, an event triggering the ERK cascade. These studies are the first to establish that a MAP kinase signal transduction pathway is initiated after phosphorylation of a peptide growth factor receptor following exposure to asbestos fibers.

Effects of anthracyclines on oxygenated and hypoxic tumor cells.

The cytotoxic effects of anthracyclines and other chemotherapeutic agents were examined in normally aerated and hypoxic Sarcoma 180 and EMT6 tumor cells in vitro. Adriamycin, daunomycin, and mitomycin C were selectively toxic to hypoxic Sarcoma 180 cells. The augmented sensitivity was not the result of an increase in susceptibility of oxygen-deprived cells toward antitumor agents in general. 1,3-Bis(2-chloroethyl)-1-nitrosourea, for example, exhibited equal cytotoxicity toward normally aerated and hypoxic cells, while streptonigrin was selectively toxic toward normally aerated cells. The cellular levels of [3H]daunomycin in both Sarcoma 180 and EMT6 cells were not different under the two conditions of oxygenation, and no greater production of either the alcohol or aglycone metabolites of daunomycin occurred in hypoxic cells, compared with their normally aerated counterparts. In addition, analysis of cellular pellets for residual drug remaining after exhaustive extraction showed no significant difference between normally aerated and hypoxic cells. The effects of reoxygenation of hypoxic cells on their sensitivity to mitomycin C and to Adriamycin were studied in both Sarcoma 180 and EMT6 cells. The enhanced efficacy of mitomycin C as a cytotoxic agent observed under hypoxia was reversed after a 2-hr reoxygenation. In contrast, the augmented toxicity of Adriamycin toward hypoxic cells was not reversible in either cell line after 2 or 4 hr of reoxygenation. The results suggest that neither the formation of a reactive oxygen species nor direct involvement of an alkylating agent generated by drug metabolism is an obligatory step in the cytotoxic action of these anthracyclines.

Temperature dependence studies of adriamycin uptake and cytotoxicity.

In order to learn whether a direct relationship exists between cellular uptake and cytotoxicity of Adriamycin, we have compared the temperature dependencies of these two processes in L1210 cells. We find that the equilibrium concentration of drug taken inside the cells varies smoothly with temperature between 37 degrees C and 0 degree C. Even at 0 degree C, however, there is still measurable uptake of the drug into cells. The cytotoxicity index (cloning in soft agar), on the other hand, does not parallel the uptake temperature dependence. Cytotoxicity rapidly diminishes as the temperature of drug exposure is lowered; at all temperatures below about 20 degrees C, Adriamycin is not active. In contrast, other cytotoxic anticancer drugs like mitomycin C, bleomycin, and ARK 73-21 (a platinum analogue) retain cytotoxic potency at low temperatures. The inability of Adriamycin to kill cells at low temperature persists even at very high drug concentrations where substantial quantities of drug enter the cells. The low temperature impotence is not a result of inoperative enzymes which could metabolize Adriamycin to an alkylating species or electron donor to oxygen, since NADH and NADPH dependent reductase activities show linear Arrhenius behavior with no indication of low temperature inactivity. Using purified L1210 plasma membranes with bound Adriamycin as a fluorescence polarization probe, we find evidence of a phase change in the cell surface occurring at the same temperature as the loss of biological activity (approximately equal to 20 degrees C). We conclude that Adriamycin induced cytotoxicity is not dictated solely by uptake, in apparent contradiction with mechanisms requiring an intracellular target. Moreover, the loss of cytotoxicity below 20 degrees C appears to be linked to a structural change in the cell surface membrane, supporting a role other than transport for this membrane in transducing Adriamycin action.

The interaction of adriamycin with small unilamellar vesicle liposomes. A fluorescence study.

The interaction of the antineoplastic agent adriamycin with sonicated liposomes composed of phosphatidylcholine alone and with small amounts (1-6%) of cardiolipin has been studied by fluorescence techniques. Equilibrium binding data show that the presence of cardiolipin increases the amount of drug bound to liposomes when the bilayer is below its phase transition temperature and when the ionic strength is relatively low (0.01 M). At higher ionic strength (0.15 M) and above the Tm (i.e. conditions which are closer to the physiological state) the binding of the drug to the two liposome types is nearly the same. Thus the differences in the interactions of adriamycin with cardiolipin-containing membranes, as opposed to those composed of phosphatidylcholine alone, are not due simply to increased binding but rather to an altered membrane structure when this lipid is present. Quenching of adriamycin fluorescence by iodide shows that bound drug is partially, but not completely, buried in the liposomal membrane. Both in the presence and absence of cardiolipin the bulk of the adriamycin is more accessible to the quencher below the Tm than above it; that is, a solid membrane tends to exclude the drug from deep penetration. Above the Tm, the presence of cardiolipin alters the nature of liposome-adriamycin interaction. Here the fluorescence quenching data suggest that the presence of small amounts of cardiolipin (3%) in a phosphatidylcholine matrix creates two types of binding environments for drug, one relatively exposed and the other more deeply buried in the membrane. The temperature dependence of the adriamycin fluorescence and the liposome light scattering reveal that cardiolipin alters the thermal properties of the bilayer as well as its interaction with adriamycin. At low ionic strength lateral phase separations may occur with both pure phosphatidylcholine and when 3% cardiolipin is present; under these conditions the bound adriamycin exists in two kinds of environment. It is notable that only adriamycin fluorescence reveals this phenomenon; thebulk property of liposome light scattering reports only on the overall membrane phase change. These data suggest that under certain conditions the drug binding sites in the membranes are decoupled from the bulk of the lipid bilayer.

Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells.

Adriamycin increases (a) the rate of agglutination of Sarcoma 180 cells by concanavalin A after brief exposure of 2-3 h and (b) membrane fluidity as measured by ESR within 30 min of exposure at concentrations of the anthracycline of 10(-7)-10(-5) M. The effect of adriamycin on agglutination is not due to an increase in the number of surface receptors for concanavalin A, since the extent of binding of the lectin is not altered by adriamycin and no change occurs in the rate of occupancy of the concanavalin A binding sites by the lectin in cells treated with the antibiotic. The order parameter, a measurement of membrane fluidity, decreases in cells exposed to adriamycin and is dose-related. The results indicate that adriamycin can induce changes in the surface membrane of Sarcoma 180 cells within a brief period of exposure to a low but cytotoxic level of this agent.

Cell surface actions of adriamycin.

Adriamycin has a vast range of reported actions on the structural and functional properties of cells. This review summarizes the literature on the ability of the drug to modulate the cell surface membrane and attempts to address the question of how such actions could be linked to cytotoxicity. In addition, we consider the use of polymer immobilization of adriamycin to separate intracellular from plasma membrane effects of the drug, and show how this approach has been helpful in interpreting the pharmacology of adriamycin. Finally, a range of biophysical and spectroscopic approaches to defining the molecular details of adriamycin-bilayer interactions is surveyed, and the results used to discuss a model for how this antineoplastic agent binds to membranes.

Cytotoxicity and differentiating actions of adriamycin in WEHI-3B D+ leukemia cells.

The monomyelocytic leukemia WEHI-3B D+ can be induced to differentiate into mature granulocytes in suspension culture when exposed to 40 nM adriamycin. Treated cells underwent approximately two divisions prior to reaching plateau phase, with approximately 55% of the cell population expressing nitro blue tetrazolium positivity (NBT+) by day 3. Decreased cellular proliferation was paralleled by a progressive increase in morphologically mature granulocytic cells. Maturation was also characterized by a 4.4-fold increase in Fc receptors on the cell surface. An increase in the size of adriamycin-treated cells occurred and correlated with residency in the G2M phase of the cell cycle. Adriamycin-induced NBT+ cells, which contained the highest levels of Fc receptors, were also found to reside in G2M. Adriamycin blocked cells in the G2M phase of the cell cycle by 8 hr (125% above control), and this arrest reached its maximum by 20 hr (194% above control). Concomitant with the block in the cell cycle was the commitment by these cells within 8 hr to the granulocytic pathway of differentiation. Fractionation of cells by centrifugal elutriation into enriched phases of the cell cycle was consistent with the hypothesis that induction of the differentiation program was initiated either in G1 or very late in the cell cycle. Immobilized adriamycin, which does not gain access to the cell interior, did not induce the maturation of WEHI-3B D+ cells, nor did it block their replication in a specific phase of the cell cycle; however, immobilized adriamycin was 30-fold more toxic to WEHI-3B D+ cells than free drug. Incubation of WEHI-3B D+ cells with the semisynthetic adriamycin analog N-trifluoroacetyl adriamycin-14-valerate (AD-32) resulted in approximately 50% of the cell population being NBT+ by day 3. The findings suggest that adriamycin must be able to enter cells to induce maturation, and that at least some portion of its toxicity is associated with an effect at the surface membrane. Furthermore, the results obtained with AD-32 imply that intercalation into DNA is not necessary for induction of the differentiated phenotype.

Activity of two novel anthracene-9,10-diones against human leukemia cells containing intercalator-sensitive or -resistant forms of topoisomerase II.

We have examined the activities of two novel aza-anthracene-9,10-diones (aza), 1-aza and 2-aza, in HL-60 human leukemia cell lines containing type II topoisomerases with different sensitivities to inhibition by other intercalating agents. The sensitive line, HL-60, was sensitive to 2-aza but not to 1-aza, whereas the resistant HL-60/AMSA was sensitive to neither agent. Measurements of 1- and 2-aza-induced, topoisomerase II-mediated DNA cross-linking in the cells revealed patterns of resistance and sensitivity that paralleled the results in the cytotoxicity assays. However, measurements of drug-induced topoisomerase II-mediated DNA cross-linking using purified HL-60 and HL-60/AMSA topoisomerase II indicated that both agents could stabilize a covalent complex between DNA and the HL-60 enzyme. HL-60/AMSA topoisomerase II resisted stabilization by either agent. This suggests that the resistance of HL-60 cells to 1-aza is not due to the inability of this drug to inhibit topoisomerase II but rather to another, undefined mechanism.

Correlation of altered tyrosine phosphorylation with methotrexate resistance in a cisplatin-resistant subline of L1210 cells.

Collateral resistance to cisplatin and methotrexate has been reported in several cell lines. A murine leukemia cell line (L1210/DDP) selected for cisplatin resistance also has been shown to be highly resistant to methotrexate. Of the mechanisms proposed for methotrexate resistance, only changes in methotrexate transport into the cells were found in an earlier report. Methotrexate enters mammalian cells via an active transport system. In the present study, we demonstrated that the transport into the cell may be impaired in the resistant cells due to altered tyrosine phosphorylation of a membrane protein with a molecular mass of 66 kDa. This alteration was manifested by altered tyrosine phosphorylation of the 66 kDa protein and may be an underlying modification that renders the cells resistant to methotrexate. These results suggest involvement of tyrosine phosphorylation in folate transport and methotrexate resistant in L1210/DDP cells.

Differential mechanisms of multidrug resistance are expressed during stepwise selection of KB-3-1 cells with adriamycin.

A major limitation of cancer chemotherapy is development of resistance. In this study, we analyzed KB-3-1 cells and adriamycin-selected multidrug resistant sublines KB-A1 and KB-A10 cells for mechanisms of resistance. KB-A10 cells are 10-fold more resistant than KB-A1 cells but have lower P-glycoprotein. Of the known mechanisms of multidrug resistance, topoisomerase II and lung-resistance-related protein were altered between the resistant cell lines. Glutathione-S-transferase activity and multidrug-resistance-related protein levels were higher in the resistant cell lines compared to the sensitive cells but were similar in KB-A1 and KB-A10 cells. Results indicate differential regulation of mechanisms of resistance with stepwise selection.

Integrated learning. Passing fad or foundation for the future?

On the surface, higher education has served the nation well. An increasing percentage of adults have attended college. Information is widely and freely available to most citizens; the economy is steady; and technological developments appear to offer ever-increasing improvements in the quality of life. Looking just beneath the surface, though, one realizes that knowledge is fragmented, access to knowledge is not universal, and there are no guarantees that past successes will sustain the future. Thus, it is worth examining how higher learning has contributed to the general prosperity and how education might evolve to address society's evident problems. This essay discusses the core approach of liberal arts in the context of the value--and obstacles--of integration across the traditional disciplines. Critical to the natural sciences is a firm grounding in the central importance of rigorous evidence, while the humanities keep us rooted in the importance of human values. Seeking linkages and connections between these realms is the lively challenge of this conference. The details of curricular design will be of less interest at this juncture than locating the critical issues and discussing how education might serve the goal of unifying knowledge and learning.