AmericanJournal ofPathology Vol. 137, No. 4, October 1990 Copyright C) American Association ofPathologists
Reversal of Colchicine-induced Mitotic Arrest in Chinese Hamster Cells with a Colchicinespecific Monoclonal Antibody
S. K. Edmond Rouan,* 1. G. Otterness,t A. C. Cunningham,t H. E. Holden,t and C. T. Rhodes* From the Department of Pharmaceutics, University of Rhode Island, Kingston, Rhode Island*; and the Department ofImmunology, and Infectious Diseases, Pfizer Central Research, Groton, Cotnnecticutt
The ability of a high-affinity colchicine-binding monoclonal antibody to reverse the effects of colchicine on Chinese hamster ovary cells was investigated. Using flow cytometry, a complete mitotic blockade was demonstrated after 16 hours with 2.5 X 10-7 mol/l (molar) colchicine. Colchicine-induced changes were reversible when equimolar antibody was added simultaneously with or up to 6 hours after colchicine. With further delay in addition ofantibody, aprogressive irreversible increase in mitotic blockade and increase in mean cell size was observed. Prolonged colchicine exposure, without antibody reversal, led to polyploidy and structural chromosome breakage. Early antibody reversal restored cells to the diploid state, whereas delayed reversal resulted in a time-dependent increase in polyploidy. Colchicine-induced polyploidy and chromosomal aberrations may be the basis for both colchicine toxicity and the time-dependent increase in irreversibility of colchicine effects. (Am JPathol 1990, 13 7:779- 787)
Colchicine is a potent drug with a narrow therapeutic range. Recent reports of colchicine-related toxicity and fatality have given rise to increasing concern about its clinical use.'2 There is no effective treatment for colchicine intoxication. Standard treatments for drug overdose, such as exchange dialysis, fail to work because of low plasma concentrations (1.5 to 2 ng/ml after a typical 1-mg oral dose) and colchicine's rapid intracellular sequestration.3 Nevertheless, colchicine continues to play a valuable role
in the treatment of acute gout and is effective in a variety of rheumatologic disorders, including familial Mediterranean fever, amyloidosis, and Behcet's disease.4 In addition, colchicine may provide some long-term benefit to patients with primary biliary cirrhosis.5 The mechanism of colchicine's therapeutic effectiveness is not completely understood, although Malawista6 has suggested that the anti-inflammatory effects are related to interference with microtubule formation in cells. Recently digoxin-specific antibody Fab (antigen-binding) fragments have been used to successfully treat lifethreatening digitalis intoxication.7 The digoxin-specific antibody acts by binding digoxin in the extracellular fluid. The consequent reduction in the extracellular free pool of drug produces a concentration gradient that promotes release of digoxin from its receptor sites. Colchicine, like digoxin, is a highly potent drug with a narrow therapeutic range. Colchicine binds to tubulin, causing disruption of the mitotic spindle apparatus and, consequently, mitotic arrest.89 Estimates of the affinity constant for colchicinetubulin interaction range from 1 X 106 to 2 X 107 mol/l (molar)-1.10"1 Thus a colchicine-specific antibody with a higher affinity constant would be expected to prevent or reverse the colchicine-tubulin interaction by lowering the effective colchicine concentration. We have recently prepared a specific high-affinity colchicine-binding monoclonal antibody.12 In the present report, we have assessed the ability of this antibody to reverse colchicine's effects in vitro. We also report the time dependent accumulation of chromosomal abnormalities associated with exposure of Chinese hamster ovary cells to colchicine. This research forms part of the doctoral dissertation of S. K. Edmond Rouan. Her work has been supported by the United States Pharmacopeial Convention Inc., Richardson Vicks Inc., and by a fellowship from the University of Rhode Island. Current address: Smith Kline and French Laboratories, 709 Swedeland Rd., King of Prussia, PA 19406. Accepted for publication April 25, 1990. Address reprint requests to Dr. I. G. Otterness, Department of Immunology and Infectious Diseases, Pfizer Central Research, Groton, CT 06340.
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Materials and Methods
DNA Staining
Reagents
Intracellular DNA content was analyzed by flow cytometry using propidium iodide as a fluorescent DNA probe.'3 Cells were harvested by treatment with trypsin (1 ml, 0.25%) for 5 minutes and collected by centrifugation. The cells then were resuspended in 2 ml culture medium and 0.4 ml of a solution of propidium iodide (250 ,ug/ml [Sigma]), and 1% vol/vol Triton X-1 00 (Sigma), prewarmed to 37°C, was added. The cells then were incubated for 15 minutes at room temperature in the dark. Stained cells were stored on ice, in the dark, and analyzed by flow cytometry within 2 hours of stain addition.
Coichicine was obtained from Sigma Chemicals, St. Louis, MO. The monoclonal anti-colchicine antibody C44 has been shown to bind colchicine with high affinity and with high specificity.12 As previously described, the hybridoma clone secreting C44 antibody was derived from fusion of the NS 1 murine myeloma cell line and a splenic lymphocyte from a BALB/c mouse immunized with a colchicine-keyhole limpet hemocyanin conjugate. The C44 hybridoma cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mmol/l (millimolar) L-glutamine, 1 mmol/l, sodium pyruvate, 100 U/ml penicillin, 100 ,ug/ml streptomycin, and 1 mmol/l minimum essential medium (MEM) nonessential amino acids solution. Ascites fluid containing high concentrations of C44 was obtained by priming BALB/c female mice with 0.5 ml pristane and at least 5 days later injecting 1 X 106 C44 hybridoma cells intraperitoneally. Ten to twenty-one days later, ascites fluid was drained every 2 days until the mice died. For these experiments, C44 antibody in ascites fluid was concentrated by precipitation with an equal volume of a saturated solution of ammonium sulfate. The concentration of colchicine-specific antibody was determined by enzyme-linked immunosorbent assay (ELISA), using a highly purified C44 antibody preparation as a standard.12 The antibody preparation used in these experiments contained approximately 8 mg/ml colchicine-specific antibody.
Cells and Culture Conditions Chinese hamster ovary cells (CHO-Kl) from the American Type Culture Collection were maintained in Ham's F12 medium (GIBCO, Grand Island, NY) supplemented with 5% vol/vol heat-inactivated fetal calf serum (GIBCO), 100 U/ml of penicillin and 100 ,ug/ml of streptomycin. For cell cycle analysis, cells in exponential growth were seeded at a density of 105 cells in 25 cm2 cell culture flasks containing 5 ml of culture medium. The cells were incubated in a humidified atmosphere containing 5% carbon dioxide for 40 hours at 370C before drug and antibody treatments. Vinblastine sulfate (1 ug/ml for 16 hours) treated cultures provided a positive control for mitotic block and untreated exponentially growing cells were the negative control for each experiment. All treatments were examined as triplicate cultures.
Flow Cytometric Analysis The FACS analyzer (Becton Dickinson, Mountain View, CA) and the FACS IV (Becton Dickinson) were used for quantitative fluorescent analyses of the cell cycle. Excitation light of 488 nm was used and emission wavelengths greater than 580 nm were monitored. Fluorescence data from the FACS analyzer was handled by determining the G1 and G2 + M peaks based on control samples of these two populations. The Gl control was a mitotic shake-off allowed to reattach for 2 hours. The G2 + M control consisted of cells arrested in mitosis by treatment with 1 ,ug/ ml vinblastine sulfate for 16 hours. Quantitation was achieved by setting a cursor at the G2 + M peak, then finding the number of cells from this point to the end of the histogram display. This number of cells is then multiplied by two and divided by the total number of cells represented by the histogram to obtain a percentage. The percentage of cells with a threshold fluorescence level greater than 1 X N (G1 peak) DNA content was determined using Disp 2D (Consort 40, Becton Dickinson) analysis of data generated by the FACS IV. Although S phase is included in this percentage, its contribution in mitotically arrested cultures is negligible. Relative cell volume changes from Coulter volume (FACS analyser) and mean small angle forward light scatter measurements (FACS IV) parameters also were quantitated from the mean channel numbers of the histogram display. A shift to higher channel number correlates with increasing cell size.
Cytologic Experiments Cells were collected by trypsinization and harvested using 1% sodium citrate for 7 minutes as hypotonic pretreatment and 3:1 methanol:acetic acid as fixative. Cells were dropped onto cold wet slides and flame dried. Cells were stained with 2% lacto-aceto-orcein for 2 hours and cover-
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slips mounted with Euperal (Asco Laboratories, Manchester, UK). At least 1000 cells were examined and the number of mitoses and the number of diploid and polyploid nuclei determined. Chromosomal aberrations also were noted.
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Results
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Relationship Between Antibody Concentration and Inhibition of Colchicineinduced Mitotic Arrest Preliminary studies demonstrated that 2.5 X 10-7 molar (0.1 jig/ml) colchicine led to a virtually complete mitotic arrest of CHO cells after 16 hours of exposure to the drug. The mitotic blockade was demonstrated in flow cytometric DNA histograms as a single peak with twice the DNA content of cells in the Gl peak of untreated controls. Therefore we examined the ability of the C44 anti-colchicine monoclonal antibody to prevent the effects of colchicine at this concentration. Initially, the effect of the anticolchicine antibody was determined by simultaneous addition of antibody and colchicine to cultures of CHO cells. The cell cultures were harvested and stained for analysis 16 hours later. The highest amount of antibody (180 jLg) was approximately equimolar with colchicine. The other antibody concentrations were twofold dilutions of the equimolar concentration. The percent inhibition of the colchicine-induced mitotic blockade follows a typical doseresponse relationship (Figure 1). An equimolar dose of antibody completely inhibited colchicine-induced mitotic blockade. At half that antibody concentration, ie, one mole of binding site per mole colchicine, approximately 60% inhibition was obtained. As the antibody was diluted, progressively less inhibition of the colchicine-induced mitotic arrest was observed. The DNA content histogram obtained from flow cytometry for cultures treated with colchicine and equimolar antibody were indistinguishable from the histogram obtained with untreated control cells.
Time Course of Recovery from Colchicine Mitotic Arrest The time course of recovery from mitotic arrest was examined by exposing cultures to 2.5 X 10-7 mol/l colchicine for 16 hours followed by the addition of equimolar antibody. The DNA profiles of triplicate cultures then were measured by flow cytofluorimetry every 2 hours after antibody addition for 16 hours. One further set of triplicate cultures was examined 32 hours after antibody addition (48 hours after colchicine). Figure 2 presents DNA and volume histograms obtained during the course of the experiment. The lower panel represents the DNA content
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Figure 1. Relationship between antibody concentration and inhibition of colchicine-induced mitotic arrest. Antibody and colchicine were added to cultures simultaneously and DNA content measured 16 hours later. The highest concentration of antibody 36 Mg/ml is approximately equimolar with colchicine. Percent Inhibition ofcolchicine mitotic arrest in the presence of antibody was calculated using thefollowingformula: G2MA4b -G2Mo % Inhibition = 1oo 1-
G2AIAh G2Mo]
G2MAb = percentage of cells in mitotic block in the presence of anti-colchicine antibody + colchicine; G2Mcol = percentage of cells in mitotic block with colchicine; G2Mo = percentage of cells in mitotic block in negative controls. Results present the mean % inhibition (±SE)for triplicate cultures.
of normal cycling cells compared with that for mitotically blocked cells. At 2 and 4 hours after antibody addition, there is no evidence for cells with 1 N DNA. Both colchicine and colchicine plus antibody-treated cells show only the single 2 N DNA peak associated with mitotic block. Only at 6 hours after antibody does the first evidence of reversal appear. A small peak with 1 N DNA content appears. This peak increased at subsequent times up to 32 hours. However, even 32 hours after antibody treatment (48 hours exposure to colchicine) there was sustained mitotic arrest within the cell population. After 48 hours' colchicine exposure, approximately 60% of the cell population was arrested in the G2 and M phases of the cell cycle with colchicine plus antibody, compared with approximately 100% with colchicine treatment alone and 40% in untreated controls. Cultures of cells treated with colchicine alone showed no sign of recovery over the entire 48 hours of colchicine exposure. Twenty-four hours after colchicine treatment, the Coulter volume histogram began to broaden, indicating an increase in the mean cell volume. The volume histogram continued to broaden for the remainder of the experiment. Additionally, there was an increase in the fraction of cells containing greater than twice the normal DNA content. DNA histograms measured 48 hours after colchicine,
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HOURS
AFTER COLCHICINE
(HOURS AFTER ANTIBODY)
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treated with antibody 16 hours after colchicine contained more cells compared with colchicine treatment alone, indicating recovery from colchicine mitotic blockade. However the presence of multinucleate cells even with antibody treatment, compared with negative controls, suggested that not all cells were able to recover from colchicine effects.
18 (2)
16 DNA VOLUME Figure 2. Reversal of colchicine-indtuced mitotic blockade with equiimolar anti-colchicin e antibody. Cells were exposed to colchicinefbr 16 hours before antibody treatment. Asshown in the first histogram, colchicine-treated cells were blocked in mitosis (-) compared with negative controls ( ) at 16hours. Histograms for DNA and Coulter volume distributions were measured at 2-hour intervals for antibody-treated cultures --- -) and for cultuirevs treated with colchicin e alone (-). ...
and not treated with antibody, showed no discrete peaks, but was a broad band, indicating different multiplicities of DNA content. These cultures, and antibody treated cultures, were examined microscopically (Figure 3). Compared with exponentially growing cell cultures, colchicine treatment for 48 hours led to a high frequency of enlarged multinucleate cells. Colchicine treatment also prevented any increase in cell number. Cultures that had been
The previous studies suggested incomplete reversal of colchicine effects when antibody addition is delayed 16 hours after colchicine. The effect of time of antibody addition after colchicine on the DNA content and cell volume was therefore examined. The FACSIV was used to quantify cell numbers arrested in mitosis and to examine the changes in cell volume found in previous experiments. Cells were exposed to colchicine for a total of 48 hours before analysis. An equimolar concentration of anti-colchicine antibody was added to one set of triplicate cultures every 2 hours up to 16 hours after colchicine addition. Finally the number of cells present in each culture at 48 hours after colchicine treatment was recorded for each set of cultures. As shown in Figure 4, when antibody was added at 2, 4, or 6 hours after colchicine, there was complete reversal of mitotic blockade. Similarly, at 2, 4, and 6 hours, no significant changes in the mean small angle forward light scatter were observed (Figure 5), indicating negligible cell volume changes.14 When antibody was added 8 hours or later after colchicine, incomplete reversal of DNA and volume changes were found. Thus, at 6, 8, 10, 12, 14, and 16 hours after colchicine there are progressively more cells with DNA content greater than 1 N and a progressive increase in cell volume. The increase in the number of cells during the 48-hour course of the experiment was dependent on the time of addition of antibody after colchicine treatment (Figure 6). However, even when antibody was added to cultures at 2, 4, or 6 hours, when negligible changes in DNA content and cell volume had been observed, there were fewer cells present relative to untreated exponentially growing cultures. The number of cells progressively declined with later time points of antibody addition. Colchicine treatment alone prevented any increase in cell number, indicating a complete block of cell division, during the 48-hour course of exposure to the drug.
Cytogenetic Effects of Colchicine Treatment Standard chromosomal staining techniques were used to examine the chromosomal effects of colchicine and the
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Figure 3. Microscopic exain /nation after 48b ours f unltreated ciultuires of cell.s (A), comnpared wvith cells treated uwith co/chic/ine ]b)r 16 hours before anitibodCyJ addition (B), anzd cells treated witb colchici/e al/one for 48 hours (C). Colchicinetreated cells were enzlaIRged an7d conItainced multiple nucleti. Antibody treatment 16' hours after colchic/ine led to ano increase in cell numn ber, although some multinucleate cells persisted. Magnificatioln, 750X.
completeness of reversal by antibody. Colchicine (2.5 X 10-7 mol/l) was added to three pairs of CHO cultures seeded as described previously. Equimolar anti-colchi-
cine antibody was added to one pair of cultures 4 hours after coichicine treatment, and to a second pair of cultures 16 hours after colchicine treatment. Cultures were har-
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X INHIBITION OF COLCHICINE
-17
CELL NUMBER -6
INDUCED
MITOTIC ARREST
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v
TIME OF ANTIBOOY ADDITION (HRS AFTER COLCHICINE)
Figure 4. Inhibition ofcolchicine-induced mitotic arrest by antibody as afunction time. Colchicine was added at time 0 and after the designatedperiod, antibody was added. DNA content was measured 48 hours after colchicine treatment. Percentage inhibition of mitotic arrest (meatn ± SE) was calculated as describedfor Figure 1.
vested 48 hours after colchicine treatment for cytogenetic analysis. Cultures exposed to colchicine for 48 hours in the absence of antibody contained a high percentage of polyploid cells. Table 1 compares the percentage of polyploid nuclei for colchicine-treated cultures and cultures treated with antibody after colchicine treatment. When antibody is added 4 hours after colchicine, the mitotic index and percentage of polyploid nuclei are low and indistinguishable from untreated controls. When antibody addition is delayed until 16 hours, there is a small increase (from 0.6 to 2.2) in mitotic index and more than two thirds of the nuclei are polyploid, relative to 2% to 4% polyploid nuclei in untreated controls. With colchicine treatment alone, there is a larger increase (from 0.6 to 10.6) in mitotic index, and essentially all nuclei are polyploid.
45-
X INCREASE IN 25/ MEAN ANGLE OF LIGHT SCATTER
2D,
15.
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4
6
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TIME OF ANTIBODY ADDITION (HRS AFTER COLCHICINE)
Figure 5. Increase in mean angle offorward light scatter as a function of delay in time ofantibody addition after the addition of colchicine to the culture. Measurements were made 48 hours after colchicine treatment. Percent Increase (mean ± SE) is measured relative to untreated control cultures.
OL
16
Figure 6 Effect of delay in time of antibody addition on reversal of colchicine effects on cell number. Colchicine was added at time 0, the delay (hours) in antibody addition is given on the abscissa anid cell numbers (Mean ± SE) were determined at 48 hours. T= OHR, number ofcells at initiation ofthe experiment (all other samples counted at 48 hours); EXPO, number of cells in conitrol, noncolchicine-treated; Col, number of cells after treatment with colchicinie alone.
In addition to the polyploidy induced by colchicine treatment, Figure 7 shows that coichicine treatment resulted in chromosomal aberrations. Aberrations observed included chromosome and chromatid breaks, rings, dicentrics, and exchanges.
Discussion When colchicine and equimolar anti-colchicine antibody are added simultaneously to cultures of CHO cells, the cellular effects of colchicine are completely inhibited. This result is consistent with the high affinity of the anti-colchicine antibody for colchicine (Ka = 1.5 X 109 mol/l-1)2 compared with estimates for the affinity of the colchicine tubulin interaction (Ka = 1 X 106 to 2 X 107 mol/1-1).10,11 Furthermore the antibody is capable of reversing estabTable 1. Effect ofAnti-colchicine Antibody on Colchicine-induced Polyploidy % diploid Mitotic nuclei indext 0.4 98 Negative controls 0.8 96 7 9.8 Colchicine (48 hours) 11.5 6 0.8 Colchicine (48 hrs) 100 + MAb 4 hours after 1.1 colchicine 99 Colchicine (48 hours) 2.3 33 + MAb 16 hours after 2.1 colchicine 28
% polyploid nuclei
2 4
93 94 0 1 67
72
The Mitotic Index represents the percent of cells in mitosis. Data from two separate cell cultures are shown. *
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I B
Figure 7. Lacto-aceto-orcein-stained metaphases from untreated controls (A), cells treated witb colchicine for 48 hours (B, C) showing dicentric chromosomes (d), and one ring chromosome (r). Magnification, X 1700.
lished colchicine-induced mitotic blockade. When CHO cells were arrested in mitotic block for 16 hours and then exposed to antibody, a population of cells were able to proceed through anaphase and return to a normal diploid state. A 6-hour delay was required before a significant number of diploid cells were observed. Part of this time
may be required for complete removal of colchicine from its cellular binding site. It is probable, however, that most of this lag represents a period of reassembly of the mitotic spindle and reorganization of the cytoskeletal apparatus for successful cell division. However, not all cells were able to recover from colchicine's effects when antibody
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treatment is delayed for 16 hours after coichicine treatment. The colchicine treatment therefore must have led to some irreversible cell changes. These studies also examined the time-dependent extent of recovery from colchicine mitotic blockade. Up to 6 hours after colchicine treatment, an essentially complete reversal of colchicine effects are observed. Our results show that even after only 2 hours' exposure to colchicine there is a reduction in the number of cells measured 48 hours later. There is further reduction in cell number with 4 and 6 hours of colchicine exposure. It is likely that these reductions in cell number reflect the length of time the exponentially growing cells were removed from their normal cell cycling. This suggests that by 2 hours the colchicine has already entered the cell, bound to tubulin, and initiated metaphase arrest in those cells entering mitosis. Previous studies of colchicine binding to purified tubulin show equilibration times of 20 to 90 minutes at 370C.10.11.15 This supports the contention that after 2 hours of colchicine exposure the drug had already bound intracellularly. The lack of colchicine-induced effects on mitosis observed with early antibody addition therefore may be considered a true reversal of colchicine-tubulin interaction. Anti-colchicine antibody added to cultures more than 6 hours after colchicine failed to completely reverse colchicine-induced cell changes. With greater delays in the time of antibody addition, there was sustained mitotic arrest, evidence of increased cellular volume, and polyploidy. Even up to 16 hours after colchicine, there was, however, a small population of cells that escaped the effects of colchicine regardless of the time at which antibody was added. Nevertheless, even if colchicine binding to tubulin remains reversible, prolonged exposure to colchicine leads to changes in the majority of cells, which no longer can be reversed by removal of the drug. Cytogenetic studies demonstrated that cells exposed to colchicine for 48 hours sustained a high frequency of chromosomal aberrations. These aberrations include structural chromosomal breakage. The clastogenic effect of demecolcine, a desacetyl analog of colchicine, has previously been described.'6 However the mechanism by which these drugs, which are known to interfere with the mitotic spindle, induce chromosomal breakage is as yet unknown. Interference with DNA synthesis has been suggested as a possible explanation for chromosomal breakage seen with demecolcine.16 The mitotic index measured after 48 hours' colchicine exposure was only about 10%. This contrasts with the flow cytometry data indicating that approximately 100% of cells contained twice or more than twice the normal DNA content. The low mitotic index therefore implies that, although cells were polyploid, these polyploid cells are not in metaphase.
Continued colchicine exposure also led to the appearance of giant multinucleate cells. Cytogenetic analysis of these cells showed them to contain up to four times the normal chromosome complement. These results indicate that synthesis of DNA continued while spindle formation, which is required for chromosomal separation, was inhibited. The high numbers of chromosomes within these cells may account for the observed changes in Coulter volume and mean small-angle light scatter. Microscopic examination of the cells showed them to be grossly enlarged and contain many packets of nuclear material. Thus, the increase in colchicine-induced polyploidy and structural chromosomal breakage may be the basis of irreversible cell damage and may explain the failure of the C44 anti-colchicine antibody to completely reverse the effect of colchicine at later times. We cannot exclude other possible mechanisms for irreversible cell damage. Colchicine has been shown to inhibit protein secretion in several cell types.17'20 It therefore is conceivable that colchicine treatment may also cause cells to fill with newly synthesized proteins. The CHO-Kl cell is not a predominately secretory cell, but this effect on protein export might be a problem in other secretory cell types. Studies employing slowly dividing cells with an easily definable protein product would further clarify this point. The results of this study demonstrate in vitro the feasibility of using an anti-colchicine monoclonal antibody for the treatment of colchicine toxicity. The CHO-Kl cell is.a rapidly dividing cell line (approximately 9-hour doubling time) and thus may provide a suitable model for the toxic effects of colchicine on other rapidly dividing cells, such as are found in the bone marrow and the gastrointestinal tract. However these studies have uncovered a potentially serious problem for the treatment of colchicine poisoning: a time-dependent increase in irreversibility of colchicine effects. The successful reversal of colchicine overdosage in vivo may depend on administration of antibody before the development of significant irreversible, and ultimately lethal, cell changes. In vivo studies are ongoing in our laboratory, examining rescue from colchicine poisoning with the anti-colchicine antibody and the significance of the time of antibody administration for successful recovery.
References 1. Roberts WN, Liang MH, Stern SH: Colchicine in acute gout: Reassessment of the risks and benefits. JAMA 1987, 257:
1920-1922 2. Wallace SL, Singer JZ: Systemic toxicity associated with the intravenous administration of colchicine. J Rheumatol 1988, 15:495-499
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3. Halkin H, Dany S, Greenwald M, Shnaps Y, Tirosh M: Colchicine kinetics in patients with familial Mediterranean fever. Clin Pharmacol Ther 1980, 28:82-87 4. Chang Y-H, Silverman SL, Paulus HE: Colchicine. In Drugs for Rheumatic Disease. Edited by H Paulus, D Furst, S Dromgoole. New York, Churchill Livingstone, 1987, pp 431 442 5. Kaplan MM, Alling D, Zimmerman HJ, Wolfe HJ, Sepersky RA, Hirsch GS, Elta GH, Glick RA, Eagen KA: A prospective trial of colchicine for primary biliary cirrhosis. N Engl J Med 1986,315:1448-1454 6. Malawista SE: Colchicine: A common mechanism for its antiinflammatory and anti-mitotic effects. Arthritis Rheum 1968, 11:191-197 7. Wenger TL, Butler VP, Haber E, Smith TW: Treatment of 63 severely digitalis toxic patients with digoxin specific antibody fragments. J Am Coll Cardiol 1985, 5:118A-123A 8. Borisy G, Taylor EW: The mechanism of action of colchicine: Binding of 3H colchicine to cellular protein. J Cell Biol 1967,
34:525-533 9. Wilson L, Friedkin M: The biochemical events of mitosis 11: The in vivo and in vitro binding of colchicine in grasshopper embryos and its possible relation to inhibition of mitosis. Biochemistry 1967, 6:3126-3135 10. Bhattacharya B, Wolff J: Anion induced increases in the rate of colchicine binding to tubulin. Biochemistry 1976, 15: 2283-2288 11. Garland DL: Kinetics and mechanism of colchicine binding to tubulin: Evidence for a ligand-induced conformational change. Biochemistry 1978,17:4266-4272 12. Edmond Rouan SK, Otterness IG, Cunningham AC, Rhodes CT: Specific, high affinity colchicine binding monoclonal anti-
bodies: Development and characterization of the antibodies. Hybridoma 1989,8:435-448 13. Taylor JW: A rapid single step staining technique for DNA analysis by flow microfluorimetry. J Histochem Cytochem
1980,28:1021-1024 14. Shapiro HM: 'Parameters and Probes' in Practical Flow Cytometry. New York, Alan R. Liss, 1985, pp 84-86 15. Deery WJ, Weisenberg RC: Kinetic and steady state analysis of microtubules in the presence of colchicine. Biochemistry 1981, 20:2316-2324 16. Satya-Prakash KL, Liang JC, Hsu TC, Johnston DA: Chromosome aberrations in mouse bone marrow cells following treatment in vivo with vinblastine and colcemid. Environ Mutagen 1986, 8:273-282 17. Antoine JC, Maurice M, Feldman G, Avraneas S: In vivo and in vitro effects of colchicine and vinblastine on the secretory process of antibody producing cells. J Immunol 1980, 125:
1939-1949 18. Patzelt C, Brown D, Jeanrenaud B: Inhibitory effect of colchicine on amylase secretion by rat parotid glands: Possible localization in the golgi area. J Cell Biol 1977, 73:578-593 19. Wright DG, Malawista SE: Mobilization and extracellular release of granular enzymes from human leukocytes during phagocytosis: Inhibition by colchicine and cortisol but not by salicylate. Arthritis Rheum 1973,16:749-758 20. Dielgelmann RF, Peterkofosky B: Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs. Proc Natl Acad Sci USA 1972, 69:892-896
Acknowledgment The authors thank Paula A. Muehlbauer for technical assistance.
Reversal of Colchicine-induced Mitotic Arrest in Chinese Hamster Cells with a Colchicinespecific Monoclonal Antibody
S. K. Edmond Rouan,* 1. G. Otterness,t A. C. Cunningham,t H. E. Holden,t and C. T. Rhodes* From the Department of Pharmaceutics, University of Rhode Island, Kingston, Rhode Island*; and the Department ofImmunology, and Infectious Diseases, Pfizer Central Research, Groton, Cotnnecticutt
The ability of a high-affinity colchicine-binding monoclonal antibody to reverse the effects of colchicine on Chinese hamster ovary cells was investigated. Using flow cytometry, a complete mitotic blockade was demonstrated after 16 hours with 2.5 X 10-7 mol/l (molar) colchicine. Colchicine-induced changes were reversible when equimolar antibody was added simultaneously with or up to 6 hours after colchicine. With further delay in addition ofantibody, aprogressive irreversible increase in mitotic blockade and increase in mean cell size was observed. Prolonged colchicine exposure, without antibody reversal, led to polyploidy and structural chromosome breakage. Early antibody reversal restored cells to the diploid state, whereas delayed reversal resulted in a time-dependent increase in polyploidy. Colchicine-induced polyploidy and chromosomal aberrations may be the basis for both colchicine toxicity and the time-dependent increase in irreversibility of colchicine effects. (Am JPathol 1990, 13 7:779- 787)
Colchicine is a potent drug with a narrow therapeutic range. Recent reports of colchicine-related toxicity and fatality have given rise to increasing concern about its clinical use.'2 There is no effective treatment for colchicine intoxication. Standard treatments for drug overdose, such as exchange dialysis, fail to work because of low plasma concentrations (1.5 to 2 ng/ml after a typical 1-mg oral dose) and colchicine's rapid intracellular sequestration.3 Nevertheless, colchicine continues to play a valuable role
in the treatment of acute gout and is effective in a variety of rheumatologic disorders, including familial Mediterranean fever, amyloidosis, and Behcet's disease.4 In addition, colchicine may provide some long-term benefit to patients with primary biliary cirrhosis.5 The mechanism of colchicine's therapeutic effectiveness is not completely understood, although Malawista6 has suggested that the anti-inflammatory effects are related to interference with microtubule formation in cells. Recently digoxin-specific antibody Fab (antigen-binding) fragments have been used to successfully treat lifethreatening digitalis intoxication.7 The digoxin-specific antibody acts by binding digoxin in the extracellular fluid. The consequent reduction in the extracellular free pool of drug produces a concentration gradient that promotes release of digoxin from its receptor sites. Colchicine, like digoxin, is a highly potent drug with a narrow therapeutic range. Colchicine binds to tubulin, causing disruption of the mitotic spindle apparatus and, consequently, mitotic arrest.89 Estimates of the affinity constant for colchicinetubulin interaction range from 1 X 106 to 2 X 107 mol/l (molar)-1.10"1 Thus a colchicine-specific antibody with a higher affinity constant would be expected to prevent or reverse the colchicine-tubulin interaction by lowering the effective colchicine concentration. We have recently prepared a specific high-affinity colchicine-binding monoclonal antibody.12 In the present report, we have assessed the ability of this antibody to reverse colchicine's effects in vitro. We also report the time dependent accumulation of chromosomal abnormalities associated with exposure of Chinese hamster ovary cells to colchicine. This research forms part of the doctoral dissertation of S. K. Edmond Rouan. Her work has been supported by the United States Pharmacopeial Convention Inc., Richardson Vicks Inc., and by a fellowship from the University of Rhode Island. Current address: Smith Kline and French Laboratories, 709 Swedeland Rd., King of Prussia, PA 19406. Accepted for publication April 25, 1990. Address reprint requests to Dr. I. G. Otterness, Department of Immunology and Infectious Diseases, Pfizer Central Research, Groton, CT 06340.
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Materials and Methods
DNA Staining
Reagents
Intracellular DNA content was analyzed by flow cytometry using propidium iodide as a fluorescent DNA probe.'3 Cells were harvested by treatment with trypsin (1 ml, 0.25%) for 5 minutes and collected by centrifugation. The cells then were resuspended in 2 ml culture medium and 0.4 ml of a solution of propidium iodide (250 ,ug/ml [Sigma]), and 1% vol/vol Triton X-1 00 (Sigma), prewarmed to 37°C, was added. The cells then were incubated for 15 minutes at room temperature in the dark. Stained cells were stored on ice, in the dark, and analyzed by flow cytometry within 2 hours of stain addition.
Coichicine was obtained from Sigma Chemicals, St. Louis, MO. The monoclonal anti-colchicine antibody C44 has been shown to bind colchicine with high affinity and with high specificity.12 As previously described, the hybridoma clone secreting C44 antibody was derived from fusion of the NS 1 murine myeloma cell line and a splenic lymphocyte from a BALB/c mouse immunized with a colchicine-keyhole limpet hemocyanin conjugate. The C44 hybridoma cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mmol/l (millimolar) L-glutamine, 1 mmol/l, sodium pyruvate, 100 U/ml penicillin, 100 ,ug/ml streptomycin, and 1 mmol/l minimum essential medium (MEM) nonessential amino acids solution. Ascites fluid containing high concentrations of C44 was obtained by priming BALB/c female mice with 0.5 ml pristane and at least 5 days later injecting 1 X 106 C44 hybridoma cells intraperitoneally. Ten to twenty-one days later, ascites fluid was drained every 2 days until the mice died. For these experiments, C44 antibody in ascites fluid was concentrated by precipitation with an equal volume of a saturated solution of ammonium sulfate. The concentration of colchicine-specific antibody was determined by enzyme-linked immunosorbent assay (ELISA), using a highly purified C44 antibody preparation as a standard.12 The antibody preparation used in these experiments contained approximately 8 mg/ml colchicine-specific antibody.
Cells and Culture Conditions Chinese hamster ovary cells (CHO-Kl) from the American Type Culture Collection were maintained in Ham's F12 medium (GIBCO, Grand Island, NY) supplemented with 5% vol/vol heat-inactivated fetal calf serum (GIBCO), 100 U/ml of penicillin and 100 ,ug/ml of streptomycin. For cell cycle analysis, cells in exponential growth were seeded at a density of 105 cells in 25 cm2 cell culture flasks containing 5 ml of culture medium. The cells were incubated in a humidified atmosphere containing 5% carbon dioxide for 40 hours at 370C before drug and antibody treatments. Vinblastine sulfate (1 ug/ml for 16 hours) treated cultures provided a positive control for mitotic block and untreated exponentially growing cells were the negative control for each experiment. All treatments were examined as triplicate cultures.
Flow Cytometric Analysis The FACS analyzer (Becton Dickinson, Mountain View, CA) and the FACS IV (Becton Dickinson) were used for quantitative fluorescent analyses of the cell cycle. Excitation light of 488 nm was used and emission wavelengths greater than 580 nm were monitored. Fluorescence data from the FACS analyzer was handled by determining the G1 and G2 + M peaks based on control samples of these two populations. The Gl control was a mitotic shake-off allowed to reattach for 2 hours. The G2 + M control consisted of cells arrested in mitosis by treatment with 1 ,ug/ ml vinblastine sulfate for 16 hours. Quantitation was achieved by setting a cursor at the G2 + M peak, then finding the number of cells from this point to the end of the histogram display. This number of cells is then multiplied by two and divided by the total number of cells represented by the histogram to obtain a percentage. The percentage of cells with a threshold fluorescence level greater than 1 X N (G1 peak) DNA content was determined using Disp 2D (Consort 40, Becton Dickinson) analysis of data generated by the FACS IV. Although S phase is included in this percentage, its contribution in mitotically arrested cultures is negligible. Relative cell volume changes from Coulter volume (FACS analyser) and mean small angle forward light scatter measurements (FACS IV) parameters also were quantitated from the mean channel numbers of the histogram display. A shift to higher channel number correlates with increasing cell size.
Cytologic Experiments Cells were collected by trypsinization and harvested using 1% sodium citrate for 7 minutes as hypotonic pretreatment and 3:1 methanol:acetic acid as fixative. Cells were dropped onto cold wet slides and flame dried. Cells were stained with 2% lacto-aceto-orcein for 2 hours and cover-
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slips mounted with Euperal (Asco Laboratories, Manchester, UK). At least 1000 cells were examined and the number of mitoses and the number of diploid and polyploid nuclei determined. Chromosomal aberrations also were noted.
80 70'LI co
Results
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82 A405
Relationship Between Antibody Concentration and Inhibition of Colchicineinduced Mitotic Arrest Preliminary studies demonstrated that 2.5 X 10-7 molar (0.1 jig/ml) colchicine led to a virtually complete mitotic arrest of CHO cells after 16 hours of exposure to the drug. The mitotic blockade was demonstrated in flow cytometric DNA histograms as a single peak with twice the DNA content of cells in the Gl peak of untreated controls. Therefore we examined the ability of the C44 anti-colchicine monoclonal antibody to prevent the effects of colchicine at this concentration. Initially, the effect of the anticolchicine antibody was determined by simultaneous addition of antibody and colchicine to cultures of CHO cells. The cell cultures were harvested and stained for analysis 16 hours later. The highest amount of antibody (180 jLg) was approximately equimolar with colchicine. The other antibody concentrations were twofold dilutions of the equimolar concentration. The percent inhibition of the colchicine-induced mitotic blockade follows a typical doseresponse relationship (Figure 1). An equimolar dose of antibody completely inhibited colchicine-induced mitotic blockade. At half that antibody concentration, ie, one mole of binding site per mole colchicine, approximately 60% inhibition was obtained. As the antibody was diluted, progressively less inhibition of the colchicine-induced mitotic arrest was observed. The DNA content histogram obtained from flow cytometry for cultures treated with colchicine and equimolar antibody were indistinguishable from the histogram obtained with untreated control cells.
Time Course of Recovery from Colchicine Mitotic Arrest The time course of recovery from mitotic arrest was examined by exposing cultures to 2.5 X 10-7 mol/l colchicine for 16 hours followed by the addition of equimolar antibody. The DNA profiles of triplicate cultures then were measured by flow cytofluorimetry every 2 hours after antibody addition for 16 hours. One further set of triplicate cultures was examined 32 hours after antibody addition (48 hours after colchicine). Figure 2 presents DNA and volume histograms obtained during the course of the experiment. The lower panel represents the DNA content
0
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z ~~ 50 a7
20-
3
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9
18
27
36
ANTIBODY (,g/mI)
Figure 1. Relationship between antibody concentration and inhibition of colchicine-induced mitotic arrest. Antibody and colchicine were added to cultures simultaneously and DNA content measured 16 hours later. The highest concentration of antibody 36 Mg/ml is approximately equimolar with colchicine. Percent Inhibition ofcolchicine mitotic arrest in the presence of antibody was calculated using thefollowingformula: G2MA4b -G2Mo % Inhibition = 1oo 1-
G2AIAh G2Mo]
G2MAb = percentage of cells in mitotic block in the presence of anti-colchicine antibody + colchicine; G2Mcol = percentage of cells in mitotic block with colchicine; G2Mo = percentage of cells in mitotic block in negative controls. Results present the mean % inhibition (±SE)for triplicate cultures.
of normal cycling cells compared with that for mitotically blocked cells. At 2 and 4 hours after antibody addition, there is no evidence for cells with 1 N DNA. Both colchicine and colchicine plus antibody-treated cells show only the single 2 N DNA peak associated with mitotic block. Only at 6 hours after antibody does the first evidence of reversal appear. A small peak with 1 N DNA content appears. This peak increased at subsequent times up to 32 hours. However, even 32 hours after antibody treatment (48 hours exposure to colchicine) there was sustained mitotic arrest within the cell population. After 48 hours' colchicine exposure, approximately 60% of the cell population was arrested in the G2 and M phases of the cell cycle with colchicine plus antibody, compared with approximately 100% with colchicine treatment alone and 40% in untreated controls. Cultures of cells treated with colchicine alone showed no sign of recovery over the entire 48 hours of colchicine exposure. Twenty-four hours after colchicine treatment, the Coulter volume histogram began to broaden, indicating an increase in the mean cell volume. The volume histogram continued to broaden for the remainder of the experiment. Additionally, there was an increase in the fraction of cells containing greater than twice the normal DNA content. DNA histograms measured 48 hours after colchicine,
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HOURS
AFTER COLCHICINE
(HOURS AFTER ANTIBODY)
48 (32)
i
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Effect of Time of Antibody Addition on Recovery from Colchicine Treatment 30 (14)
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treated with antibody 16 hours after colchicine contained more cells compared with colchicine treatment alone, indicating recovery from colchicine mitotic blockade. However the presence of multinucleate cells even with antibody treatment, compared with negative controls, suggested that not all cells were able to recover from colchicine effects.
18 (2)
16 DNA VOLUME Figure 2. Reversal of colchicine-indtuced mitotic blockade with equiimolar anti-colchicin e antibody. Cells were exposed to colchicinefbr 16 hours before antibody treatment. Asshown in the first histogram, colchicine-treated cells were blocked in mitosis (-) compared with negative controls ( ) at 16hours. Histograms for DNA and Coulter volume distributions were measured at 2-hour intervals for antibody-treated cultures --- -) and for cultuirevs treated with colchicin e alone (-). ...
and not treated with antibody, showed no discrete peaks, but was a broad band, indicating different multiplicities of DNA content. These cultures, and antibody treated cultures, were examined microscopically (Figure 3). Compared with exponentially growing cell cultures, colchicine treatment for 48 hours led to a high frequency of enlarged multinucleate cells. Colchicine treatment also prevented any increase in cell number. Cultures that had been
The previous studies suggested incomplete reversal of colchicine effects when antibody addition is delayed 16 hours after colchicine. The effect of time of antibody addition after colchicine on the DNA content and cell volume was therefore examined. The FACSIV was used to quantify cell numbers arrested in mitosis and to examine the changes in cell volume found in previous experiments. Cells were exposed to colchicine for a total of 48 hours before analysis. An equimolar concentration of anti-colchicine antibody was added to one set of triplicate cultures every 2 hours up to 16 hours after colchicine addition. Finally the number of cells present in each culture at 48 hours after colchicine treatment was recorded for each set of cultures. As shown in Figure 4, when antibody was added at 2, 4, or 6 hours after colchicine, there was complete reversal of mitotic blockade. Similarly, at 2, 4, and 6 hours, no significant changes in the mean small angle forward light scatter were observed (Figure 5), indicating negligible cell volume changes.14 When antibody was added 8 hours or later after colchicine, incomplete reversal of DNA and volume changes were found. Thus, at 6, 8, 10, 12, 14, and 16 hours after colchicine there are progressively more cells with DNA content greater than 1 N and a progressive increase in cell volume. The increase in the number of cells during the 48-hour course of the experiment was dependent on the time of addition of antibody after colchicine treatment (Figure 6). However, even when antibody was added to cultures at 2, 4, or 6 hours, when negligible changes in DNA content and cell volume had been observed, there were fewer cells present relative to untreated exponentially growing cultures. The number of cells progressively declined with later time points of antibody addition. Colchicine treatment alone prevented any increase in cell number, indicating a complete block of cell division, during the 48-hour course of exposure to the drug.
Cytogenetic Effects of Colchicine Treatment Standard chromosomal staining techniques were used to examine the chromosomal effects of colchicine and the
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Figure 3. Microscopic exain /nation after 48b ours f unltreated ciultuires of cell.s (A), comnpared wvith cells treated uwith co/chic/ine ]b)r 16 hours before anitibodCyJ addition (B), anzd cells treated witb colchici/e al/one for 48 hours (C). Colchicinetreated cells were enzlaIRged an7d conItainced multiple nucleti. Antibody treatment 16' hours after colchic/ine led to ano increase in cell numn ber, although some multinucleate cells persisted. Magnificatioln, 750X.
completeness of reversal by antibody. Colchicine (2.5 X 10-7 mol/l) was added to three pairs of CHO cultures seeded as described previously. Equimolar anti-colchi-
cine antibody was added to one pair of cultures 4 hours after coichicine treatment, and to a second pair of cultures 16 hours after colchicine treatment. Cultures were har-
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5-
X INHIBITION OF COLCHICINE
-17
CELL NUMBER -6
INDUCED
MITOTIC ARREST
x 10
T I
2
31
01-
1. ff
I
2
6
4
8
10
12
14
16
I -- ----- IT.OR EfW 2 4 6 8 10 12 14 TIME OF ANTIBODY ADDITION POST COILCHICINE TREATMENT (HOUR)
18
v
TIME OF ANTIBOOY ADDITION (HRS AFTER COLCHICINE)
Figure 4. Inhibition ofcolchicine-induced mitotic arrest by antibody as afunction time. Colchicine was added at time 0 and after the designatedperiod, antibody was added. DNA content was measured 48 hours after colchicine treatment. Percentage inhibition of mitotic arrest (meatn ± SE) was calculated as describedfor Figure 1.
vested 48 hours after colchicine treatment for cytogenetic analysis. Cultures exposed to colchicine for 48 hours in the absence of antibody contained a high percentage of polyploid cells. Table 1 compares the percentage of polyploid nuclei for colchicine-treated cultures and cultures treated with antibody after colchicine treatment. When antibody is added 4 hours after colchicine, the mitotic index and percentage of polyploid nuclei are low and indistinguishable from untreated controls. When antibody addition is delayed until 16 hours, there is a small increase (from 0.6 to 2.2) in mitotic index and more than two thirds of the nuclei are polyploid, relative to 2% to 4% polyploid nuclei in untreated controls. With colchicine treatment alone, there is a larger increase (from 0.6 to 10.6) in mitotic index, and essentially all nuclei are polyploid.
45-
X INCREASE IN 25/ MEAN ANGLE OF LIGHT SCATTER
2D,
15.
5-
2
4
6
8
10
12
14
16
18
TIME OF ANTIBODY ADDITION (HRS AFTER COLCHICINE)
Figure 5. Increase in mean angle offorward light scatter as a function of delay in time ofantibody addition after the addition of colchicine to the culture. Measurements were made 48 hours after colchicine treatment. Percent Increase (mean ± SE) is measured relative to untreated control cultures.
OL
16
Figure 6 Effect of delay in time of antibody addition on reversal of colchicine effects on cell number. Colchicine was added at time 0, the delay (hours) in antibody addition is given on the abscissa anid cell numbers (Mean ± SE) were determined at 48 hours. T= OHR, number ofcells at initiation ofthe experiment (all other samples counted at 48 hours); EXPO, number of cells in conitrol, noncolchicine-treated; Col, number of cells after treatment with colchicinie alone.
In addition to the polyploidy induced by colchicine treatment, Figure 7 shows that coichicine treatment resulted in chromosomal aberrations. Aberrations observed included chromosome and chromatid breaks, rings, dicentrics, and exchanges.
Discussion When colchicine and equimolar anti-colchicine antibody are added simultaneously to cultures of CHO cells, the cellular effects of colchicine are completely inhibited. This result is consistent with the high affinity of the anti-colchicine antibody for colchicine (Ka = 1.5 X 109 mol/l-1)2 compared with estimates for the affinity of the colchicine tubulin interaction (Ka = 1 X 106 to 2 X 107 mol/1-1).10,11 Furthermore the antibody is capable of reversing estabTable 1. Effect ofAnti-colchicine Antibody on Colchicine-induced Polyploidy % diploid Mitotic nuclei indext 0.4 98 Negative controls 0.8 96 7 9.8 Colchicine (48 hours) 11.5 6 0.8 Colchicine (48 hrs) 100 + MAb 4 hours after 1.1 colchicine 99 Colchicine (48 hours) 2.3 33 + MAb 16 hours after 2.1 colchicine 28
% polyploid nuclei
2 4
93 94 0 1 67
72
The Mitotic Index represents the percent of cells in mitosis. Data from two separate cell cultures are shown. *
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I B
Figure 7. Lacto-aceto-orcein-stained metaphases from untreated controls (A), cells treated witb colchicine for 48 hours (B, C) showing dicentric chromosomes (d), and one ring chromosome (r). Magnification, X 1700.
lished colchicine-induced mitotic blockade. When CHO cells were arrested in mitotic block for 16 hours and then exposed to antibody, a population of cells were able to proceed through anaphase and return to a normal diploid state. A 6-hour delay was required before a significant number of diploid cells were observed. Part of this time
may be required for complete removal of colchicine from its cellular binding site. It is probable, however, that most of this lag represents a period of reassembly of the mitotic spindle and reorganization of the cytoskeletal apparatus for successful cell division. However, not all cells were able to recover from colchicine's effects when antibody
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treatment is delayed for 16 hours after coichicine treatment. The colchicine treatment therefore must have led to some irreversible cell changes. These studies also examined the time-dependent extent of recovery from colchicine mitotic blockade. Up to 6 hours after colchicine treatment, an essentially complete reversal of colchicine effects are observed. Our results show that even after only 2 hours' exposure to colchicine there is a reduction in the number of cells measured 48 hours later. There is further reduction in cell number with 4 and 6 hours of colchicine exposure. It is likely that these reductions in cell number reflect the length of time the exponentially growing cells were removed from their normal cell cycling. This suggests that by 2 hours the colchicine has already entered the cell, bound to tubulin, and initiated metaphase arrest in those cells entering mitosis. Previous studies of colchicine binding to purified tubulin show equilibration times of 20 to 90 minutes at 370C.10.11.15 This supports the contention that after 2 hours of colchicine exposure the drug had already bound intracellularly. The lack of colchicine-induced effects on mitosis observed with early antibody addition therefore may be considered a true reversal of colchicine-tubulin interaction. Anti-colchicine antibody added to cultures more than 6 hours after colchicine failed to completely reverse colchicine-induced cell changes. With greater delays in the time of antibody addition, there was sustained mitotic arrest, evidence of increased cellular volume, and polyploidy. Even up to 16 hours after colchicine, there was, however, a small population of cells that escaped the effects of colchicine regardless of the time at which antibody was added. Nevertheless, even if colchicine binding to tubulin remains reversible, prolonged exposure to colchicine leads to changes in the majority of cells, which no longer can be reversed by removal of the drug. Cytogenetic studies demonstrated that cells exposed to colchicine for 48 hours sustained a high frequency of chromosomal aberrations. These aberrations include structural chromosomal breakage. The clastogenic effect of demecolcine, a desacetyl analog of colchicine, has previously been described.'6 However the mechanism by which these drugs, which are known to interfere with the mitotic spindle, induce chromosomal breakage is as yet unknown. Interference with DNA synthesis has been suggested as a possible explanation for chromosomal breakage seen with demecolcine.16 The mitotic index measured after 48 hours' colchicine exposure was only about 10%. This contrasts with the flow cytometry data indicating that approximately 100% of cells contained twice or more than twice the normal DNA content. The low mitotic index therefore implies that, although cells were polyploid, these polyploid cells are not in metaphase.
Continued colchicine exposure also led to the appearance of giant multinucleate cells. Cytogenetic analysis of these cells showed them to contain up to four times the normal chromosome complement. These results indicate that synthesis of DNA continued while spindle formation, which is required for chromosomal separation, was inhibited. The high numbers of chromosomes within these cells may account for the observed changes in Coulter volume and mean small-angle light scatter. Microscopic examination of the cells showed them to be grossly enlarged and contain many packets of nuclear material. Thus, the increase in colchicine-induced polyploidy and structural chromosomal breakage may be the basis of irreversible cell damage and may explain the failure of the C44 anti-colchicine antibody to completely reverse the effect of colchicine at later times. We cannot exclude other possible mechanisms for irreversible cell damage. Colchicine has been shown to inhibit protein secretion in several cell types.17'20 It therefore is conceivable that colchicine treatment may also cause cells to fill with newly synthesized proteins. The CHO-Kl cell is not a predominately secretory cell, but this effect on protein export might be a problem in other secretory cell types. Studies employing slowly dividing cells with an easily definable protein product would further clarify this point. The results of this study demonstrate in vitro the feasibility of using an anti-colchicine monoclonal antibody for the treatment of colchicine toxicity. The CHO-Kl cell is.a rapidly dividing cell line (approximately 9-hour doubling time) and thus may provide a suitable model for the toxic effects of colchicine on other rapidly dividing cells, such as are found in the bone marrow and the gastrointestinal tract. However these studies have uncovered a potentially serious problem for the treatment of colchicine poisoning: a time-dependent increase in irreversibility of colchicine effects. The successful reversal of colchicine overdosage in vivo may depend on administration of antibody before the development of significant irreversible, and ultimately lethal, cell changes. In vivo studies are ongoing in our laboratory, examining rescue from colchicine poisoning with the anti-colchicine antibody and the significance of the time of antibody administration for successful recovery.
References 1. Roberts WN, Liang MH, Stern SH: Colchicine in acute gout: Reassessment of the risks and benefits. JAMA 1987, 257:
1920-1922 2. Wallace SL, Singer JZ: Systemic toxicity associated with the intravenous administration of colchicine. J Rheumatol 1988, 15:495-499
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3. Halkin H, Dany S, Greenwald M, Shnaps Y, Tirosh M: Colchicine kinetics in patients with familial Mediterranean fever. Clin Pharmacol Ther 1980, 28:82-87 4. Chang Y-H, Silverman SL, Paulus HE: Colchicine. In Drugs for Rheumatic Disease. Edited by H Paulus, D Furst, S Dromgoole. New York, Churchill Livingstone, 1987, pp 431 442 5. Kaplan MM, Alling D, Zimmerman HJ, Wolfe HJ, Sepersky RA, Hirsch GS, Elta GH, Glick RA, Eagen KA: A prospective trial of colchicine for primary biliary cirrhosis. N Engl J Med 1986,315:1448-1454 6. Malawista SE: Colchicine: A common mechanism for its antiinflammatory and anti-mitotic effects. Arthritis Rheum 1968, 11:191-197 7. Wenger TL, Butler VP, Haber E, Smith TW: Treatment of 63 severely digitalis toxic patients with digoxin specific antibody fragments. J Am Coll Cardiol 1985, 5:118A-123A 8. Borisy G, Taylor EW: The mechanism of action of colchicine: Binding of 3H colchicine to cellular protein. J Cell Biol 1967,
34:525-533 9. Wilson L, Friedkin M: The biochemical events of mitosis 11: The in vivo and in vitro binding of colchicine in grasshopper embryos and its possible relation to inhibition of mitosis. Biochemistry 1967, 6:3126-3135 10. Bhattacharya B, Wolff J: Anion induced increases in the rate of colchicine binding to tubulin. Biochemistry 1976, 15: 2283-2288 11. Garland DL: Kinetics and mechanism of colchicine binding to tubulin: Evidence for a ligand-induced conformational change. Biochemistry 1978,17:4266-4272 12. Edmond Rouan SK, Otterness IG, Cunningham AC, Rhodes CT: Specific, high affinity colchicine binding monoclonal anti-
bodies: Development and characterization of the antibodies. Hybridoma 1989,8:435-448 13. Taylor JW: A rapid single step staining technique for DNA analysis by flow microfluorimetry. J Histochem Cytochem
1980,28:1021-1024 14. Shapiro HM: 'Parameters and Probes' in Practical Flow Cytometry. New York, Alan R. Liss, 1985, pp 84-86 15. Deery WJ, Weisenberg RC: Kinetic and steady state analysis of microtubules in the presence of colchicine. Biochemistry 1981, 20:2316-2324 16. Satya-Prakash KL, Liang JC, Hsu TC, Johnston DA: Chromosome aberrations in mouse bone marrow cells following treatment in vivo with vinblastine and colcemid. Environ Mutagen 1986, 8:273-282 17. Antoine JC, Maurice M, Feldman G, Avraneas S: In vivo and in vitro effects of colchicine and vinblastine on the secretory process of antibody producing cells. J Immunol 1980, 125:
1939-1949 18. Patzelt C, Brown D, Jeanrenaud B: Inhibitory effect of colchicine on amylase secretion by rat parotid glands: Possible localization in the golgi area. J Cell Biol 1977, 73:578-593 19. Wright DG, Malawista SE: Mobilization and extracellular release of granular enzymes from human leukocytes during phagocytosis: Inhibition by colchicine and cortisol but not by salicylate. Arthritis Rheum 1973,16:749-758 20. Dielgelmann RF, Peterkofosky B: Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs. Proc Natl Acad Sci USA 1972, 69:892-896
Acknowledgment The authors thank Paula A. Muehlbauer for technical assistance.
