Increased activity of the protein kinase C-delta holoenzyme in the cytoplasmic particulate fraction precedes the activation of caspases in polyomavirus-transformed pyF111 rat fibroblasts exposed to calphostin C or topoisomerase-II inhibitors

A caspase-mediated release of the 40-kDa catalytic fragment of the delta isoform (CF-delta) of protein kinase C (PKC-delta) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-delta, but cannot make the antiapoptotic Bcl-2 and Bcl-X(L) proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-delta activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101-108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737-743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-delta holoenzyme (holo-PKC-delta) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-delta fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-delta-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-delta. However, while calphostin C did not affect the delta-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the delta activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-delta activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of delta-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.     

Increased Activity of the Protein Kinase C-δ Holoenzyme in the Cytoplasmic Particulate Fraction Precedes the Activation of Caspases in Polyomavirus-Transformed pyF111 Rat Fibroblasts Exposed to Calphostin C or Topoisomerase-II Inhibitors

A caspase-mediated release of the 40-kDa catalytic fragment of the δ isoform (CF-δ) of protein kinase C (PKC-δ) is involved in apoptosis, but its actual role in apoptosis development is still unknown. In an effort to understand this role, we have used polyomavirus-transformed pyF111 rat fibroblasts, which are hypersusceptible to apoptosis as they constitutively hyperexpress PKC-δ, but cannot make the antiapoptotic Bcl-2 and Bcl-X_L proteins, while making the proapoptotic Bax protein. Calphostin C is reportedly both a specific inhibitor of PKC-δ activity (C. Keenan, N. Goode, and C. Pears, 1997, FEBS Lett. 415, 101–108) and an effective apoptogen (M. Murata et al., 1997, Cell. Mol. Life Sci. 53, 737–743). Exposure of pyF111 cells to calphostin C (75 nM) stimulated the translocation of the PKC-δ holoenzyme (holo-PKC-δ) onto the cytoplasmic particulate (CP) fraction between 15 and 45 min, which was after the release of mitochondrial cytochrome c but before the activation of cytoplasmic DEVD-specific caspases. The CF-δ fragment started accumulating only between 2 and 4 h, while apoptosis occurred mostly within 6 h. Incubating pyF111 cells with the much slower acting, apoptogenic topoisomerase-II inhibitors etoposide (VP-16) and teniposide (VM-26) also caused within 6 h a doubling of the CP-bound holo-PKC-δ-related activity but with no significant translocation of the holoenzyme to the CP fraction. Again this occurred after the release of cytochrome c but before the activation of DEVDases and the accumulation of the CF-δ. However, while calphostin C did not affect the δ-related activity in the nuclear membrane (NM) and nucleoplasmic (NP) fractions, VP-16 and VM-26 caused a prompt, large, and irreversible drop in the δ activity at the NM and a transient surge followed by a fall in the NP-associated activity. Hence, a surge of CP-anchored holo-PKC-δ activity is a common part of the signals given by various apoptogenic drugs to pyF111 cells. On the other hand, inhibition of δ-related activity, first at the NM and then in the NP fraction, is a specific feature only of the signals given by apoptogenic DNA-damaging agents.     

Protein kinase C-beta II Is an apoptotic lamin kinase in polyomavirus-transformed, etoposide-treated pyF111 rat fibroblasts

The role of protein kinase C-beta(II) (PKC-beta(II)) in etoposide (VP-16)-induced apoptosis was studied using polyomavirus-transformed pyF111 rat fibroblasts in which PKC-beta(II) specific activity in the nuclear membrane (NM) doubled and the enzyme was cleaved into catalytic fragments. No PKC-beta(II) complexes with lamin B1 and/or active caspases were immunoprecipitable from the NM of proliferating untreated cells, but large complexes of PKC-beta(II) holoprotein and its catalytic fragments with lamin B1, active caspase-3 and -6, and inactive phospho-CDK-1, but not PKC-beta(I) or PKC-delta, could be immunoprecipitated from the NM of VP-16-treated cells, suggesting that PKC-beta(II) is an apoptotic lamin kinase. By 30 min after normal nuclei were mixed with cytoplasms from VP-16-treated, but not untreated, cells, PKC-beta(II) holoprotein had moved from the apoptotic cytoplasm to the normal NM, and lamin B1 was phosphorylated before cleavage by caspase-6. Lamin B1 phosphorylation was partly reduced, but its cleavage was completely prevented, despite the presence of active caspase-6, by adding a selective PKC-betas inhibitor, hispidin, to the apoptotic cytoplasms. Thus, a PKC-beta(II) response to VP-16 seems necessary for lamin B1 cleavage by caspase-6 and nuclear lamina dissolution in apoptosing pyF111 fibroblasts. The possibility of PKC-beta(II) being an apoptotic lamin kinase in these cells was further suggested by lamin B1-bound PKC-delta being inactive or only slightly active and by PKC-alpha not combining with the lamin.     

Correlation between decreased sensitivity of the Daudi lymphoma cells to VP-16-induced apoptosis and deficiency in DNAS1L3 expression

A functional relationship between the apoptotic endonuclease DNAS1L3 and the chemotherapeutic drug VP-16 was established. The lymphoma cell line, Daudi, exhibited a significant resistance to VP-16 treatment in comparison to the lymphoma/leukemia cell line, U-937. While U-937 cells degraded their DNA into internucleosomal fragments, Daudi cells failed to undergo such fragmentation in response to the drug. Activation of both caspase-3 and DNA fragmentation factor was not sufficient to trigger internucleosomal DNA fragmentation in Daudi cells. No correlation was found between expression levels of topoisomerase-II, Pgp, Bcl-2, Bax, or Bad and decreased sensitivity of Daudi cells to VP-16. Daudi cells failed to express DNAS1L3 and ectopic expression of this protein significantly sensitized the cells to VP-16. An enhancement of caspase-3 activity and collapse of mitochondrial membrane potential underlie DNAS1L3-mediated sensitization of Daudi cells to VP-16, which may be a direct result of DNAS1L3-mediated increase in PARP-1-activating DNA breaks after VP-16 treatment. Our results suggest that DNAS1L3 plays an active role in lymphoma cell sensitization to VP-16 and that its deficiency may constitute a novel mechanism of drug resistance in these cells.     

Membrane ordering effects of the anticancer agent VM-26

The effect of the anticancer agent VM-26 on acyl chain order of cellular and model membranes was examined by electron spin resonance techniques. The order parameter for the paramagnetic probe 5-doxyl stearate was increased when VM-26 was incorporated into the bilayer of fluid-phase dimyristoylphosphatidylcholine (DMPC) or gel-phase dipalmitoylphosphatidylcholine (DPPC) liposomes at concentrations up to 4.8 mol%. The ordering effect of VM-26 in DMPC was greater than that of cholesterol on an equimolar basis. The less cytotoxic congener of VM-26, VP-16, was only one-third as active as VM-26 in its ordering effects on DMPC. Higher order parameters for 5-doxyl stearate were also noted in asolectin liposomes, Ehrlich ascites tumor cells, and CCRF-CEM cells treated with VM-26. We conclude that VM-26 has significant membrane associated activity in addition to its previously recognized nuclear effects.     

The topoisomerase II inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes.

This study shows that not only concanavalin A-stimulated proliferating lymphocytes but also unstimulated mouse splenic lymphocytes are sensitive to the topoisomerase II (topo II) inhibitor teniposide (VM-26). When unstimulated lymphocytes are pretreated with VM-26 for a 2-h period and are then incubated in drug-free medium, cell viability, as determined by trypan blue exclusion, decreases to 40% of the control by 6 h. The drug-treated cultures show two to three times the level of detergent soluble DNA than the control cultures and agarose gel electrophoresis of the soluble DNA shows the presence of oligonucleosomal-sized fragments, a feature considered to be a hallmark of apoptosis. Phase contrast microscopy, Hoechst staining for DNA, and immunofluorescence microscopy of various nuclear and cytoplasmic antigens (nucleolar fibrillarin, snRNP, ubiquitin, vimentin, tubulin) in the VM-26-treated cells characterize the morphological changes during apoptosis of these cells. The role of topo II as the mediator of the VM-26 effects is supported by pulsed field gel electrophoresis, which shows the typical topo II-induced cleavage of supercoiled DNA into loop-sized 300- and 50-kbp fragments. We conclude that the cancer chemotherapeutic agent VM-26 interacts with topo II and induces apoptosis in unstimulated lymphocytes.     

Increased nuclear translocation of catalytically active PKC-zeta during mouse colonocyte hyperproliferation

Protein kinase (PK) C-zeta is implicated in the control of colonic epithelial cell proliferation in vitro. However, less is known about its physiological role in vivo. Using the transmissible murine colonic hyperplasia (TMCH) model, we determined its expression, subcellular localization, and kinase activity during native crypt hyperproliferation. Enhanced mitosis was associated with increased cellular 72-kDa holoenzyme (PKC-zeta, 3.2-fold), 48-kDa catalytic subunit (PKM-zeta, 3- to 9-fold), and 24-kDa membrane-bound fragment (M(f)-zeta, >10-fold) expression. Both PKC-zeta and PKM-zeta exhibited intrinsic kinase activity, and substrate phosphorylation increased 4.5-fold. No change in cellular PKC-iota/PKM-iota expression occurred. The subcellular distribution of immunoreactive PKC-zeta changed significantly: neck cells lost their basal subcellular pole filamentous staining, whereas proliferating cell nuclear antigen-positive cells exhibited elevated cytoplasmic, lateral membrane, and nuclear staining. Subcellular fractionation revealed increased PKC-zeta and PKM-zeta expression and activity within nuclei, which preferentially accumulated PKM-zeta. These results suggest separate cellular and nuclear roles, respectively, for PKC-zeta in quiescent and mitotically active colonocytes. PKM-zeta may specifically act as a modulator of proliferation during TMCH.     

The effect of topoisomerase inhibitors on the expression of differentiation markers and cell cycle progression in human K-562 leukemia cells.

Treatment of human K-562-J leukemia cells for 1 h with the topoisomerase II-reactive drugs VP-16, VM-26, or mAMSA resulted in a dose-dependent inhibition of proliferation and in an increase in the percentage of cells staining positive for hemoglobin, a marker of erythroid differentiation. Staining for hemoglobin of up to about 60% of the cells was observed at 20 microM VP-16, 1 microM VM-26, and 8 microM mAMSA. Such treatment also caused a G2/M arrest in the cell cycle. Incubation of the cells with radiolabeled VP-16 indicated that the induced erythroid differentiation was not due to continuous cell exposure to a residual amount of the drug. VP-16-induced erythroid differentiation was also not affected by DNA, RNA, or protein synthesis inhibitors. Differentiation induction and the G2/M arrest evoked by VP-16, VM-26, and mAMSA were, however, reduced in the presence of novobiocin. Our results indicate that topo-reactive drugs that cause G2/M arrest in the K-562-J cell cycle can induce in these cells erythroid differentiation after a short and irreversible interaction with their target molecule(s).     

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.     

Decreased nuclear matrix DNA topoisomerase II in human leukemia cells resistant to VM-26 and m-AMSA

CEM leukemia cells selected for resistance to VM-26 (CEM/VM-1) are cross-resistant to various other DNA topoisomerase II inhibitors but not to Vinca alkaloids. Since DNA topoisomerase II is a major protein of the nuclear matrix, we asked if alterations in nuclear matrix topoisomerase II might be important in this form of multidrug resistance. Pretreatment of drug-sensitive CEM cells for 2 h with either 5 microM VM-26 or 3 microM m-AMSA reduced the specific activity of newly replicated DNA on the nuclear matrix by 75 and 50%, respectively, relative to that of the bulk DNA. However, neither VM-26 nor m-AMSA affected the relative specific activity of nascent DNA isolated from the nuclear matrices of drug-resistant CEM/VM-1 cells. The decatenating and unknotting activities of DNA topoisomerase II were 6- and 7-fold lower, respectively, in the nuclear matrix preparations from the CEM/VM-1 cells compared to parental CEM cells. Western blot analysis revealed that the amount of immunoreactive topoisomerase II in the nuclear matrices of the CEM/VM-1 cells was decreased 3.2-fold relative to that in CEM cells, but there was no significant difference in the amount of enzyme present in the nonmatrix (1.5 M salt soluble) fractions of nuclei from these cell lines. Increasing the NaCl concentration used in the matrix isolation procedure from 0.2 to 1.8 M resulted in a progressive decrease in the specific activity of topoisomerase II in matrices of CEM/VM-1 but not CEM cells, which suggested that the association of the enzyme with the matrix is altered in the resistant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C desensitization by phorbol esters and its impact on growth of human breast cancer cells

Active phorbol esters such as TPA (12-0-tetra-decanoylphorbol-13-acetate) inhibited growth of mammary carcinoma cells (MCF-7 greater than BT-20 greater than MDA-MB-231 greater than = ZR-75-1 greater than HBL-100) with the exception of T-47-D cells presumably by interacting with the phospholipid/Ca2+-dependent protein kinase (PKC). The nonresponsive T-47-D cells exhibited the lowest PKC activity. A rapid (30 min) TPA-dependent translocation of cytosolic PKC to membranes was found in the five TPA-sensitive cell without affecting cell growth. However, TPA-treatment of more than 10 hours inhibited reversibly the growth of TPA-responsive cells. This effect coincided with the complete loss of cellular PKC activity due to the proteolysis of the translocated membrane-bound PKC holoenzyme (75K) into 60K and 50K PKC fragments. Resumption of cell growth after TPA-removal was closely related to the specific reappearance of the PKC holoenzyme activity (75K) in the TPA-responsive human mammary tumor cell lines suggesting an involvement of PKC in growth regulation.     

Topoisomerase-II-mediated lesions in nascent DNA: comparison of the effects of epipodophyllotoxin derivatives, VM-26 and VP-16, and 9-anilinoacridine derivatives, m-AMSA and o-AMSA

This study compares the effects of the epipodophyllotoxin derivatives, VM-26 and VP-16, and the 9-anilinoacridine derivatives, m-AMSA and o-AMSA, on nascent and mature DNA. Two types of lesion which are putatively mediated by topoisomerase II, DNA-protein crosslinks and DNA double-strand breaks, were analyzed in drug-treated nuclei from 3H/14C labelled L1210 cells. Potassium/dodecyl sulfate precipitation assay was used to assess DNA-protein crosslinks in mature and nascent (1 min old) DNA. Both epipodophyllotoxins and m-AMSA showed a strong preference for nascent DNA. DNA double-strand cleavage induced by VM-26 and m-AMSA also showed a preference for nascent DNA as indicated by neutral elution technique. Sedimentation on neutral sucrose gradients revealed that these drugs generated highly degraded fragments (under 30 S) in nascent DNA substantially faster than in mature DNA. Lesions in nascent DNA were diminished substantially by the omission of ATP or the addition of novobiocin. The ability to induce lesions in nascent DNA correlates with cytotoxic potency of the agents studied. The results suggest that replicating DNA may constitute a preferential target for antitopoisomerase II drugs.     

Suppression of influenza A virus nuclear antigen production and neuraminidase activity by a nutrient mixture containing ascorbic acid, green tea extract and amino acids

Influenza, one of the oldest and most common infections, poses a serious health problem causing significant morbidity and mortality, and imposing substantial economic costs. The efficacy of current drugs is limited and improved therapies are needed. A unique nutrient mixture (NM), containing ascorbic acid, green tea extract, lysine, proline, N-acetyl cysteine, selenium among other micronutrients, has been shown to exert anti-carcinogenic and anti-atherogenic activity both in vitro and in vivo. Many of the constituents of NM have been shown to have an inhibitory effect on replication of influenza virus and HIV. This prompted us to study the effect of NM on influenza A virus multiplication in infected cells and neuraminidase activity (NA) in virus particles. Addition of NM to Vero or MDCK cells post infection resulted in dose-dependent inhibition of viral nucleoprotein (NP) production in infected cells. NM-mediated inhibition of viral NP was selective and not due to cytotoxicity towards host cells. This antiviral effect was enhanced by pretreatment of virus with the nutrient mixture. Individual components of NM, namely ascorbic acid and green tea extract, also blocked viral NP production, conferring enhanced inhibition when tested in combination. Incubation of cell-free virus with NM resulted in dose-dependent inhibition of associated NA enzyme activity. In conclusion, the nutrient mixture exerts an antiviral effect against influenza A virus by lowering viral protein production in infected cells and diminishing viral enzymatic activity in cell-free particles.     

Temporal and spatial localization of novel nuclear protein NP95 in mitotic and meiotic cells

Expression of novel NP95 (nuclear protein, 95 kDa), which contains a leucine zipper motif, a zinc finger motif, a putative cyclin A/E-cyclin-dependent protein kinase 2 phosphorylation site, and retinoblastoma protein-binding motifs, is associated with S-phase progression of mouse cells. It is suppressed during G1 and G2/M phases in normal thymocytes but expressed at a constantly high level irrespective of cell stage in mouse T cell lymphoma cells. NP95 was shown previously to be expressed strongly only in proliferative tissues and cells. In this immunohistochemical study, we demonstrate that NP95 is localized in S-phase nuclei as dot-like foci. Double immunostaining of NP95 and proliferating cell nuclear antigen (PCNA) showed that NP95 was co-localized with PCNA. Construction of three-dimensional images indicated that NP95 was localized with PCNA in replication sites in a somewhat distinct temporal manner. During meiosis, NP95 was present not only in proliferating spermatogonia but also in meiotic spermatocytes and differentiating spermatids which were not proliferating. The possible role of NP95 in mitotic and meiotic cells is discussed.     

Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.     

Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts

Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.     

Increased proteolysis in chromatin of terminally differentiated and quiescent cells

Endogenous proteolysis in chromatin of terminally differentiated, quiescent, and actively proliferating cells was studied by measuring the released acid-soluble radioactivity of [³H]tryptophan-prelabelled nuclear proteins, and by following the specific quantitative and qualitative changes in electrophoregrams of chromosomal proteins. The experiments suggest that the chromatin of differentiated mouse kidney and liver cells, as well as chromatin from Friend cells induced to commit terminal differentiation, exhibit increased proteolysis in comparison with that of chromatin isolated from actively proliferating cells. Enhanced proteolysis was found also for the slowly renewing and quiescent cells from adult mice. The control experiments designated to discriminate between the two possible alternatives explaining the difference—increased activity of the proteolytic enzymes associated with chromatin, or increased susceptibility of the chromosomal proteins to proteases—supported the latter alternative.     

Effects of the lipophilic anticancer drug teniposide (VM-26) on membrane transport

The epipodophyllotoxin glucopyranosides have previously been shown to interact with membrane lipids and to alter the activity of several lipid-embedded membrane proteins. To determine if these agents are acting as general membrane perturbants, we have further examined their effects on membrane processes in Ehrlich ascites tumor cells. [3H]VM-26 and [3H]VP-16 were taken up rapidly and concentrated within the cells in proportion to their lipophilicity. Neither agent was found to have any significant effect on the influx of L-[3H]leucine or alpha-[3H]aminoisobutyric acid. Likewise, these drugs had no significant effects on the hexose transporter. The nucleoside transporter, which is structurally and functionally similar to the hexose transporter, was dramatically affected, however. VM-26 was a non-competitive inhibitor of equilibrium-exchange influx of cytosine arabinoside in Ehrlich cells with a Ki of 15 microM. Equilibrium-exchange influx increased with temperature in control cells (Q10 = 2) but not in VM-26-treated cells; thus, VM-26 was a more potent inhibitor at higher temperatures. VM-26 also significantly reduced zero-trans influx in Ehrlich, P388, L5178Y, and ML-1 cells, and these effects were immediate in onset. VM-26 inhibited high-affinity binding of the nucleoside transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR), but VM-26 enhanced non-specific NBMPR binding to Ehrlich cells. The apparent specificity of the epipodophyllotoxins for the nucleoside transporter is discussed.