stability of vincristine complexes in cytosols derived from … · to receiving whole-body...

(CANCER RESEARCH 45, 3761-3767, August 1985]

Stability of Vincristine Complexes in Cytosols Derived from Xenografts ofHuman Rhabdomyosarcoma and Normal Tissues of the Mouse1

Janet A. Houghton,2 Larry G. Williams, and Peter J. Houghton

Division of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101

ABSTRACT

The selective action of vincristine (VCR) has been correlatedwith longer retention of the drug in neoplastic tissue comparedwith normal tissues of the mouse (J. A. Houghton, L. G. Williams,P. M. Terranee, and P. J. Houghton, Cancer Res., 44: 582-590,

1984). In order to examine the basis for this differential, thestability of drug-protein complexes was examined further. Thestability of drug-protein complexes formed in cytosols derived

from HxRhl 8 tumors, ileum, liver, kidney, skeletal muscle, blood,brain, spleen, lung, and bone marrow was examined. Protein-bound [3H]VCR was isolated by gel filtration of [3H]VCR-cytosol

mixtures from each tissue except for ileum and blood. Complexes formed in brain and HxRhl 8 cytosols were stable at 37Â°

for at least 2 h; all other complexes were unstable. For liver,kidney, and muscle, half-times of complexes were in a similarorder to the initial rates of elimination of [3H]VCR from thesetissues In vivo but were of shorter duration. The HxRhl 8-[3H]-VCR complex was unstable at 37Â°in the presence of cytosols

prepared from ileum, kidney, liver, and lung. Drug metabolism bythese tissues was not detected in vitro. In the presence of heat-treated extracts from ileum or kidney, [3H]VCR complex was

stable, suggesting that the destabilizing factor may be enzymic.Degradation of 125iodinated tubulin, analyzed by polyacrylamide-

sodium dodecyl sulfate gel electrophoresis, occurred in the presence of ileum but not skeletal muscle or brain cytosols. Thiscorrelated with the stability of HxRhl 8-[3H]VCR complexes. In

the presence of kidney cytosol, however, the molecular weightof 125l-tubulin remained unchanged, suggesting a different mech

anism. Based upon data obtained, cytosols from normal tissuesmay be categorized into three classes: (a) those that formedstable complexes (brain); (b) those that formed unstable complexes but also destabilized preformed complex (ileum, kidney,liver, lung); and (c) tissues that formed unstable complexes butdid not destabilize preformed complex (skeletal muscle, spleen,bone marrow, blood). The degree of instability of complexesformed in cytosols prepared from normal tissues appears tocorrelate with rapid loss of VCR from these tissues in vivo andhence may represent mechanism(s) for the selective action ofthis antineoplastic agent.

INTRODUCTION

VCR3 is one of the most effective agents for the treatment of

pediatrie RMSs (1). There are now considerable data from ourlaboratories and those of others to demonstrate that xenografts

' This work was supported by Grants CH-156 from the American Cancer Society

and CA 38933 and CA 23099 from the National Cancer Institute and by AmericanLebanese Syrian Associated Charities.

2To whom requests for reprints should be addressed.3The abbreviations used are: VCR, vincristine; RMS. rhabdomyosarcoma; SDS,

sodium dodecyl sulfate; HPLC, high-pressure liquid chromatography; CANP, calcium-activated neutral protease.

Received 12/27/84; revised 4/23/85; accepted 4/24/85.

of human cancers provide biologically and biochemically relevantmodels for specific human cancer types in humans (2-6). The

model of RMS developed in these laboratories (7) ranks clinicallyactive agents in the same order as that found in the treatmentof rhabdomyosarcoma (1).

The selective action of VCR appears to be due to specificretention of unchanged drug in RMS tissue (8) and also topersistence in cultured cells (9, 10), whereas rapid eliminationfrom normal tissues (of the mouse) occurs (8). Noble ef al. (11)also showed selective retention of vinblastine in a sensitive ratlymphoma in vivo. Bender et al. (12) reported that the level ofdrug bound at steady state correlated best with the sensitivityin vivo of a series of murine leukemias. However, although eachof these studies have indicated that selectivity may be due toselective drug retention by neoplastic tissues, studies whichhave attempted to understand the basis for this selective phenomenon have not been documented.

Vinca alkaloids have been shown to bind to the microtubulesubunit protein, dimeric tubulin, of mammalian brain (13-15) andalso of human RMS xenografts.4 Binding of these drugs in other

tissues has, however, not been documented. Because microtu-bules and thus, presumably, dimeric tubulin are constituents ofall mammalian cells, it was of interest to determine why VCR(and other dimeric Catharanthus alkaloids) was rapidly eliminatedfrom nonneoplastic tissues. Studies were therefore designed toexamine the stability of binding of [3H]VCR in tissue cytosols

and to elucidate the influence of these parameters on drugretention in vivo.

MATERIALS AND METHODS

Chemicals. [G-3H]Vincristine (specific activity, 12 Ci/mmol) was pur

chased from Moravek Biochemicals, Brea, CA and was purified by HPLCprior to use (16). 125l-Sodium iodide was obtained from Amersham Corp.,

Arlington Heights, IL. Vincristine as the pharmaceutical preparation wasobtained from the hospital pharmacy, and pure VCR was a generous giftof Eli Lilly and Co., Indianapolis, IN. SDS, polyacrylamide, and all reagentsfor gel electrophoresis were obtained from Bio-Rad Laboratories, Rich

mond, CA. All other chemicals were purchased from Sigma ChemicalCo., St. Louis, MO.

Immune Deprivation of Mice. Female CBA/CaJ mice, 4 weeks old,were immune-deprived by thymectomy followed 3 weeks later by i.p.administration of 1-/3-o-arabinofuranosylcytosine (200 mg/kg), 48 h priorto receiving whole-body irradiation (900 rads at 170 rads/min), using a137Cssource (8).

Tumor Line. Tumor HxRhl 8 was established directly as a xenograftfrom a RMS resected in a child who had received no chemotherapy. Thehuman specimen was embryonal RMS with small areas showing alveolarhistology; the xenograft was moderately differentiated, also with embryonal histology (17). Tumor fragments were implanted s.c. in the dorsalflanks of mice at 1 to 2 weeks after irradiation. Animals were used instudies after a further 3 to 4 weeks when tumors were established and

4Unpublishedobservations.

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STABILITY OF VINCRISTINE COMPLEXES

averaged 600 mg in weight.HPLC Analysis of Vinca Alkaloids in Kidney. Whole kidneys were

rapidly excised from mice and were immediately placed on ice. Theywere subsequently homogenized in 5 volumes of 10 mw K2HPO4/KH2PO4containing 10 HIM MgCI2 and 0.1 mw GTP, pH 6.8 (Buffer A) prior tocentrifugation at 12,000 x g for 10 min at 2Â°.Cytosol (225 ^l) and [3H]-VCR (38 pmol) were mixed on ice and subsequently incubated at 37Â°

for 2 h. Acidified ethanol (pH 4.9; 500 p\) was added and, after standingin ice for a further 10 min, the sample was centrifugea at 12,000 x g for10 min. To 500 p\ of supernatant, 5 ng of cold VCR were added, and200 n\ of the preparation were analyzed by HPLC. Under these conditions, VCR was stable (16). The HPLC methodology utilized has beendescribed in detail previously (8,16). Briefly, separation of [3H]VCR and

its metabolites was achieved on a Partisil 5 ODS-3 RAC column (What

man Inc., Clifton, NJ) using a linear gradient from 20% methanol in 10nriM KH2PO4, pH 4.9, to 64% methanol (pH 4.9) over a 60-min period.

The mobile phase was increased to 80% methanol by 65 min. Flow ratewas 1 ml/min; fractions (0.5 ml) were collected for 80 min into 5-ml

minivials, and radioactivity was determined.Isolation of [3H]VCR-Protein Complexes. Tumor HxRh18, ileum,

liver, kidney, skeletal muscle, brain, spleen, and lung were rapidly excisedand placed on ice. Blood was removed from anesthetized mice by cardiacpuncture into a heparinized syringe. Bone marrow was obtained byflushing the femurs and tibias from the hind limbs of 14 mice. Tissueswere washed at 2Â°in Buffer A, blotted dry, and weighed. Heal tissue

was flushed free of feces in 0.9% saline and blotted dry prior to beingslit longitudinally with scissors. The luminal surface was exposed, andsubsequent separation of the mucosa from the muscularis was effectedby scraping with a scalpel blade. Scrapings were washed twice in 0.9%NaCI solution prior to homogenization. Tissue cytosols were preparedby homogenization in cold Buffer A and centrifugation at 100,000 x gfor 60 min at 2Â°.Cytosols from blood and bone marrow were prepared

by mild sonication on ice, followed by similar centrifugation. Cytosols(1.2 ml) containing protein (4 to 6 mg/ml) for HxRh18 and protein (7 to13 mg/ml) for normal tissues were incubated (37Â°, 15 min) to steadystate with [3H]VCR at a concentration of 78 to 271 nw in foil-wrapped

tubes. Protein-bound drug was separated from free by G-25 Sephadexgel filtration of 1-ml samples on PD10 columns (1.4 x 5 cm; Pharmacia,

Inc., Piscataway, NJ) using cold Buffer A. Fractions (0.5 ml, 0.5 min)were counted, and protein was determined by the method of Lowry efa/. (18).

Stability of [3H]VCR-Protein Complexes. Peak fractions of protein-bound [3H]VCR from G-25 Sephadex gel filtration were pooled. Isolated

complex from each tissue (except ileum and blood) was incubated at37Â°in foil-wrapped tubes, and the proportion of radiolabeled complex

that could be retained on two DE-81 discs was determined at intervals

of 30 min. Aliquots (60 to 80 n\) were assayed essentially as describedby Bhattacharyya and Wolff (13), using cold Buffer A as the wash buffer.Protein-bound drug but not free [3H]VCR was retained on the filters. The

stability of complexes was calculated thus:

% of complex remaining =dpm retained at Time t

dpm retained at Time 0x 100

To determine radiolabel bound to filters, these were subsequently incubated in 2 ml of NaCI solution (1.5 M) at room temperature for 16 h toelute bound complex. Fifteen ml of ACS scintillant were added, andradioactivity was determined. Three or 4 determinations were made ateach time point over a 2-h period of incubation.

Ability of Tissues to Degrade Preformed [3H]VCR-Cytosol ComplexDerived from HxRh18 Tumors. [3H]VCR-protein complex was formed

in HxRhl 8 cytosols and separated from free drug by G-25 Sephadex gelfiltration, as described. The complex was incubated at 37Â°for periods

of up to 2 h with cytosols prepared from normal tissues, at proteinconcentrations 1.1- to 3.8-fold in excess of that of the tumor preparation.At intervals of 30 min, 80-^1 aliquots were filtered on DE-81 discs, and

the amount of bound drug was determined.

Degradation of 125lodinated Tubulin. Tubulin from pooled mouse

brains was purified by 2 cycles of polymerization according to the methodof Shelanski ef al. (19) and stored at -20Â° in 8 M glycerol prior to use.

This was subsequently radiolabeled after a third polymerization cyclewith 125Iin Buffer A using chloramine T (20). Homogenates (100,000 x

g supernatants) of mouse ileum, kidney, muscle, and brain were preparedas described in Buffer A. 125l-Tubulin was incubated with a 30-fold greaterprotein concentration of each normal tissue cytosol at 37Â°for up to 60

min; 200-Â¿Â¿laliquots were taken at intervals and boiled for 90 s with anequal volume of SDS sample buffer (21) prior to being stored at -20Â°.Effects of tissue cytosols on the stability of 125l-tubulin were examined

by 1-dimensional gel electrophoresis using the discontinuous system of

Laemmli (21). Gels were subequently stained with 0.05% Coomassieblue for 30 min and destained. The final destain solution contained 2%glycerol for 30 min prior to gel drying. The dried gel was overlayed withKodak X-Omat AR film and exposed at -20Â° for 26 to 70 days prior to

developing.

RESULTS

The concentrations of [3H]VCR achieved in HxRh18 xeno-

grafts and mouse normal tissues had been examined previouslyafter Â¡.p.administration of the drug to tumor-bearing mice at a

dose level of 3 mg/kg, the maximum tolerated dose (8). In ileum,liver, and kidney, [3H]VCR disappeared rapidly from these tis

sues, paralleling the decline in the plasma concentration. Inmuscle, there was a gradual decrease in tissue concentrationwhereas, in neoplastic tissue, the drug was tenaciously retained.Tumor levels were >5-fold higher than those observed in normal

tissues at 72 h after treatment. Previous studies had indicatedthat initial rapid loss of [3H]VCR from normal tissues was not a

consequence of VCR metabolism (8).The observation of the selective retention of [3H]VCR by

HxRh18 tumors suggested that it would be of interest to elucidate the stability of [3H]VCR-protein complexes formed in the

cytosols prepared from nonneoplastic and neoplastic tissues.Brain was also examined due to the high affinity of Vinca alkaloidsfor brain tubulin, either purified (13, 15) or in cytosols (22),although, in vivo, a clearance pattern could not be establisheddue to poor penetration through the blood-brain barrier (8, 23,

24).Initial studies were conducted to determine whether [3H]VCR

complexes could be formed in various tissue cytosols andwhether these could be isolated from unbound [3H]VCR by G-

25 Sephadex gel filtration. Data for HxRh18 xenografts areshown in Chart 1 and for ileum in Chart 2. It is clear that, inneoplastic tissue, protein-bound [3H]VCR eluted from the columnwith the protein fraction and was separated from free [3H]VCR.[3H]Vincristine-cytosol complexes were also isolated from brain,

liver, kidney, skeletal muscle, lung, bone marrow, and spleen(data not shown). In ileum, however, no complex could beisolated and, after incubation of cytosol with [3H]VCR at 37Â°for

15 min, all radioactivity coeluted with free VCR. Similarly, complexes could not be isolated from supernatants of whole bloodprepared by sonication (data not shown).

The stability of [3H]VCR-cytosol complexes isolated by gelfiltration were examined after incubation at 37Â°, by periodicfiltration through DE-81 discs (Chart 3). The retention of protein-bound [3H]VCR by the discs was linear for at least 325 ng of

protein added, and all studies were conducted in this range.Filtration efficiencies for HxRhl 8 xenografts and brain were 95.3


3762




EQ.

30Fraction Number

Chart 1. Isolation of [3H]VCR-cytosol complexes formed in HxRh18 tumors.Cytosol (100,000 x g supernatant) prepared in Buffer A (2Â°)and containing protein(5.6 mg/ml) was incubated with [3H]VCR (198 nM) for 15 min at 37" to steady

state. One ml of the mixture was eluted with Buffer A under gravity from a G-25Sephadex (1.4 x 5 cm) gel filtration column. Fractions (0.5 ml) (0.5 min) werecollected, and radioactivity and protein content were determined. â€¢,protein (mg/ml); O, protein-bound [3H]VCR (dpm); D, [3H]VCR alone (dpm). The flattened protein

peak is indicative of pooling of Fractions 6 to 8.

- 4,000

- 2,000

0

Fraction NumberChart 2. G-25 Sephadex filtration of [3H]VCR-cytosol mixtures in ileum. Cytosol

(100,000 x g supernatant) containing protein (10.8 mg/ml) was incubated for 15min at 37Â°with [3H]VCR (78 nM) prior to gel filtration of a 1-ml sample. â€¢,protein(mg/ml); O, protein-bound [3H]VCR (dpm); D, [3H]VCR alone (dpm). The flattened

protein peak indicates the pooling of Fractions 6 to 8.

and 75.0%, respectively; for muscle, kidney, and liver, thesewere lower, ranging from 43.9 to 53.2% for the studies presented. Retention of [3H]VCR alone was 1.9%. Complexes

formed from neoplastic and brain tissues were stable for at least2 h examined. In liver and kidney, however, [3H]VCR-cytosol

complexes were unstable, with f% values of 36 and 38 min,respectively. The complex formed in muscle cytosol also declined, with a fvi of 150 min, the rate of loss being less than thatobserved for liver and kidney. It was of note that the stability ofcomplexes in these tissues was also reflected in their filtrationefficiencies through DE-81 discs. Complexes formed with lung,

200 r

100

50

oo>

10

O 1 2

Time (hr.)Chart 3. Stability of [3H]VCR-protein complexes in neoplastic and normal tis

sues. Cytosols (1.2 ml) prepared in Buffer A were incubated (37Â°, 15 min) with[3H]VCR at a concentration of 185 to 271 nM. After isolation of protein-bound drugby gel filtration, complexes were incubated at 37Â°,and 60- to 80-^1 aliquots werefiltered on DE-81 discs to determine the stability of each complex. Assays wererepeated at 30-min intervals over a 2-h period with 3 or 4 determinations at eachtime point for each tissue. Results are the mean; oars, SD. â€¢,HxRh18 tumors; O,brain; â€¢liver; D, kidney; A, skeletal muscle. Cytosols derived from blood and ileumdid not form complexes that could be isolated by gel filtration after incubation with[3HJVCR.

bone marrow, and spleen were also unstable, demonstrating f.*values during the first h of 36, 36, and 21 min, respectively (datanot shown).

Because the stability of [3H]VCR-protein complexes formed in

different tissues varied so considerably, it was of interest todetermine whether cytosols from these tissues could destabilize"stable complex" formed in HxRhl 8 cytosol. Protein-bound [3H]-

VCR was isolated from tumor cytosol and incubated in thepresence of cytosols derived from nonneoplastic tissues (Chart4). In the presence of cytosol derived from skeletal muscle, brain,bone marrow, spleen, and blood, the HxRh18-[3H]VCR complex

was stable. However, when incubated with cytosols derivedfrom ileum and kidney (which formed unstable complexes), thef%values for the HxRh18-[3H]VCR complex were 25 and 32 min,

respectively. Liver cytosol also caused dissociation of tumorcomplex (f%= 132 min). Lung tissue, which formed an unstablecomplex with [3H]VCR, also had an effect on the HxRh18-[3H]-

VCR complex (f./z= 236 min).The half times for retention of [3H]VCR in vivo and for the

dissociation of protein-bound [3H]VCR complexes are shown inTable 1. In liver, kidney, and skeletal muscle, where [3H]VCR-

cytosol complexes could be isolated, the latter demonstratedshorter tv, values than the initial half-times observed for elimination of [3H]VCR from these tissues in vivo; however, in both

studies, the fVÃ®values were in the order of skeletal muscle >kidney > liver. [3H]VCR bound in HxRh18 tumors was stable

both in vitro and in vivo. The stability of complexes formed incytosols was therefore similar to the retention of [3H]VCR by

tissues in vivo. Kidney and Â¡lealcytosols were capable of reducing


3763




200

100

50

0>

10

Time (hr)Chart 4. Stability of [3H]VCR-cytosol complex derived from HxRh18 tumors in

the presence of cytosol prepared from normal tissues. [3H]VCR-HxRh18 complexprepared in cytosols and isolated by gel filtration was incubated at 37Â° in the

absence (â€¢)or presence of cytosols derived from brain (O), liver (â€¢).kidney P),skeletal muscle (A), blood (A), and ileum (T). The amount of [3H]VCR-HxRh18complex remaining was determined at 30-min intervals over a 2-h period using theDE-81 filter disc assay. Results represent the mean of 3 or 4 determinations foreach time point; bars, SO.

Table 1Half-times (t*) of 13H]VCR in vivo and of [3H]VCR-cytosol complexes (min)

Invivo"Tissue

iÂ»0HxRhl

8 14400Plasma 22Blood NDIleum 100Liver 74Kidney 96Skeletal muscle 576Brain ND/â€¢**ND"

0.97ND0.960.960.941.00NDIn

vitrobtv,

Of [3H]-VCR-cytosolcomplexesStable

NDtf

3638

150Stabletv.

Of[3H]-VCR-HxRh18

complex inthe presenceof normal tissuecytosolsND

NDStable

25132

32Stable

Stable*Tumor-bearing mice were given injections i.p. with [3H]VCR at a dose level of

3 mg/kg (data derived from Ref. 8).6 The stability of [3H]VCR-cytosol complexes was determined over a period of

c Initial fvÂ»values were determined during the first 1 h for plasma, 2 to 4 h for

ileum, liver, and kidney, and over 48 h in skeletal muscle.a r* = correlation coefficient.8 ND, not determined.' Complexes could not be isolated by gel filtration.

the stability of HxRh18-[3H]VCR complexes. This complex was

also unstable in the presence of liver cytosol, although the tv,was longer. It was of interest that [3H]VCR was also rapidly

eliminated from Â¡lealtissue in vivo.The stability of protein-bound [3H]VCR in HxRh18 tumor cy

tosols was examined in the presence of extracts from kidneyand ileum that had been heated to 100Â°for 3 min (Table 2). In

both instances, complexes were completely stable.These results suggested that the [3H]VCR-cytosol complex

derived from HxRhl 8 tumors may be induced to dissociate by asoluble, heat-labile factor from ileum and kidney but not from

brain and muscle tissues. Consequently, the ability of thesetissue cytosols to degrade purified 125l-tubulin was examined.

Autoradiographs derived from the dried gels after overlaying withphotographic film are shown in Figs. 1 and 2.125l-Tubulin incubated alone at 37Â°was stable. In the presence of brain andskeletal muscle preparations, 125l-tubulin was also stable. How

ever, when the radiolabeled substrate was incubated with ilealcytosol, there was a rapid degradation of the protein, effectivewithin the first 10 min. Low-molecular weight degradation prod

ucts could be detected. These observations correlated with thestability of HxRh18-[3H]VCR complexes in the presence of brain,muscle, and ileal cytosols. After incubation of 125l-tubulin with

kidney cytosol, however, no change in the molecular weight ofthe substrate was detected, which was of interest, since kidneypreparations had been found to cause instability of the HxRhl 8-[3H]VCR complex.

The ability of kidney cytosols to cause dissociation of protein-bound [3H]VCR in HxRhl 8 cytosols due to metabolism of the

drug was examined (Chart 5). However, after 2 h of incubationof cytosol with [3H]VCR at 37Â°,85% of the drug remained in the

form of the parent compound. Degradation or dissociation of theHxRhl 8-[3H]VCR complex could therefore not be explained on

the basis of drug metabolism.

DISCUSSION

In the mouse, pharmacokinetics of VCR have been characterized by rapid association of drug in normal tissues where tissuelevels greatly exceeded plasma levels (8). However, unlike neo-

plastic tissue, VCR was rapidly eliminated from normal tissues,a factor that appeared not to be a consequence of drug metabolism. We (8) and others (11) have suggested that the basis fortherapeutic selectivity may be a consequence of selective retention of Vinca alkaloids in neoplastic tissues. However, the biochemical basis for the differential retention in normal and neoplastic tissues remains to be elucidated. It is this aspect, thefailure of normal tissues to retain VCR, that we have addressedin the current investigation.

The rapid elimination of [3H]VCR from ileum, kidney, liver, and

also skeletal muscle but not HxRhl 8 tumors in vivo suggestedthe possibility that Vinca complexes in normal tissue cytosolsmay not be formed or, Â¡fformed, they may be unstable. Indeed,no complex could be isolated in ileal cytosols by gel filtration.The half-times for dissociation of complexes formed in cytosols

derived from skeletal muscle, liver, and kidney correlated withthe order of f%values for elimination of [3H]VCR in these tissues

in vivo, although the values derived in cytosols were of shorterduration. One possibility may be the utilization of lower concentrations of [3H]VCR in in vitro studies (185 to 271 nrvi), in

comparison to the intracellular concentrations achieved in vivo[17 fiM in ileum, 1.5 UMin skeletal muscle (Ref. 8)]. Thus, in vivo,there would be a more rapid rate of reassociation of free [3H]-

VCR, giving a lower apparent rate of net dissociation of complex.Brain tissue, which has been shown to possess a high affinityfor VCR (15), also formed a stable complex with the drug, as didHxRhl 8 tumors.

Based upon in vitro studies, it was found that normal tissuescould be divided into 3 groups: (a) those that formed stable [3H]-


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Table2Incubation ol cytosols from kidney and ileum after heat treatment with HxRhlB-fHJVCR complex

% of complexremaining8Tissue

cytosolHxRh18-[3H]VCRcomplex

Complex + kidneyComplex + kidney + heatc

Complex + ileumComplex + ileum + heat00.5

h97.3Â±9.0*

77.9 Â±6.8101 .6 Â±11.9

39.9 Â±2.7NO"1

h114.3Â±

11.138.2 Â±1.592.2 Â±9.417.4 Â±2.1

110.7 Â±9.51.5

h113.8

Â±13.69.7 Â±0.4

89.1 Â±10.49.4 Â±0.5

ND2h126.3

Â±2.37.9Â±0.4

84.9 Â±8.06.9 Â±1.1

110.8 Â±10.1

nesuus represem o ur


100 I

Chart 5. Metabolism of [3H]VCR by kidneycytosol was examined by HPLC as describedin "Materials and Methods." The retention time

of VCR was 60 min and, of the minor peaks, itwas 69 and 71 min.

mco

o.d "O

40-

20-

010 20 30 40 50

Time (min)

60 70 80

involved in the regulation of the turnover of mammalian tubulin,which is a substrate for the enzyme(s) (29). Specific degradationproducts have been identified from the action of CANP onmyofibrils, namely Â«-actininand troponin C (28,29). A "staircase"

of breakdown products has also been observed in the degradation of vimentin and desmin (32). In our study, intermediates inthe degradation of tubulin by Â¡lealcytosols were not identified,which may be due to the rapid cleavage observed. Low-molec

ular weight degradation products have also been identified in thecleavage of neurofilaments by a purified CANP (20). Alternatively,this may be due to the use of crude cytosolic preparations, whichmay result in the further degradation of tubulin. This has beendemonstrated for a-actinin in studies with myofibrils (28), where

further degradation is due to a factor unrelated to the activity ofthe CANP. The activity of the currently unidentified enzymicfactor in ileal preparations may therefore relate to the inability toisolate vVnca-protein complexes by gel filtration and also to therapid elimination of [3H]VCR from this tissue in vivo.

In kidney cytosols, the situation may be more complex. Itwould appear that factors other than the direct proteolysis oftubulin may be involved. Cleavage of only a few amino acidsfrom 125l-tubulin by kidney cytosol, changes in the ratio of dimeric

to monomeric tubulin, or alterations in the phosphorylation stateof the molecule could conceivably lead to dissociation of [3H]-

VCR from the tubulin dimer in HxRh18 supernatants. Theseevents would not be detected by a change in the molecularweight of 125l-tubulin examined by polyacrylamide-SDS gel elec-

trophoresis. It is of interest that irreversible loss of tubulin polymerization involving calcium-dependent proteolytic action specificfor high-molecular weight microtubule-associated proteins has

been identified (36) and could lead to a change in the ratio oftubulin dimers:monomers within the tubulin pool. Also, a CANP,identified in kidney (30), is capable of activating a protein kinasethat phosphorylates histone and protamine. It is conceivable thata similar situation may exist with tubulin. The effects observedin kidney, therefore, may be more subtle than events observedwith ileal preparations.

Factors that influence the selective action of VCR in the

treatment of human cancer would appear complex and requiremore detailed exploration. However, the stability of protein-bound [3H]VCR in HxRhl 8 cytosols but not in many other normaltissues has correlated with the selective action of [3H]VCR in

human RMS growing as xenografts. The expression of tubulinisoforms in different tissues may influence drug binding and,hence, drug retention. Human RMS xenografts and normal tissues that do not influence the stability of HxRh18-[3H]VCR

complex, such as brain, skeletal muscle, spleen, and bone marrow, may fall into this category. In ileum and kidney, these alsomay be important considerations. Alternatively, the stability of[3H]VCR-tubulin complexes may be mediated by additional fac

tors that may relate to the regulation of the turnover of tubulinin vivo.

ACKNOWLEDGMENTS

The authors wish to acknowledge Ruby Cook and Pamela Lutz for technicalassistance in the preparationof tumor-bearing immune-deprivedmice.

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2. Giulani, F. C., Zirvi, K. A., Kaplan, N. 0., and Goldin, A. Chemotherapy ofhumancolorectal tumor xenografts in athymic mice with clinicallyactivedrugs:5-fluorouracil and 1,3-bis-(2-chloroethyl)-1-nitrourea(BCNU):comparison withdoxorubicin derivatives,4'-deoxydoxorubicin and 4'-O-methyldoxorubicin. Int.J. Cancer,27:5-13,1981.

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4. Houghton, J. A. and Houghton, P. J. The xenograft as an intermediatemodelsystem. In: B. T. Hill and P. P. Dendy, (eds.), Human Tumor Drug SensitivityTesting in Vitro:Techniquesand ClinicalApplications,pp. 179-200. New York:Academic Press, Inc., 1983.

5. Povlsen,C., and Jacobsen,J. Chemotherapyof a humanmalignantmelanomatransplanted in the nude mouse. Cancer Res., 35: 2790-2796,1975.

6. Shorthouse, A. J., Peckham, M. J., Smyth, J. F., and Steel, G. G. Thetherapeutic response of bronchial carcinoma xenografts: a direct patient-xenograft comparison. Br. J. Cancer,41: 142-145, 1980.

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1985;45:3761-3767. Cancer Res Janet A. Houghton, Larry G. Williams and Peter J. Houghton of the MouseXenografts of Human Rhabdomyosarcoma and Normal Tissues Stability of Vincristine Complexes in Cytosols Derived from

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