Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium.

Negative regulation of diacylglycerol kinase theta mediates adenosine-dependent hepatocyte preconditioning.

In liver ischemic preconditioning (IP), stimulation of adenosine A2a receptors (A2aR) prevents ischemia/reperfusion injury by promoting diacylglycerol-mediated activation of protein kinase C (PKC). By concerting diacylglycerol to phosphatidic acid, diacylglycerol kinases (DGKs) act as terminator of diacylglycerol signalling. This study investigates the role of DGK in the development of hepatocyte IP. DGK activity and cell viability were evaluated in isolated rat hepatocytes preconditioned by 10 min hypoxia followed by 10 min re-oxygenation or by the treatment with the A2aR agonist, CGS21680, and subsequently exposed to prolonged hypoxia. We observed that after IP or A2aR activation, a decrease in DGK activity was associated with the onset of hepatocyte tolerance to hypoxia. CGS21680-induced stimulation of A2aR specifically inhibited DGK isoform theta by activating RhoA-GTPase. Consistently, both siRNA-mediated downregulation of DGK theta and hepatocyte pretreatment with the DGK inhibitor R59949 induced cell tolerance to hypoxia. The pharmacological inhibition of DGK was associated with the diacylglycerol-dependent activation of PKC delta and epsilon and of their downstream target p38 MAPK. In conclusion, we unveil a novel signalling pathway contributing to the onset of hepatocyte preconditioning, which through RhoA-GTPase, couples A2aR to the downregulation of DGK. Such an inhibition is essential for the sustained accumulation of diacylglycerol required for triggering PKC-mediated survival signals.

Diacylglycerol kinase-alpha phosphorylation by Src on Y335 is required for activation, membrane recruitment and Hgf-induced cell motility.

Diacylglycerol (DAG) kinases (Dgk), which phosphorylate DAG to generate phosphatidic acid, act as either positive or negative key regulators of cell signaling. We previously showed that Src mediates growth factors-induced activation of Dgk-alpha, whose activity is required for cell motility, proliferation and angiogenesis. Here, we demonstrate that both hepatocytes growth factor (HGF) stimulation and v-Src transformation induce tyrosine phosphorylation of Dgk-alpha on Y335, through a mechanism requiring its proline-rich C-terminal sequence. Moreover, we show that both proline-rich sequence and phosphorylation of Y335 of Dgk-alpha mediate: (i) its enzymatic activation, (ii) its ability to interact respectively with SH3 and SH2 domains of Src, (iii) its recruitment to the membrane. In addition, we show that phosphorylation of Dgk-alpha on Y335 is required for HGF-induced motility, while its constitutive recruitment at the membrane by myristylation is sufficient to trigger spontaneous motility in absence of HGF. Providing the first evidence that tyrosine phosphorylation of Dgk-alpha is required for growth-factors-induced activation and membrane recruitment, these findings underscore its relevance as a rheostat, whose activation is a threshold to elicit growth factors-induced migratory signaling.

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx.

Anticancer drugs generally have intracellular targets, implicating transport over the plasma membrane. For amphiphilic agents, such as the anthracycline doxorubicin, this occurs by passive diffusion. We investigated whether exogenous membrane-permeable lipid analogues improve this drug influx. Combinations of drugs and lipid analogues were coadministered to cultured endothelial cells and various tumour cell lines, and subsequent drug accumulation in cells was quantified. We identified N-hexanoyl-sphingomyelin (SM) as a potent enhancer of drug uptake. Low micromolar amounts of this short-chain sphingolipid, being not toxic itself, enhanced the uptake of doxorubicin up to 300% and decreased its EC(50) toxicity values seven- to 14-fold. N-hexanoyl SM acts at the level of the plasma membrane, but was found not incorporated in (isolated) lipid rafts, and artificial disruption or elimination of raft constituents did not affect its drug uptake-enhancing effect. Further, any mechanistic role of the endocytic machinery, membrane leakage or ABC-transporter-mediated efflux could be excluded. Finally, a correlation was established between the degree of drug lipophilicity, as defined by partitioning in a two-phase octanol-water system, and the susceptibility of the drug towards the uptake-enhancing effect of the sphingolipid. A clear optimum was found for amphiphilic drugs, such as doxorubicin, epirubicin and topotecan, indicating that N-hexanoyl-SM might act by modulating the average degree of plasma membrane lipophilicity, in turn facilitating transbilayer drug diffusion. The concept of short-chain sphingolipids as amphiphilic drug potentiators provides novel opportunities for improving drug delivery technologies.

Sphingolipids related to apoptosis from the point of view of membrane structure and topology.

Sphingolipids and their metabolites are implicated in signal transduction, but the mechanisms are still poorly understood. In particular, the presumed function of ceramide as a second messenger remains controversial. Here, we emphasize the importance of both ceramide and sphingomyelin for membrane structure. The effects of sphingolipid turnover in the induction and effector phases of apoptosis are explained by their impact on membrane microdomains that are relevant for cell signalling or changes in morphology. The topology of sphingolipid metabolism is important because of their limited transbilayer and inter-membrane movement. For instance, glycosylceramide synthase converts de novo synthesized ceramide to glycosylceramide, but it is neither a general attenuator of ceramide accumulation at the plasma membrane, nor of the apoptotic process. Synthetic alkyl-lysophospholipids modulate membrane-lipid composition and, therefore, apoptosis sensitivity.

Alkyl-lysophospholipids as anticancer agents and enhancers of radiation-induced apoptosis.

Synthetic alkyl-lysophospholipids (ALPs, also referred to as ether-phospholipids) have been studied as antitumor agents for more than a decade. Classical examples of these ALPs include 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); Edelfosine) and hexadecylphosphocholine (HePC; Miltefosine). Unlike most currently available chemotherapeutic drugs that target the nuclear DNA, ALPs exert their action at the plasma membrane level, where they interfere with mitogenic signal transduction pathways. Whereas malignant cells are highly sensitive to the lethal action of ALPs, normal cells remain relatively unaffected, illustrating the potential selective antitumor properties of this class of drugs. Recently, ALPs have regained interest because of their capacity to induce apoptosis in various tumor cell lines. Moreover, in combination with other (conventional) anticancer regimens, ALPs seem to cause an additive and sometimes synergistic cytotoxic effect. These biologic properties make ALPs attractive drugs for further clinical evaluation. The present review discusses recent insights into the mode(s) of action of ALPs, their interaction with ionizing radiation, and clinical application.

Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis.

We previously showed that ceramide (Cer) formed during the execution phase of apoptosis is derived from plasma membrane sphingomyelin (SM), most likely by a neutral sphingomyelinase activity (Tepper et al., J. Cell Biol. 150, 2000, 155-164). In this study, we investigated the involvement of a cloned putative human neutral sphingomyelinase (nSMase1) in this process. Site-directed mutagenesis of predicted catalytic residues (Glu(49), Asn(180), and His(272)) to Ala residues abolished the catalytic activity of nSMase1. Jurkat cells were retrovirally transduced with either wildtype or inactive (with all three point mutations) Myc-tagged nSMase1. Cells overexpressing wildtype nSMase1 showed dramatically elevated in vitro nSMase activity. However, nSMase1 gene transduction (wildtype or mutant) did not alter steady-state levels of SM, Cer, or glucosylceramide. Moreover, the Cer response and apoptosis sensitivity to ligation of the CD95/Fas receptor in cells overexpressing wildtype or mutant nSMase1 were identical to vector-transduced cells. We conclude that not nSMase1 but a different, yet to be identified, nSMase accounts for the generation of Cer during the execution phase of death receptor-induced apoptosis.

Properties and functions of diacylglycerol kinases.

Diacylglycerol kinases (DGKs) phosphorylate the second-messenger diacylglycerol (DAG) to phosphatidic acid (PA). The family of DGKs is well conserved among most species. Nine mammalian isotypes have been identified, and are classified into five subgroups based on their primary structure. DGKs contain a conserved catalytic domain and an array of other conserved motifs that are likely to play a role in lipid-protein and protein-protein interactions in various signalling pathways dependent on DAG and/or PA production. DGK is therefore believed to be activated at the (plasma) membrane where DAG is generated. Some isotypes are found associated with and/or regulated by small GTPases of the Rho family, presumably acting in cytoskeletal rearrangements. Others are (also) found in the nucleus, in association with other regulatory enzymes of the phosphoinositide cycle, and have an effect on cell cycle progression. Most DGK isotypes show high expression in the brain, often in distinct brain regions, suggesting that each individual isotype has a unique function.

Glucosylceramide synthase does not attenuate the ceramide pool accumulating during apoptosis induced by CD95 or anti-cancer regimens.

Ceramide (Cer) accumulating during the execution phase of apoptosis is generated from plasma membrane sphingomyelin (SM), which gains access to a sphingomyelinase due to phospholipid scrambling (Tepper, A. D., Ruurs, P., Wiedmer, T., Sims, P., Borst, J., and van Blitterswijk, W. J. (2000) J. Cell. Biol. 150, 155-164). To evaluate the functional significance of this Cer pool, we aimed to convert it to glucosylceramide (GlcCer), by constitutive overexpression of glucosylceramide synthase (GCS). Jurkat cells, retrovirally transduced with GCS cDNA, showed a 10-12-fold increase in GCS activity in vitro and a 7-fold elevated basal GlcCer level in vivo. However, Cer accumulating during apoptosis induced by ligation of the death receptor CD95, treatment with the anti-cancer drug etoposide, or exposure to gamma-radiation was not glycosylated by GCS. Likewise, Cer liberated at the plasma membrane by bacterial SMase was not converted by the enzyme. Thus, GCS, located at the Golgi, is topologically segregated from Cer produced in the plasma membrane. In contrast, de novo synthesized Cer as well as an exogenously supplied cell-permeable Cer analog were efficiently glycosylated, apparently due to different Cer topology and distinct physicochemical behavior of the synthetic Cer species, respectively. Exogenous cell-permeable Cer species, despite their conversion by GCS, effectively induced apoptosis. We also observed that GCS activity is down-regulated in cells undergoing apoptosis. In conclusion, GCS can convert de novo synthesized Cer but not SM-derived Cer, and, therefore, the ability of GCS overexpression to protect cells from possible detrimental effects of Cer accumulation is limited.

Sphingomyelin hydrolysis to ceramide during the execution phase of apoptosis results from phospholipid scrambling and alters cell-surface morphology.

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-sphingosylphosphorylcholine (C(6)-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca(2+) ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-beta-cyclodextrin, which is indicative of a loss of tight SM-cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C(6)- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.

Protein kinase C activation by acidic proteins including 14-3-3.

14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-zeta and facilitate coupling of PKC-zeta to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-zeta activity. Using recombinant 14-3-3 proteins and purified PKC-zeta in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-zeta activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-zeta by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-zeta with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-zeta. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-zeta activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-zeta, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.

Diacylglycerol kinases in signal transduction.

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. A family of nine mammalian isotypes have been identified. Their primary structure shows a diverse array of conserved domains, such as a catalytic domain, zinc fingers, pleckstrin homology domains and EF-hand structures, known to interact with other proteins, lipids or Ca2+, in signal transduction processes. DGK is believed to act in the phosphoinositide cycle in which DAG is enriched with arachidonoyl moieties, but the majority of DGK isotypes do not show specificity for this DAG species in vitro. This could imply that DGKs may also have other functions in the cell. DGK activity is not only found in membranes, but also in the nucleus and at the cytoskeleton. Agonist-induced translocations of DGK to or from these subcellular sites are known to occur. Some isotypes are contained in signaling complexes in specific association with members of the Rho family of small GTP binding proteins, suggesting that they are involved in Rho-mediated processes such as cytoskeletal reorganization.

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.

Alkyl-lysophospholipids activate the SAPK/JNK pathway and enhance radiation-induced apoptosis.

Alkyl-lysophospholipids (ALPs) represent a new class of antitumor drugs that induce apoptotic cell death in a variety of tumor cell lines. Although their precise mechanism of action is unknown, ALPs primarily act on the cell membrane, where they inhibit signaling through the mitogen-activated protein kinase (MAPK) pathway. Because stimulation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway is essential for radiation-induced apoptosis in certain cell types, we tested the effect of ALPs in combination with ionizing radiation on MAPK/SAPK signaling and apoptosis induction. Here, we present data showing that three ALPs, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, hexadecylphosphocholine, and the novel compound octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate (D-21266) induce time- and dose-dependent apoptosis in the human leukemia cell lines U937 and Jurkat T but not in normal vascular endothelial cells. Moreover, in combination with radiation, ALPs strongly enhance the induction of apoptosis in both leukemic cell lines. All tested ALPs not only prevented MAPK activation, but, like radiation, stimulated the SAPK/JNK cascade within minutes. A dominant-negative mutant of c-Jun inhibited radiation- and ALP-induced apoptosis, indicating a requirement for the SAPK/JNK pathway. Our data support the view that ALPs and ionizing radiation cause an enhanced apoptotic effect by modulating the balance between the mitogenic, antiapoptotic MAPK, and the apoptotic SAPK/JNK pathways. This type of modulation of specific signal transduction pathways in tumor cells may lead to the development of new therapeutic strategies.

Ordering of ceramide formation, caspase activation, and mitochondrial changes during CD95- and DNA damage-induced apoptosis.

To evaluate the role of ceramide (Cer) in apoptosis signaling, we examined Cer formation induced by CD95, etoposide, or gamma-radiation (IR) in relation to caspase activation and mitochondrial changes in Jurkat T cells. The Cer response to all three stimuli was mapped in between caspases sensitive to benzoyloxycarbonyl-VAD-fluoromethylketone (zVAD-fmk) and acetyl-DEVD-aldehyde (DEVD-CHO). Cer production was independent of nuclear fragmentation but associated with the occurrence of other aspects of the apoptotic morphology. Caspase-8 inhibition abrogated Cer formation and apoptosis induced by CD95 but did not affect the response to etoposide or IR, placing CD95-induced Cer formation downstream from caspase-8 and excluding a role for caspase-8 in the DNA damage pathways. CD95 signaling to the mitochondria required caspase-8, whereas cytochrome c release in response to DNA damage was caspase-independent. These results indicate that the caspases required for the Cer response to etoposide and IR reside at or downstream from the mitochondria. Bcl-2 overexpression abrogated the Cer response to etoposide and IR and reduced CD95-induced Cer accumulation. We conclude that the Cer response to DNA damage fully depends on mitochondrion-dependent caspases, whereas the response to CD95 partially relies on these caspases. Our data imply that Cer is not instrumental in the activation of inducer caspases or signaling to the mitochondria. Rather, Cer formation is associated with the execution phase of apoptosis.

Diacylglycerol kinase theta binds to and is negatively regulated by active RhoA.

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. To date, very little is known about the regulation of DGK activity. We have previously identified the DGKtheta isotype, which is predominantly expressed in brain (Houssa, B., Schaap, D., van der Wal, J., Goto, K., Kondo, H., Yamakawa, A., Shibata, M., Takenawa, T., and Van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422-10428). We now report that DGKtheta binds specifically to activated RhoA in transfected COS cells as well as in nontransfected neuronal N1E-115 cells. Binding is abolished by a point mutation (Y34N) in the effector loop of RhoA. DGKtheta does not bind to inactive RhoA, nor to the other Rho-family GTPases, Rac or Cdc42. Like active RhoA, DGKtheta localizes to the plasma membrane. Strikingly, the binding of activated RhoA to DGKtheta completely inhibits DGK catalytic activity. Our results suggest that DGKtheta is a downstream effector of RhoA and that its activity is negatively regulated by RhoA. Through accumulation of newly produced diacylglycerol, RhoA-mediated inhibition of DGKtheta may lead to enhanced PKC activity in response to external stimuli.

Protein kinase Czeta is a negative regulator of protein kinase B activity.

Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKCzeta but not PKCalpha and PKCdelta can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKCzeta was also found in COS-1 cells transiently expressing PKB and PKCzeta, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with platelet-derived growth factor (PDGF) resulted in a decrease in the PKB-PKCzeta interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGF-induced activation of PKCzeta was not affected by co-expression of PKB. However, both PDGF- and p110-CAAX-induced activation of PKB were significantly abolished in cells co-expressing PKCzeta. In contrast, co-expression of a kinase-dead PKCzeta mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by co-expression of PKCzeta. A clear inhibitory effect of PKCzeta was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKCzeta in vivo and that PKCzeta acts as a negative regulator of PKB.

Radiation-induced apoptosis--the ceramide-SAPK signaling pathway and clinical aspects.

Apoptosis, or programmed cell death is an important regulatory mechanism that is involved in a variety of homeostatic processes. Decreased cellular sensitivity or inappropriate responses to apoptotic stimuli may be important factors in tumorigenesis and resistance to anticancer treatments. It is generally accepted that all mammalian cells constitutively express the biochemical machinery to execute apoptosis. It is, however, not clear which signal transduction pathways are involved, or to which extent various stimuli activate independent or partially overlapping pathways. In this paper we discuss the involvement of a ceramide-mediated stress-activated protein kinase (SAPK) signaling cascade in radiation-induced apoptosis. Furthermore, examples are presented of pharmacological intervention in specific signal transduction pathways that lead to modulation of the apoptotic response. Finally, data are presented to illustrate the potential clinical relevance of apoptosis.

The role of the stress-activated protein kinase (SAPK/JNK) signaling pathway in radiation-induced apoptosis.

Ionizing radiation, like a variety of other cellular stress factors, initiates apoptosis, or programmed cell death, in many cell systems. This mode of radiation-induced cell kill should be distinguished from clonogenic cell death due to unrepaired DNA damage. Ionizing radiation not only exerts its effect on the nuclear DNA, but also at the plasma membrane level where it may activate multiple signal transduction pathways. One of these pathways is the stress-activated protein kinase (SAPK) cascade which transduces death signals from the cell membrane to the nucleus. This review discusses recent evidence on the critical role of this signaling system in radiation- and stress-induced apoptosis. An improved understanding of the mechanisms involved in radiation-induced apoptosis may ultimately provide novel strategies of intervention in specific signal transduction pathways to favorably alter the therapeutic ratio in the treatment of human malignancies.