alkaline phosphatase substrate specificities in cultured ... · species. it was our intention to...

Alkaline Phosphatase Substrate Specificities in Cultured

INTRODUCTION

It has been reported (1) that adenosine tnphosphate or muscle adenylic acid gave greaterblackening than yeast adenylic acid or sodiumglycerophosphate when used as substrates in theGomoni test (4) for alkaline phosphatase on tissuecultures of embryonic mouse skin and mousesarcomas T@41 and Ma387. This finding has nowbeen followed by more detailed studies on avariety of tumors and normal tissues from severalspecies. It was our intention to determine whetheror not malignant cells showed any consistentdeviation from normal cells in the relative substrate specificity of their alkaline phosphatase.For this purpose, results with muscle adenylicacid were compared to those obtained with yeastadenylic acid and sodium glycerophosphate.

Muscle adenylic acid is preferentially dephosphorylated by the enzyme 5-nucleotidase, described by Reis (11, 1@). Gulland and Jackson (6),as well as Reis, observed that extracts rich in5-nucleotidase activity rapidly attacked muscleadenylic acid or inosinic acid but split such materials as yeast adenylic acid or glycerophosphatemuch more slowly. The purine niboside-5-phosphate grouping found in muscle adenylic acid isthe specific substrate of the group II alkaline phosphatase enzymes of Newman and collaborators(9). It has been observed by Gomoni (5) that alkaline phosphatase tests with muscle adenylic acidgive different results from those with glycerophosphate in paraffin sections of certain organs.Hence, the studies reported below may be regarded as comparisons of the relative activities ofnonspecific phosphomonoesterase and 5-nucleotidase.

The exactness of localization of alkaline phosphatase in the histochemical test has recently beenquestioned (3, 7), with evidence brought forward

* This work was carried out with the aid of grants fromthe American Cancer Society and the National CancerInstitute, of the National Institutes of Health, Public HealthService.

Received for publication October 9, 1950.

for the diffusion of the enzyme and its adsorptionon nuclear structures, resulting in a possibly falsepicture of its distribution. With this in mind, onemay interpret the results reported here in twoways : The intensity of staining on nuclear structures may be regarded either as bearing some nelation to enzymatic activities on those structures inlife or as reflecting activity of diffusible enzymeoriginating elsewhere in the tissue and adsorbed onthe nuclear structures after death. That is, theinterpretation is related to the tissue culture as awhole, and with some doubt it may apply tolocalized structures as well.

It was found that the various tissues, whencultured and fixed, showed different predilectionsfor the substrates employed. Most notable was theconsiderably greater splitting of muscle adenylicacid than of the other substrates by Sarcoma 180cells. However, there was no correlation betweenmalignancy and the differential ability to attackany given substrate. Results of tests for phosphatase activity in mitotic chromosomes wereparalleled by results in resting nuclei, in whichthe nucleolar complex of plasmosome and nucleolus-associated heterochromatin was specificallyunder consideration.

MATERIALS AND METHODS

Cultures were made of tissues from threespecies. The mouse tissues included sarcoma T@41,lung tumor Ma387, Crocker sarcoma 180, Ridgway osteogenic sarcoma, and Carcinoma 10@5, aswell as four embryonic tissues : heart, skeletalmuscle, liver, and brain. The rat tissues plantedwere sarcoma R39 and embryonic skin. Thechicken tissues included Rous sarcoma cells takenfrom cultures on the chonioallantoic membrane,and embryonic skeletal muscle, brain, and intestine.'

Small fragments of these tissues about 1 or@mm. in diameter were planted from the animal oncoverslip inserts in flattened roller tubes. Each

â€˜Weare indebted to Drs. K. Sugiura and D. A. Karnofskyfor certain of these materials.

174

Normal and Malignant Cells of Mouse, Rat, and Fowl*

JOHN J. BIESELE AND ANNE YATES WILSON

(From the Sloan-Kettering Institute for Cancer Research, New York @1,N.Y.)

on March 2, 2020. © 1951 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

BIESELE AND WILSONâ€”Alkaline Phosphatase in Tissue Culture 175

roller tube held two coverslip inserts, and therewere two or three explants on each coverslip,usually of different tissues. The explants werecovered with a thin chicken plasma clot, andclotted plasma was also used to hold the coverslipsto the roller tube wall. The incubation medium,1 ml. per roller tube, was composed of 4 partsGey's salt solution,@ parts chick embryo extract,1 part human placental serum, and 3 parts horseserum, with initial pH at 7.4â€”7.8. This mediumcontained @25units each of penicillin and streptomycin per milliliter. The cultures were incubatedat 37Â°C. for 1 or@ days (i.e., until a sufficientgrowth had been obtained) in a drum rotating 10times per hour.

After being rinsed in physiological saline, cultures were fixed in a solution suggested by Danielli(s). The fixativewascomposedof 70partsethylalcohol, @0parts pyridine, 5 parts 37 per centformaldehyde, and 5 parts water. The fixative wasmade up fresh and chilled to â€”@0Â°C. before use.The cultures were held in the fixative at this temperature overnight or for several days before theGomori phosphatase test was carried out. Beforethe test, the explants were removed from thecoverslips with fine forceps, with the zones ofgrowth being left.

For the test, the coverslip cultures were incubated in Columbia staining dishes holding 10 ml.of distilled water containing sodium barbital,

@.1m@&/l; calcium chloride, 15 m@/l; magnesiumchloride, @0nmi/l; and the substrate in concentrations ranging in fivefold steps from 0.0003 to 5.0mM/l. Control cultures were incubated in thismedium without an organic phosphate substrate.The pH of the medium was adjusted to 9 wherenecessary. Incubation was for 19 hours at 37Â°C.Thereafter, the coverslips were passed through 4changes of 1 per cent calcium chloride and werethen exposed to@ per cent cobalt chloride for 5minutes, a running distilled water rinse for 30seconds, a 1 per cent dilution ofammonium sulfideby volume for 3 minutes, and a second runningdistilled water rinse for@ minute. After beingdehydrated in alcohol and cleared in xylene, thepreparations were mounted in a toluene-solubleresin.

An attempt was made to estimate the extent ofblackening by means of a microphotometer. Thisapparatus was patterned after that describedby Pollister and Ris (10) and made use of aPhotovolt photometer model 51@ with a photomultiplier tube. Illumination of KÃ¶hler type wasfurnished by a tungsten ribbon filament lamp andwas filtered through a glass water cell and a Corning blue daylight glass. The microscope condenser

diaphragm was opened to admit only axial illumination through a circle 3 mm. in diameter. Readings were made with the slide under oil immersion.A diaphragm beneath the photocell opening admitted only that light passing through a circle ofapproximately @20square j@ area in the objectplane.

In twenty nonmitotic cells taken at random ina given culture, readings were made of the lightpassing through a cylindrical portion of the cell,including a nucleolus and associated heterochromatin. The nuclear membranes and a thin sheet ofcytoplasm above and another below the nucleuswere also in the path of light, it must be remembered. With each such reading an additional reading through an adjacent portion of the clot free ofcells was taken. The data in the tables are ratiosof the galvanometer deflection found with lightpassing through the nucleus to that found in theclot reading.

Mean and standard error were determined forthe twenty observations through resting nuclei perculture. In addition, readings in irregular numberswere taken through mitotic figures at stages ofgreat chromosome condensation, namely, metaphase, anaphase, and early telophase.

Microphotometry was employed with reservations, which are mentioned in the discussion. Serving somewhat as a shorthand substitute for anextensive photographic record, the photometricdata are to be regarded only as indications, withlittle quantitative significance, of the extent ofblackening with the cobalt suffide precipitate.This, in turn, may reflect only crudely the actualenzyme activity. Significant differences may wellbe those restricted to extensive blackening on theone hand and light browning or none on the other.

RESULTS

Results of some 5,000 observations with restingnuclei are summarized in Tables 1â€”4.Mean ratiosof light transmission through a cylindrical portionof cell including some of the nucleus containing anucleolus are given for various concentrations ofthe three substrates, together with standarderrors. Results with the lowest concentrations inmany sets are omitted from the tables to savespace, because they agree with control values.

The mouse tissues could be grouped into roughly three classes with respect to their blackeningwith the substrates. Crocker mouse sarcoma 180was outstanding in the far greater ease with whichphosphatase activity could be demonstrated inits fixed cells with muscle adenylic acid than withyeast adenylic acid or glycerophosphate. Somewhat less pronounced in this same respect were



TABLE 1

MousE NORMALTissuEs; RATIOSOF TRANSMISSIONTHROUGHNUCLEUS(NuCLEOLUS) TO TnAT T}IROUGH ADJACENT CLOT

Muscix Yx&sT GLYCSROADaNYLIC ACID ADENTLIC ACID PEOSPEATS No aosam@x

CONC. Mean St. err. Mean St. err. Mean St. err. Mean St. err.

MOUSE EMBRYO SKIN: 0.92Â±0.0085.0 ruM/I 0.28Â±0.015 0.48Â±0.020 0.68Â±00281.0 0.50 0.029 0.70 0.027 0.77 0.0180.2 0.44 0.019 0.82 0.014 0.92 0.0050.04 0.46 0.041 0.92 0.007 0.92 0.0090.008 0.94 0.007 0.96 0.005 0.89 0.010

MOUSE EMBRYO BRAIN: 0.88 0.017

0.04 0.39 0.030 0.47 0.045 0.88 0.0130.008 0.88 0.017 0.82 0.011 0.95 0.007

MOUSE EMBRYO HEART: 0.89 0.016

5.0 0.48 0.020 0.49 0.0241.0 0.41 0.019 0.69 0.022 0.53 0.0280.2 0.43 0.023 0.62 0.084 0.44 0.0810.04 0.55 0.026

MousE EMBRYOLIVER: 0. 85 0.009

5.0 0.51 0.080 0.49 0.021 0.47 0.0271.0 0.45 0.028 0.66 0.040 0.62 0.0310.2 0.61 0.020 0.90 0.009 0.74 0.0210.04 0.71 0.026 0.84 0.024 0.92 0.0110.008 0.88 0.011 0.86 0.013 0.80 0.023

TABLE 2

MOUSE TUMOR TISSUES; RATIOS OF TRANSMISSION THROUGH NUCLEUS

(NUCLEOLUS) TO THAT THROUGH ADJACENT CLoT

Mmcix Yz.&ST GLYCaROADENYLIC ACID AD@NTLIC ACID PHOSPHATS No svssra&@x

CONC. Mean St. err. Mean St. err. Mean St. err. Mean St. err.CROCKER SARCOMA 180: 0.78 Â±0.018

5.0mM/I 0.57Â±0.020 0.70Â±0.0161.0 0.17Â±0.006 0.80 0.009 0.77 0.0110.2 0.21 0.009 0.84 0.019 0.84 0.0820.04 0.21 0.010

SARC0MAT241: 0.88 0.0155.0 0.15 0.011 0.31 0.017 0.40 0.0251.0 0.16 0.012 0.60 0.028 0.58 0.0250.2 0.18 0.015 0.65 0.018 0.75 0.0110.04 0.24 0.018 0.70 0.011 0.78 0.0160.008 0.78 0.019 0.73 0.014 0.78 0.015

LUNG TUMOR MA387: 0.88 0.0115.0 0.28 0.011 0.50 0.0221.0 0.30 0.018 0.65 0.019 0.72 0.0160.2 0.39 0.012 0.81 0.014 0.89 0.0090.04 0.61 0.036 0.81 0.017

CARCINOMA 1025: 0.90 0.0115.0 0.37 0.028 0.49 0.024 0.56 0.0301.0 0.48 0.025 0.59 0.020 0.72 0.0210.2 0.70 0.017 0.63 0.0250.04 0.76 0.024 0.93 0.008

RIDGWAY OSTEOGENIC SARCOMA: 0.86 0.0155.0 0.87 0.026 0.60 0.025 0.46 0.0250.2 0.57 0.028 0.50 0.027 0.54 0.0220.04 0.78 0.099 0.67 0.022 0.89 0.0120.0016 0.83 0.018 0.91 0.007 0.91 0.008



BIESELE AND WILSONâ€”Alkaline Phosphatase in Tissue Culture 177

sarcoma T@41, lung tumor Ma387, Carcinoma10@5, embryo skin, and embryo brain. All threesubstrates were split with approximately equalease in the third group, which included mouseembryo heart, liver, and skeletal muscle (for whichno photometric data were collected), as well asRidgway osteogenic sarcoma.

Fixed cultures of rat sarcoma R39 and ratembryo skin each split the two adenylic acids toabout the same extent and glycerophosphatenearly so.

Fixed cultures of chick embryo brain, intestine,and skeletal muscle showed slightly but irregularly greater blackening with the adenylic acids thanwith glycerophosphate. Cultures of Rous sarcomacells exhibited only weak alkaline phosphataseactivity and a slight predilection, if any, for yeastadenylic acid among the three substrates.

The above results with phosphatase reactions inthe nucleolus and associated chromatin in resting

nuclei were paralleled in great measure by resultswith mitotic chromosomes. Photometric determinations were made on sets of cultures of mouseembryo skin, heart, and brain, and of Sarcoma 180,Carcinoma 10@5,and chick skeletal muscle. In theresults given in Table 5, each entry is the meanof determinations on 3â€”15different mitotic figures.

The parallelism of results between nucleoli withassociated heterochromatin in resting nuclei, onthe one hand, and mitotic chromosomes, on theother, seen in a comparison of Table 5 with theprevious tables, appeared to extend to all tissuesplanted, according to visual observations.

Most striking were results with Sarcoma 180chromosomes. As with resting nuclei, Sarcoma180 chromosomes were greatly blackened by cobaltsulfide after incubation with muscle adenylic acidbut only weakly stained after incubation with theother substrates. Figures 1â€”3are photomicrographs made under similar conditions of fixed cells

TABLE 3

RAT TISSUES;RATIOSOFTRANSMISSIONTHROUGHNUCLEUS(NUCLEOLUS)TO THAT THROUGH ADJACENT CLOT

No SUBSTRATEMean St.err.

0 .92Â±0.014

0.94 0.007

MUSCLEYEASTGLTCEROADENTLIc ACIDADENTLICACIDPUOSPRATECONC.

Mean St. err.Mean St. err.Mean St.err.RAT

EMBRYOSKIN:5.OmM/l0.57Â±0.0940.75Â±0.0221.0

0.51 0.0230.73Â±0.0260.590.0230.20.63 0.0250.53 0.0260.930.0070.040.93 0.0060.91 0.0091.000.002RAT

SARCOMA1189:5.00.26 0.0980.26 0.0980.470.0401.00.85 0.0410.26 0.0890.370.0420.20.53 0.0110.38 0.0420.560.0420.040.57 0.0320.62 0.0550.990.0020.0080.91 0.0120.92 0.008TABLE

4

CHICKTissuEs; RATIOSOFTRANSMISSIONTHROUGHNUCLEUS(NUcLEOLUS) TO THAT THROUGH ADJACENT CLoT

MuscixADE:NYLIc ACID

Conc. Mean St. err.CHICK EMBRYO BRAIN:

5.0mM/I 0.26Â±0.0251.0 0.32 0.0280.2 0.54 0.0390.04 0.65 0.0290.008 0.82 0.012

CHICKEMBRYOINTESTINE:5.0 0.40 0.0321.0 0.44 0.0130.2 0.61 0.0250.04 0.94 0.007

CHICK EMBRYO SKELETAL MUSCLE:5.01.0 0.63 0.0180.2 0.92 0.008

ROUS SARCOMA FROM CHICK EMBRYOS:5.0 0.65 0.020 0.461.0 0.71 0.026 0.520.2 0.96 0.008 0.97

YEAST Gaycmo

ADnrruc ACID PHOSPHATEMean St. err. Mean St. err.

No SUBSTRATEMean St. err.

0.86Â±0.0120 .54 Â±0.0350.65 0.0300.52 0.0410.84 0.0170.89 0.012

0.42 0.0200.51 0.0260.53 0.0080.93 0.005

0.56 0.0280.82 0.0170.71 0.017

0.41Â±0.0280.56 0.0220.65 0.0080.92 0.009

0.46 0.0910.68 0.0160.87 0.0080.91 0.005

0.71 0.0290.81 0.0210.95 0.009

0.51 0.0260.95 0.0060.90 0.012

0.95 0.007

0.93 0.007

0.94 0.0120. 0370.0190.004



178 Cancer Research

at room temperature or with water at 80Â°C. for10 minutes.

DISCUSSION

Increased objections have arisen of late to theuse of the histochemical test for alkaline phosphat&se activity (3, 7) because of the possibilitythat the enzyme or an intermediate product in thereaction is not securely localized. If it were to beaccepted that much of the staining of nuclei inthis histochemical procedure is an artifact causedby post flwrtefll adsorption of diffusible enzyme onnuclear structures (3), then interpretation of ourresults would be considerably restricted. The dataon nuclear blackening would be meaningless asfar as nuclei are concerned and would serve toindicate only crudely the relative amounts of thediffusible enzymes originally present in the cultures, subject to possible differences in adsorptive

No capacity of the nuclei of the several tissues withSUBSTRATE

Mean respect to any one enzyme.

0.84 However, it is noteworthy in our material that

the various tissues retained their characteristicactivity patterns despite the fact that cultures of

0 .88 several different tissues were customarily plantedon each coverslip within a few mm. of one another.Indeed, the substrate specificities supplemented

0 .77 purely morphological means of detecting cells fromone culture that had wandered into the growth zoneof another (Fig. 19). A fixation artifact involvingmovement of enzyme within the individual nucleuswas evident in cells on the coverslip beneath the

0.80 original explant, which was removed after fixa

tion. In these nuclei, there was a movement ofphosphatase-positive material in the presumed

0 .87 direction of movement of the fixative toward thecenter of the explant. The enzyme movement wasapparently halted by the nuclear membrane (Fig.

@0).This artifact was not evident in cells of thegrowth zone, where our interest lay. It would appear that the movement of enzyme in our materialcould not have been extreme under the conditionsemployed.

The microphotometric apparatus served as aconvenient means of assessing the approximatedensity of the stain. The results are to be regardedat best as only coarse indications of relative enzyme activity. In many cases the several highestconcentrations of substrate gave equivalent smalldeflections of the galvanometer, much as several ofthe lowest concentrations caused equivalent greatdeflections ; with these lowest concentrations, aswith controls, light loss apparently occurredlargely because of structural light scattering andbecause of the staining of preformed phosphate.Frequently, only a few intermediate concentrations caused an intermediate deflection of the

of Sarcoma 180 cultures incubated with the sameconcentration of the three substrates. The processing of the three photographs was identical.

A sharp contrast is furnished by the photomicrographs of embryonic mouse heart cells inFigures 4â€”6.These cultures were on the samecoverslips as were the Sarcoma 180 cultures ofFigures 1â€”3,yet the heart cultures were nearlyequivalent in staining after all substrates.

Figures 7â€”18illustrate the effect of change ofsubstrate upon some of the other tissues. Eachset of three figures from each tissue was prepared

TABLE 5CUROMOSOMES; RATIOS OF TRANSMISSION THROUGH CoN

DENSED MIT0'rlc FIGmu@s TO THATTHROUGH ADJACENT CLOT

by similar photographic processes. The differencesin density of background within each set areprobably related to differences in clot thicknessand staining, some of which may depend ondifferences in quantity of diffusible phosphatasein the clot derived from the cells.

Experiments of both groups I and II of Newman and collaborators (9) on inhibitors indicatedthe presence of enzymes in tissue cultures of Sarcoma 180 and mouse embryo skin. The splittingof muscle adenylic acid was hindered less than wasthat of yeast adenylic acid by 10 nmi/l sodiumcyanide in the incubation medium. There was lessdifference in this respect in the presence of @50mM/l glycine. There were equally great inhibitionswith both adenylic acids after treatment of thefixed cultures with 5 per cent trichloroacetic acid

Mmcxx YEASTADENYLIC

ACID ACID

Co'ic. Mean Mean

MousE EMBRYOSKIN:5.OmM/l 0.891.0 0.41 0.630.2 0.43 0.57

MousE EMBRYOHEART:5.0 0.291.0 0.35 0.580.2 0.85 0.28

MousE EMBRYOBRAIN:0.04 0.24 0.250.008 0.59 0.72

CRIcK EMBRYO SKELETAL MUSCLE:5.0 0.601.0 0.730.2 0.68

MoUSE SARcOMA180:5.0 0.611.0 0.140.2 0.16 0.84

MousE CARCINoMA1025:5.0 0.21 0.291.0 0.31 0.680.2 0.54 0.500.04 0.61 0.89

GLTCERO

PEOSPRATS

Mean

0.620.540.74

0.400.860.46

0.710.69

0.650.770.80

0.800.710.77

0.480.47



FIG. 1.â€”Mouse Sarcoma 180 culture after alkaline phosphatase reaction with muscle adenylic acid, 0.2 mM/i. Metaphase at arrow. Mag. X600.

FIG. 2.â€”Sarcoma 180 culture after alkaline phosphatase reaction with yeast adenylic acid, 0.2 mM/i. An early anaphase,

not discernible, is at arrow. Mag. X600.

FIG. 3.â€”Sarcoma 180 culture after alkaline phosphatasereaction with sodium glycerophosphate, 0.2 mM/I. Anaphaseat arrow. Mag. X600. Photographic treatment was identicalwith that of Figures 1 and 2.

FIG. 4.â€”AKm mouse embryo heart culture 011same coverslip as Sarcoma 180 culture of Figure 1, after alkaline phosphatase reaction with muscle adenylic acid, 0.2 mM/i. Noteanaphase. Mag. X600.

FIG. 5.â€”AKm mouse embryo heart culture on same coverslip as Sarcoma 180 culture of Figure 2, after alkaline phosphatase reaction with yeast adenylic acid, 0.2 mM/I. Metaphase at arrow. Mag. X600.

FIG. 6.â€”AKm mouse embryo heart culture on same coverslip as Sarcoma 180 culture of Figure 3, after alkaline phosphatase reaction with sodium glycerophosphate, 0.2 mM/i.Anaphase at arrow . Photographic treatment was the same asfor Figures 1â€”5.Mag. X600.

FIG. 7.â€”Mouse (â€˜arcinoma 1025 culture after alkaline phosphatase reaction with muscle adenylic acid, 1.0 mM/i. Metaphase at arrow. Mag. X600.

FIG. 8.â€”Mouse Carcinoma 102.5 culture after alkaline phosphatase reaction with yeast adenylic acid, 1.0 hiM/i. Notetripolar telophase. Mag. X600.

FIG. 9.â€”('arcinoma 1025 culture after alkaline phosphatasereaction with sodium glycerophosphate, 1.0 mM/L Note tripo-.

lar telophase. Photographic treatment was the same as for Figures 7 and 8. Mag. X600.

FIG. 10.â€”AKm mouse embryo skin culture after phosphatase reaction with muscle adenylic acid, 1.0 mM/i. Note anaphase. Mag. X600.

FIG. 11.â€”AKIn IflOU5@ embryo skin culture after phosphatase reaction with yeast adenylic acid, 1.0 mM/i. Noteanaphase. Mag. X600.

FIG. 12.â€”AKm mouse embryo skin culture after phosphatase reaction with sodium glycerophosphate, 1.0 mM/i.Prometaphase at arrow. Mag. X600. Photographic treatmentwas similar for Figures 10, 11, and 12.

FIG. 13.â€”Chick embryo skeletal muscle culture after phosphatase reaction with muscle adenylic acid, 1.0 mat/i. Telophase at arrow. Mag. X600.

FIG. 14.â€”Chick embryo skeletal muscle culture after phosphatase reaction with yeast adenylic acid, 1.0 mM/i. Metaphase at arrow. Mag. X600.

FIG. 15.â€”Chick embryo skeletal muscle culture after phosphatase reaction with sodium glycerophosphate, 1.0 mM/i.Metaphase at arrow. Photographic treatment was the same forFigures 13, 14, and 15. Mag. X600.

FIG. 16.â€”Rous sarcoma cultured from chick embryo, afterphosphatase reaction with muscle adenylic acid, 1.0 mM/i.Mitosis at arrow. Mag. X600.

FIG. 17.â€”Rous sarcoma culture after phosphata.se reactionwith yeast adenylic acid, 1.0 mM/i. Mitosis at arrow. Mag.X600.

FIG. i8.â€”Rous sarcoma culture after phosphatase reactionwith sodium glycerophosphate, 1.0 mM/I. Photographic treatmelit was the same for Figures i6, 17, and 18. Mag. X600.



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FIG. 19.â€”Sarcoma 180 cells (arrows) set off from cells ofembryonic mouse skin, among which they had wandered, bygreater blackening after alkaline phosphatase reaction with

0.2 mM/i muscle adenylic acid. Mag. X600.

FIG. 20.â€”Fixation artifact in nuclei of AKm mouse skillcells lying beneath original explant (now removed) when fixed.Arrow points toward center of original explant and in directionof presumed fixative movement. Phosphatase-active materialseems to have been halted iii its movement by the nuclearmembrane. The cells at right edge were not beneath the ex

plant. Muscle adenylic acid, 0.2 mM/I. Mag. X600.



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BIESELE AND WILSONâ€”Alkaline Phosphatase in Thsue Culture 179

galvanometer. Possibly this situation might havebeen improved if a translucent dye had beenformed, instead of an opaque precipitate, as mdicator of enzyme activity (8).

The biochemical studies of 5-nucleotidase (6,11, 1@) have been followed by publications inhistochemistry indicating that such an enzyme isan entity separate from nonspecific alkalinephosphomonoesterase (5, 9). Our present studiessupport this view, by and large, with the mostcogent evidence being provided by the greatersplitting of muscle adenylic acid by fixed cells ofSarcoma 180.

The differentiation with respect to favored substrate cuts across groupings of neoplastic and embryonic cells. It appears that it is not possible toseparate normal from malignant cells on the basisof substrate specificity (so far as tested) of theenzyme or enzymes demonstrated by means ofthe histochemical reaction for alkaline phosphatase.

SUMMARY

1. Fixed cultures of various embryonic andmalignant tissues of the mouse, rat, and fowl havebeen subjected to histochemical tests for alkalinephosphatase.

@.The organic phosphates used as substratesin these tests were muscle adenylic acid, yeastadenylic acid, and sodium glycerophosphate.

3. The tissues differed from one another in theextent of blackening in the resting nucleus (nucleolus) with the various substrates.

4. Mitotic chromosomes gave results similar tothose obtained with resting nuclei.

5. Crocker mouse sarcoma 180 cells were outstanding in their high dephosphorylation of muscleadenylic acid and near failure to split yeastadenylic acid and glycerophosphate.

6. Substrate specificity of alkaline phosphatase(or relative activities of nonspecific alkalinephosphatase and 5-nucleotidase) did not serve todistinguish all malignant cells from all normalcells.

REFERENCES

1. Bisssas, J. J. Phosphatases of the Mitotie Apparatus inCultured Normal and Malignant Mouse Cells. ProceedingsFirst National Cancer Conference, American CancerSociety and the National Cancer Institute of the PublicHealth Service, pp. 34-41, 1949.

2. D@isau, J. F. A Critical Study of Techniques for Determining the Cytological Position of Alkaline Phosphatase. J. Exper. Biol., 22: 110â€”17,1946.

3. FaiGn@r,I.; Woir, A.; and KABAT, E. A. HistochemicalStudies on Tissue Enzymes. VI. A Difficulty in the Histochemical Localization of Alkaline Phosphatase in Nuclei.Am. J. Path., 26:647â€”59,1950.

4. GoMoal, G. MicrotechnicalDemonstration of Phosphatasein Tissue Sections. Proc. Soc. Exper. Biol. & Med., 42:28-26, 1939.

5. . Further Studies on the Histochemical Specificityof Phosphatases. Ibid., 72:449â€”50,1949.

6. GULLAND,J. M., and JACKSON,E. M. 5-Nucleotidase.Biochem. J., 32:597â€”601, 1938.

7. JACOBY,F., and M@utTn@,B. F. The Histochemical Test forAlkaline Phosphatase. Nature, 163:875â€”76, 1949.

8. MANHEIMER,L. H., and [email protected],A. M. Improvement inthe Method for the Histochemical Demonstration of Alkaline Phosphatase and Its Use in a Study of Normal andNeoplastic Tissues. J. Nat. Cancer Inst., 9: 181â€”99,1948.

9. Nswa@, W.; FEIGIN,I.; Woir, A.; and KABAT,E. A.Histochemical Studies on Tissue Enzymes. IV. Distribution of Some Enzyme Systems Which Liberate Phosphateat pH 9.2 as Determined with Various Substrates andInhibitors; Demonstration of Three Groups of Enzymes.Am. J.Path.,26:257â€”305,1950.

10. POLLISTER,A. W., and Ris, H. Nucleoprotein Determination in Cytological Preparations. Cold Spring HarborSymp. Quant. Biol., 12: 147â€”57,1947.

11. Reis, J. tYberdie spezifische Phosphatase der Nervengewebe. Enzymologia, 2: 110â€”16,1987.

12. . tYber die AktivitÃ¤t der 5-Nukleotidase in dentierischen mid menschlichen Geweben. lUd., pp. 183-90.



1951;11:174-179. Cancer Res John J. Biesele and Anne Yates Wilson and Malignant Cells of Mouse, Rat, and FowlAlkaline Phosphatase Substrate Specificities in Cultured Normal

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