Harvest and storage of adult human retinal pigment epithelial sheets

PURPOSE: To describe a method for the harvesting and storing of intact viable sheets of adult human retinal pigment epithelial (RPE) cells. METHODS: Adult human RPE cells were harvested as intact sheets from 21 cadaver eyes, using the enzyme Dispase. The sheets were embedded in 50% gelatin containing 300 mM sucrose and stored at 4 degrees C. The viability of the cells, as well as their ability to proliferate in vitro, was studied for 96 hours after harvesting. Light microscopy (LM), transmission (TEM) and scanning electron microscopy (SEM) were performed to determine the integrity and ultrastructural features of the cells. Microbiologic culture of the harvested sheets was performed to exclude contamination. RESULTS: LM, TEM and SEM showed intact RPE cells with well-developed microvilli, basal infoldings and intercellular connections. The initial viability of intact RPE sheets was 86%, with a progressive decline in viability with increased storage time. Cells harvested within 24 hours after death maintained greater viability than those harvested after 24 hours (p < 0.05). Harvested RPE cells were free of microbial contamination and rapidly proliferated when cultured in vitro. CONCLUSION: Intact sheets of adult human RPE can be isolated using the enzyme Dispase. The cells appeared suitable for retinal transplantation if harvested within 24 hours of death and maintained 82% viability for as long as 48 hours if stored at 4 degrees C.     

Protein processing and auxin response in transgenic tobacco harboring a putative cDNA of zeatin O-xylosyltransferase from Phaseolus vulgaris

Exogenous angiogenin undergoes rapid nuclear translocation in cultured human umbilical artery endothelial cells at 37 degrees C but not at 4 degrees C. Treatment of cells with colchicine, nocodazole and taxol, which disrupt the microtubule system, does not affect the nuclear translocation process of angiogenin, suggesting that cells transport internalized angiogenin in a microtubule independent fashion. Lysosomal inhibitors, chloroquine and leupeptin, neither inhibit nor enhance the nuclear translocation of angiogenin, indicating that lysosomal targeting and processing are not required for, and do not compete with, the nuclear translocation. Moreover, treatment of cells with a tyrosine kinase antagonist, genistein, does not change the ability of the cells to translocate angiogenin into the nucleus. We suggest that exogenous angiogenin is translocated to the nucleus by a mechanism that does not require activation of tyrosine kinase, but includes receptor-mediated endocytosis, microtubule and lysosome independent transport across the cytoplasm, and nuclear localization sequence-assisted nuclear import.     

Chimeric Wnt proteins define the amino-terminus of Wnt-1 as a transformation-specific determinant

PURPOSE: To determine whether hepatocyte growth factor (HGF) receptor (HGFR) is expressed in retinal pigment epithelial (RPE) cells and to test whether RPE cells are responsive to HGF. To evaluate expression of HGFR in human donor eyes and in several epiretinal membranes associated with proliferative vitreoretinopathy and idiopathic epiretinal membranes. METHODS: HGF-dependent migration and proliferation in primary and simian virus (SV) 40-transformed human RPE cells was studied using a Boyden chamber and [3H]thymidine uptake, respectively. The expression and tyrosine phosphorylation of HGFR protein was evaluated in RPE cells by immunoprecipitation and western blot analysis. Expression of HGFR in human donor eyes and in several epiretinal membranes associated with proliferative vitreoretinopathy (PVR) and idiopathic epiretinal membranes was analyzed by immunohistochemistry. RESULTS: HGFR was expressed in RPE cells and was tyrosine-phosphorylated in response to HGF. Whereas HGF was a potent motogen for RPE cells, it induced only a modest, dose-dependent uptake of [3H]thymidine. Evaluation of human donor eyes showed that the RPE monolayer was the major cell type that was strongly positive for HGFR. HGFR was uniformly and readily detected in the cellular component of epiretinal membranes associated with PVR, whereas little or no HGFR was found in idiopathic epiretinal membranes. CONCLUSIONS: HGFR is expressed in cultured RPE cells, in the RPE monolayer in human donor eyes, and in epiretinal membranes obtained from patients with PVR. Furthermore, HGF is a potent chemoattractant for cultured human RPE cells. These observations suggest a role for HGF and HGFR in normal function of RPE cells and in RPE-related disease such as PVR.     

Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: a possible mechanistic basis for its antitumor action

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (Kd, approximately 30 microM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.     

Mechanism of action cryptophycin. Interaction with the Vinca alkaloid domain of tubulin

Cryptophycin is a potent antitumor agent that depletes microtubules in intact cells, including cells with the multidrug resistance phenotype. To determine the mechanism of action of cryptophycin, its effects on tubulin function in vitro were analyzed. Cryptophycin reduced the in vitro polymerization of bovine brain microtubules by 50% at a drug:tubulin ratio of 0.1. Cryptophycin did not alter the critical concentration of tubulin required for polymerization, but instead caused substoichiometric reductions in the amount of tubulin that was competent for assembly. Consistent with its persistent effects on intact cells, cryptophycin-treated microtubule protein remained polymerization-defective even after cryptophycin was reduced to sub-inhibitory concentrations. The effects of cryptophycin were not due to denaturation of tubulin and were associated with the accumulation of rings of microtubule protein. The site of cryptophycin interaction with tubulin was examined using functional and competitive binding assays. Cryptophycin blocked the formation of vinblastine-tubulin paracrystals in intact cells and suppressed vinblastine-induced tubulin aggregation in vitro. Cryptophycin inhibited the binding of [3H]vinblastine and the hydrolysis of [gamma32P]GTP by isolated tubulin, but did not block the binding of colchicine. These results indicate that cryptophycin disrupts the Vinca alkaloid site of tubulin; however, the molecular details of this interaction are distinct from those of other antimitotic drugs.     

Radiation response of porcine RPE cells in vitro

PURPOSE: To investigate the antiproliferative effect of ionizing radiation on retinal pigment epithelial (RPE) cells that are supposed to play a major role in the pathogenesis of proliferative vitreoretinopathy (PVR). METHODS: RPE cells from pig eyes were irradiated with doses ranging from 4 to 16 Gy (1 Gray = 1 Joule/kilogram). Cells were counted at 1, 2, 3, and 4 weeks (Experiment 1) or 1, 2, 4, and 6 weeks (Experiment 2) after treatment. In Experiment 3, cells were trypsinized 24 h after radiation and seeded again. Colonies were counted 10 days later, and the surviving fraction was determined. RESULTS: The numbers of cells and colonies were inversely correlated to the doses applied. In Experiment 2, cell numbers of radiated cultures remained stable during the time of follow-up, whereas, in Experiment 1, significant proliferation occurred in treated cultures as well as in controls. This may be due to the higher growing rate that was found in the cultures of Experiment 2, compared to those of Experiment 1, at the time of radiation. In Experiment 3, a D0 value of 0.72 Gy was found. CONCLUSIONS: Proliferation of RPE cells can be suppressed by irradiation in a dose-dependent manner. Therefore, radiotherapy may be useful in the treatment of PVR. Its effect probably depends on the stage or activity of PVR at the time of radiation.     

Paclitaxel inhibits arterial smooth muscle cell proliferation and migration in vitro and in vivo using local drug delivery

BACKGROUND: The antineoplastic compound paclitaxel (Taxol) causes an increased assembly of extraordinarily stable microtubules. The present study was designed to characterize the effects of paclitaxel on proliferation and migration of human arterial smooth muscle cells (haSMCs) in vitro and on neointima formation in an in vivo experimental rabbit model. METHODS AND RESULTS: Both monocultures of haSMCs and cocultures with human arterial endothelial cells (haECs) were used. Cell growth after 4, 8, and 14 days was determined in the absence or presence of platelet-derived growth factor-AB (PDGF-AB), basic fibroblast growth factor (bFGF), or thrombin. Nonstop paclitaxel exposure, as well as single-dose applications of paclitaxel for 24 hours or even 20 minutes (0.1 to 10.0 micromol/L), caused a complete and prolonged inhibition of haSMC growth up to day 14, with an IC50 of 2.0 nmol/L. Mitogens or cocultures with stimulating haECs did not significantly attenuate paclitaxel-induced effects. Immunohistochemistry showed characteristic cytoskeletal changes predominantly in the microtubule network. Additionally, in 20 male New Zealand White rabbits, intimal plaques were produced by electrical stimulation. In 10 animals, paclitaxel was locally applied by use of microporous balloons. Histologically, the intima wall area, wall thickness, and degree of stenosis were reduced significantly in paclitaxel-treated animals compared with controls. CONCLUSIONS: Our data show that paclitaxel inhibits haSMC proliferation and migration in a dose-dependent manner in monocultures and cocultures even in the presence of mitogens. Furthermore, paclitaxel prevents neointima formation in rabbits after balloon angioplasty. The long-lasting effect after just several minutes' exposure time makes this lipophilic substance a promising candidate for local antiproliferative therapy of restenosis.     

Role of microtubules in milk secretion--action of colchicine on microtubules and exocytosis of secretory vesicles in rat mammary epithelial cells

Effect of colchicine on microtubules was studied in mammary epithelial cells treated both in vivo and in vitro with the alkaloid. Three hours after the intramammary infusion of colchicine, secretory activity of mammary epithelia ceased, milk constituents accumulated and were randomly distributed within the cytoplasm, sometimes leaking into the perialveolar connective tissue, and autophagic vacuoles were prevalent. It appeared that an accelerated involutionary process was occurring. No microtubules were observed after this treatment. In vitro treated cells appeared to be less affected by the alkaloid. Although numerous casein-containing secretory vesicles accumulated in the cytoplasm, lipid droplet accumulation was less, and fewer autophagic vacuoles were observed, although lysosomes were commonly observed. Occasionally, obliquely sectioned microtubules were found in cells treated with low concentrations of colchicine but were absent at higher colchicine concentrations; however, paracrystalline inclusions (tubulin aggregates) were observed in some cells at all concentrations of the drug. These observations provide evidence that drugs which interfere with microtubule integrity reduce the secretory activity in mammary epithelia. This evidence is consistent with the concept of an association of the microtubular system and the secretory process.     

The relationships between seasonal variations in the concentration of urea in bulk milk and the production and fertility of dairy herds

The retinal pigment epithelium (RPE) is clinically involved in diverse ocular inflammatory diseases. Because perturbed RPE cells produce a variety of inflammatory substances, RPE cells may play an integral part in these diseases. Interleukin-1 (IL-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are pleiotropic cytokines with the ability to trigger numerous inflammatory responses. This report shows that cultured human RPE cells synthesize interleukin-1 beta (IL-1 beta) and GM-CSF in response to the potentially inflammatory cytokine, IL-1 alpha, but not to E. coli endotoxin. Control RPE cells made little or no mRNA or protein for either IL-1 beta or GM-CSF. Upon stimulation of the cells by IL-1 alpha, both IL-1 beta and GM-CSF mRNAs were readily apparent by 3 hours, persisted for over 24 hours, and were translated into immunologically detectable proteins. GM-CSF protein was secreted into the culture medium, whereas IL-1 beta protein remained cell associated. The IL-1 alpha-induced mRNA and protein production were inhibited by dexamethasone. These observations provide additional evidence that RPE cells are capable of playing a pivotal role during ocular inflammation.     

Cultured human retinal pigment epithelial cell produced and secreted epidermal growth factor: its bioactivity and clinical significance

The epidermal growth factor bioactivity from cultured primary human and monkey retinal pigment epithelial (RPE) cell systems was detected by using the radioreceptor assay. We report that the cultured human and monkey RPE cells produce and secrete the EGF bioactively to the media as demonstrated by radioreceptor binding assay. The EGF bioactivities secreted by human and monkey RPE cells were at peak of 48 hours (human RPE cells secreted 2.11 +/- 0.46 ng/ml vs monkey RPE was 1.56 +/- 0.12 ng/ml) in the serum-free media. The results indicate that the RPE is one of important sources for EGF in the eye. The RPE cells may play much important roles in the development of proliferative retinal diseases through the autocrine or paracrine mechanism. This new discovery will be helpful to elucidate the pathogenesis of proliferative retinal diseases and also provide an important basis for the treatment of such diseases.     

Scaling of the limb long bones to body mass in terrestrial mammals

In their normal state, RPE cell are strongly adherent to Bruch's membrane. Certain pathological conditions such as retinal detachment cause an injury-type response (probably augmented or induced by the local accumulation of a variety of substances which modulate cell behaviour) in which RPE begin to dissociate from the membrane. This RPE-Bruch's membrane separation may be mediated by proteins with counter-adhesive properties and proteolytic enzymes, partly derived from the RPE themselves. Concomitant with the RPE disassociation, the cells begin to lose tertiary differentiation characteristics and gain macrophage-like features. When the "free" RPE arrive at the surface of the neuroretina, they may attach to or create a provisional matrix. Some of the cells adopt a fibroblast-like phenotype. This phenotype is similar to that of the dermal fibroblast during cutaneous wound repair and the fibroblastic RPE synthesise the types of matrix components found in healing skin wounds. Many of these molecules in turn further modulate the activities of the cells via several families of cell surface receptors, while the RPE continue to remodel the new matrix with a range of proteolytic enzymes. The resulting tissue (or membrane) has many of the features of a contractile scar and is the hallmark of the condition known as proliferative vitreoretinopathy (PVR). Thus the development of PVR, and the resulting tractional distortion of the neuroretina, appears to be dependent on RPE-matrix interactions. The interactions present a number of potential therapeutic targets for the management of the disorder.     

Paclitaxel and nocodazole differentially alter endocytosis in cultured cells

PURPOSE: Microtubule-based transport facilitates the endocytosis of exogenous macromolecules. We have determined how microtubule accumulation and disassembly alter endocytosis. METHODS: The effects of paclitaxel, which promotes microtubule assembly, and nocodazole, which promotes microtubule disassembly, on fluid-phase and receptor-mediated endocytosis were measured using uptake of horseradish peroxidase and 125I-transferrin, respectively. Changes in membrane and microtubule organization were examined by fluorescence microscopy. RESULTS: Neither paclitaxel (4 microM, 60 min pretreatment) nor nocodazole (1 microgram/ml, 60 min pretreatment) significantly inhibited fluid-phase endocytosis. However, paclitaxel caused a redistribution of fluorescent fluid-phase marker to the periphery. Both paclitaxel and nocodazole treatment significantly (p < or = 0.05) reduced the initial uptake of 125I-transferrin at 5 min to approximately 50% of control. Despite the similarity of the effects on initial endocytic uptake, the effects on steady state accumulation of 125I-transferrin were quite distinct. Exposure of CV-1 cells to paclitaxel for an additional 30, 60 or 90 min also showed reduced accumulation of 125I-transferrin up to a maximum significant (p < or = 0.05) inhibition of 48% +/- 10% of control at 90 min. In contrast, nocodazole caused an initial significant (p < or = 0.05) increase in 125I-transferrin accumulation after 30 min (159% +/- 13% of control), while by 90 min 125I-transferrin accumulation had returned to control levels. Microtubule content, particularly of stable microtubules, was increased in CV-1 cells by paclitaxel, but abolished by nocodazole treatment. CONCLUSIONS: Our data show that changes in the microtubule array can alter the dynamics of receptor movement through the endosomal pathway. However, microtubule assembly versus disassembly have different effects.     

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

A novel retinal pigment epithelial protein suppresses neutrophil superoxide generation. I. Characterization of the suppressive factor

In acute intraocular inflammation, neutrophils release a variety of agents, that are potentially toxic to the surrounding tissues. The reactive oxygen metabolites, including superoxide are among these injurious agents. In the present study, retinal pigment epithelial (RPE) cells were found to inhibit by 70% to 80% the production of superoxide by neutrophils that were stimulated either by the receptor-coupled activator, N-formyl-methionyl-leucyl-phenylalanine, or by the non-receptor-coupled activator, phorbol myristate acetate. The inhibition is effective with a relatively small number of RPE cells (0.65 x 10(5)) mixed in a large pool of neutrophils (7.6 x 10(5)). The transduction of this effect does not require a neutrophil-RPE cell surface contact since the RPE culture supernatants also effectively inhibit the superoxide production. This protein is secreted specifically by cultured and noncultivated intact RPE cells, but not by fibroblasts, corneal epithelial cells or intact choroidal tissues. The protein is not cytotoxic to the neutrophils, and the inhibitory effect occurs in a dose-dependent manner up to the concentration tested. This factor is unrelated to either transforming growth factor-beta, or transferrin. There was no evidence of RPE scavenging of superoxide generated enzymatically by hypoxanthine-xanthine oxidase system, indicating that this factor does not have superoxide dismutase activity. When RPE cells were preincubated with 10 micrograms ml-1 cycloheximide, 60% of the activity was lost, suggesting that a de novo protein synthesis is required for the activity and that the protein is a significant steady-state product of RPE cells. Incubation of the released RPP with thermolysin (10 micrograms ml-1 15 min, 37 degrees C) also eliminated the activity. The degradation of activity by protease and inhibition of RPP activity by cycloheximide, therefore, confirmed the protein nature of the suppressive factor. This protein from RPE cells appears to act directly on the neutrophils, reducing the release of oxygen metabolites during activation, and thus limiting tissue injury during inflammation.     

5-Fluorouracil interferes with paclitaxel cytotoxicity against human solid tumor cells

Paclitaxel, a naturally occurring antimitotic agent, has shown efficacy in the treatment of certain solid tumors, particularly metastatic breast carcinoma and drug-refractory ovarian cancers. Recent studies have demonstrated that paclitaxel, in addition to its effects on microtubules and cell cycle arrest, possesses significant cell-killing activity in solid tumor cells by the induction of apoptosis. However, the mechanism by which paclitaxel leads to cell death and its relationship with paclitaxel-induced mitotic arrest is presently unclear. In this study, we attempted to determine whether pre-arresting tumor cells at other phases of the cell cycle could affect paclitaxel-induced apoptosis. We found that 5-fluorouracil (5-FU), another antineoplastic agent that usually arrests tumor cells at the G1-S phase of the cell cycle, could significantly repress the cell-killing activity of paclitaxel in solid tumor cells, even when it was added simultaneously with paclitaxel. Further studies indicated that 5-FU actually inhibits the cytotoxic effects of paclitaxel on both mitotic arrest and apoptotic cell death, suggesting that 5-FU might interfere with paclitaxel cytotoxicity at an early stage, probably by preventing tumor cells from entering G2-M phase. Because recent clinical trials have used a combination of paclitaxel and 5-FU in the treatment of metastatic breast cancers, our results also suggest that the combination of these two drugs might not be as valuable in clinical chemotherapy.     

Mechanism of action of the unusually potent microtubule inhibitor cryptophycin 1

Cryptophycin 1 is a remarkably potent antiproliferative compound that shows excellent antitumor activity against mammary, colon, and pancreatic adenocarcinomas in mouse xenographs. At picomolar concentrations, cryptophycin 1 blocks cells in the G2/M phase of the cell cycle by an apparent action on microtubules. The compound binds to tubulin, inhibits microtubule polymerization, and depolymerizes preformed microtubules in vitro. Its exceptionally powerful antitumor activity (many-fold greater than paclitaxel or the vinca alkaloids) raises important questions about its mechanism of action. By quantitative video microscopy, we examined the effects of cryptophycin 1 on the dynamics of individual microtubules assembled to steady state from bovine brain tubulin. At low nanomolar concentrations, in the absence of net microtubule depolymerization, cryptophycin 1 potently stabilized microtubule dynamics. It reduced the rate and extent of microtubule shortening and growing and increased the frequency of rescue. The results suggest that cryptophycin 1 exerts its antiproliferative and antimitotic activity by binding reversibly and with high affinity to the ends of microtubules, perhaps in the form of a tubulin-cryptophycin 1 complex, resulting in the most potent suppression of microtubule dynamics yet described.     

Inhibition of rabbit lens epithelial cell proliferation

OBJECTIVE: To study the ability of homoharringtonine, 5-fluorouracil and adriamycin on inhibiting the proliferation of rabbit lens epithelial cells (RLEC) and the prevention of after cataract by using homoharringtonine. METHODS: RLEC were isolated and cultured. (1) The passage RLEC were placed in 24-well tissue culture plates and incubated for 48 hours, then exposed to different concentrations of homoharringtonine, 5-fluorouracil and adriamycin for 24 and 72 hours; (2) The passage RLEC and homoharringtonine, 5-fluorouracil, adriamycin were placed and cultured for 24 hours to investigate the rate of attached cells; (3) The morphological changes of RLEC were studied under light microscope. RESULTS: The ID50 of homoharringtonine, 5-fluorouracil and adriamycin exposed to RLEC for 24 hours were 0.84 micrograms, 0.58 micrograms and 4.50 ng/ml and those for 72 hours were 0.49 micrograms, 0.33 micrograms and 3.85 ng/ml respectively. In the homoharringtonine group, the rate of attached cells was less than that of 5-fluorouracil and adriamycin groups. The study of the morphological changes showed that the different concentrations of antiproliferative drugs affected on RLEC at different regions. CONCLUSION: The authors consider that homoharringtonine may be more effective for the prevention of after cataract than 5-fluorouracil and adriamycin.     

Enhanced separation of antidepressant drugs using a polymerized nonionic surfactant as a transient capillary coating

PURPOSE: To investigate the role of the P-glycoprotein (P-gp) drug efflux pump in the intracellular disposition of colchicine and vinblastine. METHODS: Uptake and efflux kinetics were studied in vitro in human lymphocytes and in HL-60 cells with or without the P-gp modulator, verapamil. RESULTS: In human lymphocytes, colchicine was slowly taken up (uptake half-life was 18.9+/-1.1 hr.) and verapamil increased colchicine uptake by 37%, whereas it did not modify colchicine efflux from cells. In HL-60 cells, colchicine uptake was non-linear and slower than that of vinblastine, the colchicine uptake half-life (11.1+/-0.5 hr.) being 25-fold longer than that of vinblastine at 25 nM. Verapamil did not significantly modify colchicine uptake half-life, but increased its intracellular accumulation by 23% and that of vinblastine by 81%. Immuno-flow cytometry showed that P-gp expression in HL-60 cells increased significantly from 24 hr. following colchicine or vinblastine exposure. The significant increase in colchicine uptake induced by verapamil at 24 hr. was correlated with this enhanced P-gp expression. The drug efflux half-life was 11.5-fold higher for colchicine (23+/-0.9 hr) than vinblastine, indicating a much slower elimination of colchicine from cells that could be related to its longer dissociation half-life from the tubulin receptor. Verapamil treatment did not modulate either colchicine or vinblastine efflux kinetics, suggesting that the intracellular drugs are not available to the transmembrane P-gp binding sites. CONCLUSIONS: P-gp may not be the main reason for the slowness of colchicine uptake. It may be more efficient at controlling entry of colchicine and vinblastine through the plasma membrane than at mediating their efflux from HL-60 cells.