1Thisinvestigationwassupported

[CANCER RESEARCH 33, 1889-1897, August 1973]

Distribution and Regulation of Deoxythymidine Kinase Activity in Differentiating Cells of Mammalian Intestines'

Josephine S. Salser and M. Earl Balis

Division of Cell Metabolism, Sloan-Kettering Institute for Cancer Research, New York, New York 10021

SUMMARY TdR kinase (ATP-thymidine 5'-phosphotransferase, EC 2.71.21) activity, which decreases markedly when epithelial The distribution of deoxythymidine kinase activity has cells leave the crypts of rat jejunum on the lower one-third of been reexamined in rat and human intestinal mucosa human colonic mucosa, is generally associated with rapid separated by a planing technique into cells in different growth and increased DNA synthesis ( I2, 32). The pen stages of differentiation. When epithelial cells leave the odicity of this enzyme during the mitotic cycle is well crypts, a decrease in kinase activity was observed in both established but the specific mechanisms that tie the levels of resuspended pellet and supernatant fractions. Commercial kinase activity to the rates of DNA synthesis and cell preparations of phospholipase C added to the assay system division are far from well understood. Synthesis of new resulted in remarkable stimulation of kinase activity, an enzyme rather than change in turnover rate of enzyme effect that was more pronounced in extracts from rat tissues protein could explain the sharp increases in activity ob and in pellet fractions of all the tissues studied. Although served in regenerating liver (2, 9, 28), the S phase of pellet activity in preparations from human mucosa was synchronized cultures of mammalian cells ( 1, 26, 29, 39), primarily associated with the nuclear fraction, significant and virus-infected tissue culture cells (24, 25, 33). The peak activity was also found in the mitochondrial fraction. The of TdR kinase activity, however, often occurs somewhat effects of phospholipase C and/or detergents varied consid later than the peak of DNA synthesis (6, 7, 3 1). The erably with the various subcellular fractions. The degree of subsequent rapid decline in kinase activity in regenerating stimulation by phospholipase C appeared to be inversely liver (2, 9) and intestinal epithelium (22) has been attributed related to the level of deoxythymidine kinase activity to the short half-life of the enzyme. present under standard assay conditions. The implications In all the foregoing studies, only the supernatant fractions of some aspects of these observations have been discussed. after high-speed centnifugation of aqueous homogenates of the tissues have been examined. Although the greater proportion of TdR kinase activity is indeed present in this INTRODUCTION fraction, significant amounts have been found associated with the particulate fractions of mouse fibroblasts (1), The activities of a number of enzymes involved in human lymphocytes (31), rat thymus (14), rat liver (5, 37), intermediary metabolism of purine and pyrimidine deriva and mouse liver (2). Moreover, appreciable activity has been tives change as intestinal epithelial cells, which proliferate recently demonstrated in typically nonproliferating tissues rapidly in the crypts, differentiate and migrate in an orderly and in various subccllulan fractions of rat liver using an fashion up to the surface of the mucosa. Although some assay system containing phospholipase C (37). These obser enzymes, e.g., adenylate deaminase and thymidylate phos vations have led to a reexamination of the TdR kinase phatase, are uniformly distributed in the mucosal cells of rat activity in differentiating epithelial cells of human and rat jejunum, there are increases in other enzymes, e.g., adeno intestines, using a mild aqueous isolation technique and a sine deaminase, nucleoside phosphorylases, and purine special planing method that enables the separation of cells phosphonibosyltransferases, and a decrease in TdR' kinase of known ages from the mucosa (4, 22, 4 1). The results and in the cells of the villus tip (22). In differentiating human possible implications of some aspects of these studies are colonic mucosal cells, there are increases in purine phos presented in this paper. phoribosyltransferases and decreases in both TdR phospho rylase and TdR kinase (41). Differential turnover of en zymes and their templates (23) and synthesis of new MATERIALS AND METHODS enzymes which, although functionally similar, differ consid erably in other properties (4) have been suggested as some of Materials. TdR and 2-mercaptoethanolwerepurchased the regulatory systems involved in the control of enzymic from Calbiochem, Los Angeles, Calif.; TdR-2-'4C (57 patterns characteristic of this differentiative process. mCi/mmole) was from Amensham/Seanle, Arlington 1ThisinvestigationwassupportedbyUSPHSResearchGrantCA08748.Heights, Ill.; 3-phosphoglycenic acid, ATP, Tnis, and phos 2 The abbreviation used: TdR, deoxythymidine. pholipase C (a crude preparation from Clostridium we!chii, Received September 27, 1972; accepted May 3, 1973. 2 units/mg) was from Sigma Chemical Co., St. Louis, Mo.;

AUGUST 1973 1889

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1973 American Association for Cancer Research. Josephine S. Salser and M. Earl Ba/is and sucrose (absolute grade) was from Research Plus, usually stored frozen (â€”20Â°)and assayed on the following Denville, N. J. day. There was no apparent loss ofTdR kinase activity even Male CFN rats (Carworth Farms, New City, N. Y.), when these samples were stored frozen for as long as 2 weighing between 180 and 200 g, were fed commercial months. laboratory chow ad libitum until they were killed by cervical The purity of the subcellular fractions was monitored by dislocation. Segments of colon, duodenum, and jejunum assaying for specific marker enzymes in the various frac were removed, slit lengthwise, and washed gently with cold tions: NAD pyrophosphonylase (EC 2.7.71) for the nuclear 0.9% NaCI solution to remove intestinal contents. Mucosal fraction (36), cytochnome c oxidase (EC I.9.3. 1) for the epithelial cells were separated with a tissue planing appana mitochondnial fraction (3), acid phosphatase (EC 3.1.3.2) tus (22) either as a single composite layer on as 5 successive for the lysosomal-enniched fraction (3), cytochnome c sections, designated as tip, upper middle, lower middle, reductase (EC 1.6.99.3) for the microsomal fraction (15), upper crypt, and lower crypt (4). The morphological and glucose 6-phosphate dehydrogenase (EC 1. I. 1.49) for characteristics of these sections were confirmed by histolog the postmicrosomal supernatant fraction ( I8). ical examination. In general, samples from 3 animals were EnzymeAssay. The standard reaction systemfor human pooledto obtainsufficientexperimentalmaterial. mucosa contained enzyme (30 to l20 @gprotein), 6.57 An adaptation of this planing method (41) was used to nmoles TdR-2-â€•C (100,000 dpm), 12 zmoles Tnis-HCI (pH separate epithelial cells from surgical specimens of adult 8.0, 37Â°), 1.8 izmolcs ATP, I .5 Mmoles 3-phosphoglyccnic human colon (kindly provided by the Gastric and Mixed acid, and 1.8 @tmolesMgCl2 in a total volume of 0. 15 ml. Tumor and the Rectal Services, Department of Surgery, More TdR (9 nmoles, 100,000 dpm) as well as 0.6 @tmole Memorial Hospital) into 3 layers corresponding to cells in NaF were present in the reaction mixture when rat mucosal different stages of differentiation and maturation. These fractions were assayed. To study the effect of phospholipase layers are referred to as upper third, middle third, and lower C on TdR kinase activity, 25 @tgofthe crude commercial third. Histologicalexaminationsof theselayersconfirmed preparation were added to the above systems. Whole earlier observations ( 14) that 60 to 70% of the proliferative homogenates, resuspended pellets, supernatant fractions, cells were found in the lower third. The remainder was and recombined pellet and supernatant fractions were essentially in the middle third with very few (less than 5%), if assayed. The protein concentration of the enzyme extracts any, in the upper third. For larger scale isolation of TdR was determined by the method of Lowry et a!. (27), using kinase, autopsy specimens obtained from Memorial Hospi bovine serum albumin as standard. tal were used. Samples were taken only from cadavers in Incubation was carried out for 30 mm at 37Â° in a which there had been no diseases of the gastrointestinal metabolic shaker. The reaction was stopped by immersing tract. Specimens of this type were cleaned and freed from the capped reaction tubes in a boiling water bath for 3 mm. underlying tissues. The mucosal cells were removed as a After cooling in an ice bath and centnifugation at 3,000 x g single layer with either a razor blade or a glass slide and for 15 mm, aliquots (20 @l)ofthe protein-free supernatants stored frozen for subsequent extraction and assay. There wereappliedonto DEAE-cellulose(WhatmanNo. DE 81) was no significant loss in TdR kinase activity in samples squares, which were washed as described by Bresnick and stored over 4 months at â€”20Â°orover I year at â€”70Â°. Karjala (8), dried, and transferred to counting vials. The TissueExtractsandSubcellularFractions.Mucosalepi washing procedure effectively removed all unreacted thelial cells were suspended (20% w/v) in 0.23 M sucrose TdR-2-'4C while retaining all labeled deoxynibonucleotides. 0.007 M Tris-HCI (pH 8.2, 25Â°)-0.005 M KC1-0.003 M The radioactivity was determined in an Intertechnique 2-mercaptoethanol and homogenized in a Potter-Elvehjem liquid scintillation spectrometer with a scintillation solution homogenizer by 10 strokes with a wide clearance Teflon containing 5 g of PPO and 100 mg of Dimethyl POPOP per glass pestle rotating at 1,200 rpm. All steps were carried out liter of toluene. at 0-4Â°. Homogenates were centrifuged either once at Under the above assay conditions, the reaction was linear 127,000 x g for 60 mm or successively to yield: nuclear with respect to time and the amount of enzyme present. fraction at 700 x g, 10 mm; mitochondnial fraction at TdR kinase activity is expressed either in unit(s)/mg protein 10,000 x g, 15 mm; lysosomal-enniched fraction at 25,000 (specific activity) or in unit(s)/100 mg homogenate protein xg,10mm;andmicrosomalandpostmicrosomalsupenna(total activity). A unit is defined as the enzyme phos tant fractions at 127,000 x g, 60 mm (5). Centnifugation phorylating I zmole TdR per mm at 37Â°. steps at 25,000 x g or less were performed in a Sorvall RC 2-B while centnifugation at 127,000 x g was carried out in a Spinco L-2 preparative ultracentnifuge. Pellets of the van RESULTS ous fractions were washed twice by resuspending in the homogenizing mixture with glass rods and resedimented Preliminary studies with mucosa obtained from human with centrifugal forces identical with those used for the colon indicated that there was an appreciable amount of initial sedimentation. The washed pellets were finally resus TdR kinase activity associated with the pellet fraction. pended by gentle homogenizing in the same volume of Enzyme extracts used in these comparative studies were medium as the original hc@mogenate from which they were prepared by homogenizing mucosal epithelial cells in su prepared. Due to the time involved in preparing the tissues crose medium containing 2-mercaptoethanol. These prepa and the various fractions of the homogenates, samples were nations were consistently more active than those prepared

I890 CANCER RESEARCH VOL. 33

by more vigorous homogenization procedures, e.g., sonic phospholipase C. The amount of AMP formed at the end of disruption on various high-speed blendors. Moreover, the the reaction, which was used as a measure of ATPase pattern of distribution in a particular tissue did not vary activity, was also negligible. In the standard assay system, qualitatively from sample to sample. The microsomal AMP could not be substituted for ATP. When phospholi marker enzyme present in the supernatant fraction was less pase C was present, AMP had some activity as an alternate than 1% and the residual supernatant marker enzyme in the phosphatedonorbut wasonly 20% asefficientas ATP. washed pellet fraction, less than 2%. Differences were observed in both total -and specific When dTMP-2-â€•C was substituted for TdR-2-â€•C in the activities of TdR kinase present in homogenates prepared standard assay system, there was no apparent degradation from autopsy samples ofhuman and rat intestines (Table 1). of the labeled deoxynibonucleotide. Addition of NaF (as Under standard assay conditions, the greater proportion of high as 2.4 smoles) to the assay medium had no effect on the kinase activity in human colon and rat intestines was the kinase activity in human mucosal preparations. The foundin thesupennatantfraction.In humansmallintestine, level of 3-phosphoglycerate (1 .0 mM) used in the kinase however, the enzyme was apparently distributed equally assay was more than adequate completely to inhibit dTMP between the pellet and the supernatant fractions. The phosphatase activity present in the samples (1 to 2 specific activities of kinase in the supernatant fractions from nmoles/mg supernatant protein and 3 to 5 nmoles/mg pellet rat tissues were considerably greater than those of the protein). However, when these studies were extended to corresponding pellet-associated enzyme. In preparations include rat tissues, modifications were necessary to optimize from human intestines, the latter enzyme was at least as kinase activity, e.g., higher TdR concentration and addition active as its counterpart in the supernatant fraction. of NaF to the incubationmedium.The latter wasaddedas Addition of phospholipase C increased the apparent supplemental phosphatase inhibitor since the concentration kinase activity in all the mucosal samples examined. A of 3-phosphoglycerate(I .5 mM) necessaryto inhibit phos greaten stimulatory effect was consistently observed in the phatase activity present in rat mucosa (about twice that pellet fraction of a given tissue, e.g., over 250-fold in the found in human mucosa) also caused about 20% reduction pellet and only about 20-fold in the supernatant fraction of in kinase activity. rat duodenum. Although activation by phospholipase C was Higher concentrations of NaF, which is a potent inhibitor always found in human mucosal preparations, it was never of ATPase as well as dTMP phosphatase, had no apparent quite as pronounced as those seen with rat samples. The effect on the phosphorylation ofTdR even in the presence of pattern of stimulation of TdR kinase activity in homoge

Table I mucosaMucosal Effect of PC' on TdR kinase activity in human and rat intestinal pelletfractions epithelial cells from autopsy samples were removed and homogenized. Supernatant and washed resuspended two.TdR were from a single centnifugation at I27,000 x g and reconstituted whole homogenates, from recombination of the inâ€œMaterialskinase activity was assayed under STD and in the presence of phospholipase C (PC). Experimental details are given mghomogenateand Methods.â€• Specific activity is expressed as units x I0'/mg protein and total activity as units x lO'/lOO protein. All values are averagessamples.Specific of replicate assays of 2 different

activity(units xl06/mgprotein)

Total activity totalhomog-Whole (units x lO'/l00 Fraction of theenate Super- Recon- mg homogenate activity in supernatantHumanColonSTD Pellet natant stituted protein)

0.67PC 8.6 10.0 9.7 8.7 80 0.38Small 20.2 64.5 18.8 19.3 188 intestineSTD 0.53PC 7.1 12.3 6.0 7.4 89 0.52RatColonSTD 25.8 59.7 23.8 26.4 321

0.90PC 6.5 1.4 11.9 6.6 87 0.40DuodenumSTD 63.0 104.2 70.8 67.2 839

0.88PC 2.6 1.0 4.6 2.7 7 0.36JejunumSTD 102.3 255.7 94.1 102.2 272

0.78PC 1.8 1.0 2.3 1.9 7 17.2 46.3 48.9 17.6 67 0.61

a PC, phospholipase C-containing system; STD, standard conditions.

AUGUST 1973 1891

nates of human colonic mucosa was unchanged when were arbitrarily designatedas nuclear, mitochondnial, lyso surgical specimens (cf. Table 2) were used instead of somal enriched, microsomal, and postmicrosomal superna autopsy samples. tant, the purity of these fractions was determined by When human colonic mucosa was separated into 3 layers assaying for specific marker enzymes. The nuclear fraction corresponding to cells in different stages of differentiation contained essentially no lysosomal-enniched, microsomal, (41), the greater proportion ofthc kinase activity was found and postmicrosomal supernatant (less than 0.5% of each of in the lower third (Chart I). Activation of whole homoge these) marker enzymes and about 3% of the mitochondnial nates and pellet fractions by phospholipase C, however, was marker. However, the differences in stimulation by phos 3 to 4 times greater in the upper third than in either of the pholipase C and inhibition by detergents (see â€œDiscussionâ€•) other 2 layers (Table 2). This apparently resulted in a more argue against the kinase activity being attributable to the uniform distribution of the kinase activity. When various small amounts of the other fractions present. Each of the fractionswere recombined,theactivitiesof the individual remaining subcellular fractions contained no significant components were additive under standard conditions of amount of any marker enzyme other than the one specific assay but not when phospholipase C was also present in the for it. About 90% of the pellet activity was associated with system (Chart I). the nuclear and mitochondrial fractions under standard For evaluation of the pellet-associated TdR kinase activi assay conditions and only 60% when phospholipase C was ty, the intracellular distribution of the enzyme in human present. This shift in distribution was apparently due to intestinal mucosa has been examined (Table 3). Although considerably greater activation of the kinase activity in the various fractions obtained by differential centnifugation lysosomal-enniched and microsomal fractions. The separate activities of these subcellulan fractions were not additive, ouuA50(Standard especially when they were recombined to reconstitute either total pellet or whole homogenate and assayed in the @ C400 Phospholipose presence of phospholipase C. Neither were they additive when pellet fractions were added back either individually on in combination with others to the supernatant fraction 30( (Chart 2). In general, the inhibitory effect was greater in @2@X samples containing the lysosomal-enniched and/or the mi crosomal fraction. When ionic (sodium lauryl sulfate or sodium deoxycho ,iuâ€” late) or non-ionic (Tween 80 or Triton X-lOO) detergents WP5RW WPSRW I@ZP)3W. â€˜S as ts were added to give a final concentration of 0.25% (w/v) in Experimental Calculated the assay medium, there were considerable differences in the SINGLE RECONSTITUTEDFROM extent of inhibition of TdR kinase activity observed in the SCRAPING SEPARATED THIRDS various subcellular fractions of human colon. Only the kinase activity of the nuclear fraction was inhibited by all 3cx100;@@- these detergents. Inhibition of the whole homogenate was remarkably similar to that of the supernatant fraction. Among these detergents, only sodium lauryl sulfate added @20( to the standardassaysystemresultedin markedinhibition of the enzymein all the subcellularfractions,e.g., 50% in cc the lysosomal-enriched and about 100% in both the mito â€˜P chondnial and the supernatant fractions. Although there werealsoratherlargedecreasesinthestimulatoryeffectof phospholipase C on all the resuspended pellet fractions whensodiumlaunylsulfatewaspresent,theactivationofthe LI UML UML UML UML. U@1 supernatant fraction was essentially unchanged. In contrast Experi to wholehomogenatesofrat liver(37), sodiumdeoxycholate mental Calculated did not appreciably decrease the activation by phospho WHOLE PELLET SUPER- RECONSTITUTED HOMO- NATANT WHOLE lipase C and non-ionic detergents did not potentiate this GENATE HOMOGENATE stimulatory effect. Chart I. Effect of phospholipase C on the total TdR kinase activity in When rat duodenum and jejunum segments were sepa epithelial cells from surgical samples of human colon. A, comparison of rated by a planing technique into tip, upper middle, lower single total scraping and samples reconstituted from the separated thirds middle, upper crypt, and lower crypt sections, stimulation given in B. Values given are for whole homogenates ( W), pellet (P), was greater in each of the pellet fractions. The values for supernatant (S), reconstituted whole (R), and arithmetic summation (@ P total activity (Charts 3 and 4) and specific activity (Table 4) + S) B, comparison of the upper (U), middle (M) and lower (L) thirds separated by a special planing technique. For experimental details, see have been summarized. Activation was greatest in the tip

â€˜.Materials and Methods.â€• Total TdR kinase activity is expressed as units and upper middle sections of the duodenum (300- to x I0'/lOO mg homogenateprotein.All valuesare averagesofreplicate 400-fold) and the upper middle of the jejunum (130-fold) assays of a typical sample. and lowest in the lower crypt of both these segments. The

1892 CANCER RESEARCH VOL. 33

Table 2 Efftct of PCâ€•onthe specific activity of TdR kinase in human colonic mucosa The specific activities of TdR kinase in the various fractions of human colonic mucosa have been calculated from data obtained in the experiment summarized in Chart I. Specific activity is expressed as units x l0'/mg protein. All values are averages of replicate assays of a typical sample.

lO'/mgprotein)Wholeactivity (units x

HomogenatePelletSupernatantSTD@

PCSinglescrapingSampleSpecific PCSTDPCSTD

28.3 30.3 Separated into thirds Upper 9.5 60.1 8.0 206.7 12.9 36.0 Middle 11.0 37.2 9.1 77.1 16.3 48.l Lower11.3 11.4 23.611.6 10.241.6 63.815.4 18.1 30.6

a PC, phospholipase C-containing system; STD, standard conditions.

Table 3 5004. HUMANCOLON0 Efftct of PC@on the intracellular distribution of TdR kinase activity in human intestinal mucosa Standard4000 CEiiii700â€”aPhospholipose Whole homogenates of mucosa from human autopsy samples of colon and small intestine were divided into 3 parts: A, whole homogenate; B, single centnifugation; and C, differential centrifugation (cf â€œMaterials and Methodsâ€•). TdR kinase activity was assayed under STD and in the I presence of PC. Total activity is expressed as units x lO'/l00 mg homogenate protein. All values given are averages of replicate assays of 2 different samples. Summation of the activities of the component parts is designated as @.

Total activity (units x l0'/ 100protein)SmallColon mg homogenate INTESTINE600500â€¢100]j@ddIi-HUMANSMALL

intestineSTDÂ° I

PCA.Whole PC STD

321B.Single homogenate80 188 89 centrifugationPellet32 190Supernatant64 206 41 203Reconstituted 124 47 whole87 31596 186 89 393C.Differential 330 88 centnifugationPelletâ€œNuclearâ€•19 EC EC CC EC EC CC EC EC EC EC @ SUPERNATANT _ __x @x@x xx@@ 143Mitochondnial6 110 18 â€˜i@ucLEAR11 â€”x x Mff0@H0NDRIAL - - X XX- 138Lysosomal-enriched> 81 15 1YSc@0MAL-ENRKHEDâ€”â€” â€” x 2x â€” â€” â€” x x x â€” 101Microsomal2 I 66 2 MKROSOMAL â€”â€”â€”â€”â€”X2X4XXXX 108Reconstituted 72 2 WHOLEHOMOGENATE x 201@;28 pellet33 216 44 Chart 2. Effect of pellet fractions on the total TdR kinase activity in 490Supernatant71 329 37 194Reconstituted 130 45 supernatant fractions from human mucosa. Resuspended pellet fractions whole84 32599 200 86 from the differential centnifugation of whole homogenates of human colon 684a 459 82 and small intestine (cf. Table 3) were added back to their respective supernatant fractions at the original cellular concentration (x), twice this PC, phospholipase C; STD,standard conditions. concentration (2x), or 4 times this concentration (4x). The experimental values (E) are averages of replicate assays of 2 different samples. The calculated values (C) are obtained by the summation of the individual greater proportion of the activity was found in the superna activities given in Table 3. tant fraction when standard conditions of assay were used (Chants 3 and 4B). The relative distribution between 2 fractions was reversed when phospholipase C was added to lower crypt. The dramatic drop observed in the upper crypt the assay system. was followed by relatively small decreases in the subsequent An examination of the S successive sections shows that sections. Stimulation by phospholipase C resulted in a under standard conditions most of the activity was in the somewhat more gradual decline in the kinase levels of

AUGUST 1973 1893

Josephine S. Sa/ser and M. Earl Ba/is

duodenal sections. The specific activity ofTdR kinase in the B lowercryptwasgenerallygreaterthanthatofkinaseinthe tip unless phospholipase C was present (Table 4). 8.1

@25Oh DISCUSSION 6.1 @2OO@ @ The results presented in this paper confirm earlier 1 observations that TdR kinase activity is not found exclu 4.( sively in the high-speed supernatant fraction of mammalian

tissues (1, 2, 5, 14, 31). These reports are, however, highly 2C conflicting with respect to the localization and the relative @ _____ â€” B I- ,@ 4 u34$ 1fl4$ I@34$ 12145 1234$

â€” Cd@ m@ @ @NGLE @cONSTrruTEDFROM @wPELLET RE@N@flflED sai@@@; @RATEDLAYtRS HOMO- P@TN4T @lOLE GEN@TE HOM@t @ I 2 )6O@ I i@L@ % 11w@p,1$ 12345 1234$ 2343 12343 1234$ ,- E@p@i â€” Co@oIed m@IC@aio@d @ioc* @1 SINGLE REcONS11TUTEDFROM @lOLEPELLETSUPER- @cONSTITUTED S@RAPlNG SEPARATED LAYERS HOMO- NATANT *IOLE mat GENATE HOMOGENATE @ec@ I 1@t @ 21â‚¬@ 2345 2345 12343 13345 234$ j! Ei@@sn I @Â° p m@ C@d @aE PELLET &FE@ @NSTffUTED HOMO- NATN@T @lOLE GEP4ATE HOMOGENATE Chart 4. Effect of phospholipase C on the total TdR kinase activity in @i â€˜@1. @ ; 8O@ p ,.@ rat jejunal mucosa. All the designations are the same as those given in : J@ r@ Chart 3. Mucosa from rat jejunum has been used instead of that from rat @ :@ .â€˜4@ c@1'1i@ @â€˜U@ duodenum.

@ 2c@ amounts of the enzyme associated with the particulate (&IU, fractions. In mouse fibroblasts (1) and human lymphocytes @ 4 o Ii :ll!@@ i!@i z 234$ 1234$ 12343 1234$ 12345 (31), most ofthc total cellular kinase was found in the crude E@er'@ mer4oi C@culoId nuclear fraction. In contrast, most ofthe kinase activity was @1O(.EPELLET SUPER- REtONSTITUTED recovered in the supernatant fraction of rat thymus (14) and HOMO- NATANT WHOI@E GENATE HOMOGENATE regenerating liver of both rats (5) and mice (2). The Chart 3. Effect of phospholipase C on the total TdR kinase activity in particulate-associated enzyme was entirely nuclear in thy rat duodenal mucosa. Mucosa from rat duodenum was scraped either as a mus, whereas it was mostly microsomal in rat liver and single layer or after separation into 5 layers by a planing technique almost entirely mitochondrial in mouse liver. Most of the described in the text. Pooled samples from 3 rats were used in these studies. enzyme in human and rat intestinal mucosa was also in the Experimental details are given in â€œMaterials and Methods.â€• A, compani supernatant fraction. In larger-scale preparations, TdR son of single total scraping and samples reconstituted from the 5 separated kinaseactivityin thepelletfractionfroma singlecentnifuga layers in B and C. Values given are for whole homogenate ( W), pellet (P), supernatant (S), reconstituted whole (R), and arithmetic summation (X P tion at 127,000 x g remained essentially unchanged even @ + 5). B and C, comparison ofthe 5 layers. 1, tip; 2, upper middle; 3, lower after 3 reextractions and Conclusive middle; 4, upper crypt; 5, lower crypt under standard assay conditions (B) evidence for the existence of the particulate enzyme as an and in the presence of phospholipase C (C). Total activity is expressed as entity completely distinct from the soluble kinase awaits units x I0'/ 100 mg homogenate protein; all experimental values are averages of replicate assays of 2 different samples. 3J. S. Salser and M. E. Balis, unpublished observations.

1894 CANCER RESEARCH VOL. 33

Table 4 Effect of PC@on the specific activity of TdR kinase in rat duodenal and jejunal mucosa The specific activities of TdR kinase in the various fractions of rat duodenal and jejunal mucosa have been calculated from data obtained in the experiments summarized in Charts 3 and 4. Specific activity is expressed as units x l0/mg protein.

l0/mgprotein)WholeWholeHomogenatePelletSupernatantHomogenatePelletSupernatantSTDDuodenum (units x lO'/mgprotein)Jejunum(units x

PCSinglescraping2.6 PCSTD PCSTD PCSTD PCSTD PCSTD

47.8Separated 104.71.0 271.64.6 93.11.8 17.11.0 45.92.3 layersTip1.9into 24.6Uppermiddle2.2 266.91.2 400.83.5 108.51.1 41.01.9 129.21.3 40.7Lowermiddle2.6 195.81.1 431.73.4 94.31.3 32.20.7 95.11.8 35.9Uppercrypt2.8 172.42.1 500.34.6 97.91.5 16.31.0 47.22.5 55.0Lowercrypt7.7 144.02.1 458.24.7 76.02.2 30.11.4 70.73.9 82.01.9 90.011.6 94.74.0 27.51.5 48.97.2 33.8

a PC, phospholipase C; STD, standard conditions. purification of the enzymes from the 2 fractions. The absence of any enhancement of kinase activity by NaF lend particulate-associated activity was primarily in the nuclear further support to this contention. fraction although somewhat smaller amounts were also Data from recombination of various enzyme preparations found in the mitochondnial fraction. indicate that there were interactions between fractions from When proliferating intestinal epithelial cells mature and total mucosa as well as those from separated mucosal differentiate as they migrate progressively toward the sections. This suggest that inhibitor(s) may be present in mucosal surface, there is a sharp decline in TdR kinase someof the fractions.Inhibitorsof TdR kinasein tissue activity. Such a decrease was observed not only in supenna extracts have been previously described in mammalian tant fractions (17, 22, 41) but also in the corresponding systems (20, 2 1, 30). The nature of such inhibitor(s) is far washed and resuspended pellet fractions. Moreover, there from well characterized. It is conceivable that competitive was a 15- to 20-fold difference in the kinasc levels ofthe tip enzymes could fall into this category. There was relatively and the lower crypt sections of both rat duodenum and little deviation from the predicted values when supernatant jejunum. The kinase level in the jejunum was highest in the and unfractionated pellets were recombined unless phos lower crypt and not in the upper crypt as was previously pholipase C was also present in the assay medium. How reported (22). This apparent discrepancy may be due to the ever, when either pellets fractionated by differential centnif more refined tissue planing technique (4) as well as the ugation or fractions from successive sections of the mucosa completely different extraction and assay methods used in were recombined, recovery of kinase activity in the samples these studies. The present findings agree with labeling was considerably less than the sum of their individual experiments in vivo (10, 11) which indicated that, as activities. The magnitude of the changes observed was epithelial cells migrate up the crypt, there is a progressive sufficiently large to exclude mere physical loss during reduction in the number of cells that are capable of further recombination. cell division. There appears to be an inverse relationship between the TdR kinase activity in human colonic mucosa was level of normal kinase activity and the degree of activation primarily in the lower third, the region with the greatest by phospholipase C. The degree of stimulation in intestinal proportionof rapidly proliferatingcells(14, 41). A sharp mucosa increases when epithelial cells mature and migrate decrease occurred in the middle third as was expected but toward the lumen. Stimulation of the pellet fraction was the kinase activity ofthe upper third was still relatively high. consistently greater than that of either the corresponding This is not too surprising since surgical specimens, which supernatant fraction or the original whole homogenate. are generally obtained from an area near a tumor, often The remarkable effect of commercial phospholipase C appear to be histologically normal mucosa and yet may preparations form C. we/chii was essentially independent of contain some cells with the capacity to proliferate during their lecithinase activity as has also been reported by others their entire life-span ( I3). Although this could also account (3 1). It is conceivable that the active component is a for the higher specific activities of TdR kinase from these nucleoside phosphotnansferase similar to (or identical with) samples compared to the values reported by others (19), it that recently identified as the protein responsible for the cannot explain the higher specific activities of kinase from stimulatory effect of culture filtrates of Clostridium autopsy samples. Apparently, the degradation of dTMP perfringens (34). Increases in the phosphorylation ofTdR in was minimized by using an extraction method that avoided adult rat liver were apparently attributable to the concerted rupturing the microsomes and liberating dTMP phospha action of liver ATPase and the nucleoside phosphotransfer tase (16, 19) and an assay system containing 3-phosphoglyc ase from clostridia (34, 40). The relative insensitivity of crate, which proved to be an effective inhibitor of this intestinal mucosa preparations to NaF (which is also a enzyme as was previouslyreportedby others(38). The potentinhibitorof ATPase),thenegligibleATPaseactivity

AUGUST 1973 1895

in the assaysystems,the lower efficiencyof AMP as a 6. Bianchi, P. A., Crathorn, A. R., and Shooter, K. V. Thymidine phosphatedonor, and the activation retained by TdR Kinases and DNA Synthesis in Normal and Regenerating Liver. preparations that had been purified over 500-fold from Biochim. Biophys. Acta, 61: 728-735, 1962. human colon and had no detectable ATPase activity' 7. Brent, T. P., Butler, J. A. V., and Crathorn, A. R. Variations in Phosphokinase Activities during the Cell Cycle in Synchronous indicate that such a mechanism cannot account for the Populations of HeLa Cells. Nature, 207: 176-177, 1965. stimulation of intestinal TdR kinase activity by phospho 8. Bresnick, E., and Karjala, R. J. End Product Inhibition of Thymidine lipase C preparations. Moreover, in contrast to the lack of Kinase Activity in Normal and Leukemic Human Leucocytes. Cancer stimulation of regenerating rat liver by culture filtrates of C. Res.,24:84l-846, 1964. perfringens (37), the kinase activity of rapidly proliferating 9. Bresnick, E., Williams, S. S., and Mosse, H. Rates of Turnover of cells in the intestinal crypt and that of regenerating rat liver' Deoxythymidine Kinase and of Its Template RNA in Regenerating were enhanced by phospholipase C. and Control Liver. Cancer Res., 27: 469-475, 1967. During the orderly and rapid maturation accompanying 10. Cairnie,A. B., Lamerton,L. F., andSteel,G. G. Cell Proliferation the migration of intestinal epithelial cells toward the lumen, Studies in the Intestinal Epithelium ofthe Rat. I. Determination of the Kinetic Parameters. Exptl. Cell Res., 39: 528-538, 1965. regulatory mechanisms are activated to decrease their I I . Cairnie, A. B., Lamerton, L. F., and Steel, G. G. Cell Proliferation proliferative activity and induce differentiation. Lower Studies in the Intestinal Epithelium of the Rat. II. Theoretical incorporation of TdR into cellular DNA and decline in the Aspects. Exptl. Cell Res., 39: 539-553, 1965. level of TdR kinase activity are manifestations of some of 12. Cleaver, J. E. Thymidine Metabolism: Pathways of Incorporation and the enzymatic changes occurring at this time. In precancer Degradation. Frontiers Biol. 6: 43-69, 1967. ous lesions of human colon, however, TdR kinase activity 13. Deschner,E.,Lipkin, M., andSolomon,C. Studyof HumanRectal remains relatively high; some of the morphologically nor Epithelial Cells in Vitro. II. H'-Thymidine Incorporation into Polyps mal cells develop and maintain an enhanced capacity to and Adjacent Mucosa. J. Nail. Cancer Inst., 36: 849-857, 1966. proliferate throughout their entire life-span. The data 14. Durham, J. P., and Ives, D. H. Intracellular Distribution of Pyrimi presented here suggest that the decrease in the apparent dine Nucleoside Phosphorylating Activities in Rat Thymus. Exptl. Cell Res., 70: 97-105, 1972. level of TdR kinase activity could be partly due to inhibition 15. Ernster, L., Siekevitz, P., and Palade, G. E. Enzyme-Structure of the enzyme rather than solely to its degradation. Relationships in the Endoplasmic Reticulum of Rat Liver. A Morpho Consequently, the increase in kinase activity in mature cells, logical and Biochemical Study. J. Cell Biol., 15: 541 -562, 1962. when proliferation is stimulated by disease, wounding, 16. Fiala, S., Fiala, A., Tobar, G., and McQuilla, H. Deoxynucleotidase starvation, or irradiation, is not merely attributable to Activity in Rat Liver and Certain Tumors. J. NatI. Cancer Inst., 28: synthesis of new enzyme. On the contrary, the present 1269-1289,1962. findings suggest that these increases in kinase activity could 17. Fortin-Magna, R., Hurwitz, R., Herbst, J. J., and Kretchmer, N. be partly due to inactivation of an inhibitor, which itself is Intestinal Enzymes: Indicators of Proliferation and Differentiation in labile to phospholipase C preparations or, alternatively, the Jejunum. Science, 167: 1927-1928, 1970. activation of a latent enzyme by a mechanism that phos 18. Glock, G. E., and McLean, P. Further Studies on the Properties and Assay of Glucose-6-Phosphate Dehydrogenase and 6-Phosphoglucon pholipase C mimics. Both of these might well be expressions ate Dehydrogenase of Rat Liver. Biochem. J., 55: 400-408, 1953. of the sameaction. 19. Gordon, H. L., Bardos, T. J., Chmielewicz, Z. F., and Ambrus, J. L. Comparative Study of the Thymidine Kinase and Thymidylate Kinase ACKNOWLEDGM ENTS Activities in Normal and Neoplastic Human Tissue. Cancer Res., 28: 2068-2077,1968. The authors wish to thank Stanley Zeichner for his capable technical 20. Gray, H. J. Nucleotides and Nucleotide Metabolism. In: H. Busch assistance. (ed), Methodsin CancerResearch,Vol.3, pp. 243-389. New York: Academic Press, Inc.. 1967. 21. Gray, E. D., Weissman, S. M., Richards, J., Bell, D., Keir, H. M., REFERENCES Smellie, R. M. S., and Davidson, J. N. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1973 American Association for Cancer Research. Distribution and Regulation of Deoxythymidine Kinase Activity in Differentiating Cells of Mammalian Intestines

Josephine S. Salser and M. Earl Balis

Cancer Res 1973;33:1889-1897.

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