[CANCER RESEARCH 45, 4301-4307, September 1985]

Comparison of the Morphology and Enzyme Activity of Mononuclear Cells from

Fischer 344 Rats with Either Spontaneous or Transplanted Leukemia

Michael P. Dieter, Robert R. Maronpot, and John E. French

National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

ABSTRACT

Mononuclear cell (MNC) leukemia was identified in 26-month-

old F344 rats by splenomegaly, reduced red blood cell counts,and elevated white blood cell counts. Atypical MNC were predominant in spleen and blood with acentric nuclei and red cyto-

plasmic granules. Rentóse shunt, glycolytic, and Krebs cycleenzyme activities were elevated 2- to 11-fold in the enrichedMNC fraction (Ficoll-Paque density gradients, 1.077 g/ml) iso

lated from spleen. A leukemic MNC line was derived from one ofthe spontaneously leukemic donors and then maintained in vivoby s.c. transfer of 2 x 107 spleen cells into 7-8-week-old syn-

geneic recipients. In these serial transplantation experimentsleukemia that was clinically and morphologically indistinguishablefrom spontaneous leukemia in 104-week-old rats was inducedin 22-24-week-old rats. Enhanced enzyme activity in MNC was

not essential to maintain the phenotypic expression of Fischerrat leukemia. The pattern of biochemical response in spleenMNC from transplanted cases was the opposite of that previously noted in spontaneously leukemic rats, with 50-70% de

creases in the specific activities of pentose shunt enzymes andmalate dehydrogenase. Reversal of the expression of theseenzymes in MNC may be related to a difference in the growthrate of the tumors or to selective proliferation of the transplantedleukemic cells. In addition acetylcholinesterase activity decreased 35-85% in MNC of spleen and blood. Transplantable

MNC from F344 rats provide abundant tumorigenic material witha novel biochemical expression that may be useful in the studyof chemotherapeutic intervention.

INTRODUCTION

Fischer rat leukemia was described by Moloney (1) as aspontaneous MNC1 leukemia originating in the spleen (2) with

secondary involvement of many tissues in the body. The incidence of spontaneous leukemia in the Fischer strain was 11 of43 in females and 10 of 43 in males, averaging 24.4% (1, 3). Theduration of the disease after appearance of leukemic cells inperipheral blood averaged 5 weeks; age at death ranged between 14 and 30 months with a median of 25 months. Otherstrains of rats vary in their incidences of spontaneous leukemiafrom 17.9% in the Wistar-Furth strain (4) to 2% in Wistar rats (5)to 0.03% recently reported in Sprague-Dawley rats (6).

Three laboratories have studied the morphological, clinical,and immunological characteristics of spontaneous F344 rat MNCleukemia and have suggested that the cells were morphologically

' The abbreviations used are: MNC, mononuclear cell; PCV, packed cell volume;HB, hemoglobin; G6PD, glucose-6-phosphate dehydrogenase; 6-PGD, 6-phos-

phogluconic dehydrogenase; HMS, hexose monophosphate shunt pathway; ICDH,isocitric dehydrogenase; MDH, malate dehydrogenase; TCA, tricarboxylic acidcycle; ACHE, acetylcholinesterase; HBSS, Hanks' balanced salt solution.

Received 10/15/84; revised 4/17/85; accepted 6/7/85.

similar to large granular lymphocytes (7-12) and therefore may

be of lymphoid cell origin, while others favor a reticuloendothelialcell lineage (4, 6). These cells have been described as heterogeneous with respect to cell surface antigens, strongly esterasepositive, and with some of the immunological characteristics ofnatural killer cells (8, 12). Losco and Ward (13) subsequentlytransplanted tumor or spleen tissue fragments from nine 20-35-month-old rat donors into 3-month-old syngeneic recipients

which developed leukemia between 91 and 292 days posttransplantation.

None of these recent studies have addressed the enzymaticcharacteristics of the leukemic cells. Extensive research on thebiochemistry of human leukemic cells was performed in the1950s by Beck and Valentine (14,15) and by Beck (16-18), who

attempted to elucidate metabolic derangements associated withleukemogenesis. No consistent pattern of glycolytic enzymeresponses in leukemic cells was found, but it was suggestedthat decreases in glucose 6-phosphate concentration and hex-

okinase activity in leukemic cells accounted for the invariableincrease in the percentage of glucose metabolized by the hexosemonophosphate shunt pathway in leukemic cell preparations(18).

Beck was constrained to use heterogeneous populations ofleukocytes isolated from patients that had been diagnosed withdifferent types of leukemia and that had received treatment suchas radiation. In addition phytohemagglutinin was used in the cellisolation procedures and most of the metabolic measurementswere made on cultured cells or cell homogenates during 2-3 h

of in vitro incubation. A review of this early work by Hayhoe (19)concluded that there was no clear evidence of disorders ofcarbohydrate metabolism in the leukemic cells studied, probablybecause of the heterogeneity of the cell populations used.

In a later study Brody ef a/. (20) incubated a heterogeneouspopulation of lymphocytes obtained from patients receivingchemotherapy or X-irradiation treatment for chronic lymphocytic

leukemia and reported a decrease in hexose monophosphateshunt utilization and decreased glycolytic activity in comparisonto normal lymphocytes.

We have reevaluated the cellular biochemistry of leukemiautilizing discrete populations of MNC derived from spontaneouscases of Fischer rat leukemia. An in vivo cellular transplant modelwas subsequently developed that compressed the time coursefor expression of leukemia to 103-112 days, thereby enabling

production of large amounts of biological material in a reasonablelength of time to carefully validate the model and to determinethe value of these cellular biochemical responses as markers forleukemia. Toward this end we have separated, quantitated, andidentified an enriched MNC population from the peripheral bloodand spleens of F344 rats and report here a comparison of themorphology and enzyme activities of cells derived from hostswith either spontaneous or transplanted MNC leukemia.

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ENZYME ACTIVITY IN LEUKEMIC MONONUCLEAR CELLS

MATERIALS AND METHODS

One of our contract laboratories, Southern Research Institute inBirmingham, AL, assisted us in identifying leukemic rats by preparingblood smears from control F344 rats being used in 2-year carcinogenicity

tests. Fifty control rats were sampled and by hematological examinationwe selected six to eight positive or negative cases for leukemia thatwere subsequently verified at necropsy. At termination 4-ml heparinized

blood samples from the inferior vena cava and single cell suspensionsfrom the spleen were collected from each rat, placed on ice, andtransported by plane to the National Institute of Environmental HealthSciences, Research Triangle Park, NC, for further processing the following day. In addition body weights, organ weights, spleen impressions,and selected tissues for histopathological analysis were collected fromeach of the rats.

The whole spleen from controls or about 2 g of leukemic spleens werecrushed in a Retri dish with heparinized HBSS and the slurry was passedthrough gauze. Single cell suspensions were prepared by expressing thecoarsely filtered material through a 25-gauge needle. Either 3 ml of thesecell suspensions or 3 ml of blood were layered on the top of Ficoll-Paquegradients and centrifugea at 400 x g in a swinging bucket rotor at 5°C

for 20 min, and the enriched MNC fraction at a density of 1.077 g/mlwas removed, washed twice in HBSS, counted, and resuspended atappropriate concentrations.

MNC were identified by light microscopy as normal or leukemic inblood smears, spleen smears, and in concentrated single cell cytospinpreparations after Wright-Giemsa staining. Cells for transplantation orenzymatic analyses were counted on an Ortho ELT-8 hematology ana

lyzer.Cell suspensions from the blood and spleen of one selected leukemic

rat were washed, counted, and suspended on Ficoll-Paque gradients

and an enriched MNC fraction was separated, identified, and quantitated.Syngeneic 6-7-week-old male F344 rats were given s.c. injections of

0.25 ml of 2 x 107 of these leukemic mononuclear spleen cells in HBSS.

When animals exhibited unequivocal signs of leukemia, including weightloss, pale eyes, and icterus, they were sacrificed and an aliquot from apool of their leukemic mononuclear spleen cells was used for transplantation into the next group of syngeneic recipients. The original leukemicmononuclear spleen cell line has thus been maintained by serial transplantation. The remainder of these cells, as well as mononuclear bloodcells from leukemic rats, were cryopreserved at -70°C at each trans

plantation passage.RBC and WBC, PCV, and HB concentration were obtained on the

hematology analyzer. Serum chemistry was determined on a Centrifi-

chem centrifugal analyzer with automatic pipetor utilizing commercialreagent kits for glucose, serum enzymes, and total bilirubin. The methodsfor these assays have been reported previously (21).

Tissues from leukemic rats were sectioned and stained with hematox-

ylin and eosin and examined by light microscopy; spleen and liver smearswere prepared and examined by the same procedures.

Discrete MNC aliquots from spleen or peripheral blood were held at-70°C and were used after quickly thawing the cells at 30°C. Samples

(1.5 ml) were sonicated with a Kontes cell disrupter for 10 s with 50 n\0.1% Triton X added to ensure complete disruption of membranes.Samples were centrifuged for 5 min at 17,500 x g and the supernatantswere assayed for protein and enzyme activities. Protein concentrations

were measured by the Bradford-Coomassie blue dye-binding method(22) with a commercial reagent (Bio-Rad Laboratories, Richmond, CA)

and run against a standard curve of bovine serum albumin. The cellularsupernatant was assayed for enzyme activities that included G6PD (EC1.1.1.49) and 6-PGD (EC 1.1.1.43) from the HMS pathway, pyruvate

kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) from theEmbden-Meyerhof pathway, ICDH (EC 1.1.1.42) and MDH (EC 1.1.1.37)from the TCA cycle, and the membrane-bound enzyme ACHE (EC

3.1.1.7).All of the enzyme activities were measured under linear conditions so

that activities were proportional to amount of added supernatant andelapsed time. If possible enzymes were analyzed in a batch mode utilizinga centrifugal analyzer with automatic pipetor (Centrifichem; Baker Chemical Co.), but, when necessary, enzymes with lower activities weremeasured on an automated recording Model 250 spectrophotometer(Gilford Instruments Corp.). Within the limits of sensitivity enzyme activities attained with the two instruments were identical. All of the enzymeassays except ACHE used coenzyme-linked standard methods (23) thatmeasured the oxidation or reduction of NAD-NADP at 340 nm. ACHE

was measured at 405 nm using the method of Ellman ef al. (24). Enzymeactivities were related to protein concentration in the supernatant andwere expressed as specific activities (nmol substrate transformed permin per mg protein). Data were analyzed for statistical significance usingStudent's f test.

RESULTS

Spontaneous Leukemia

Body Weight, Spleen Weight, and Hematology. The bodyweight of rats developing spontaneous leukemia was less than10% below that of normal rats, but absolute spleen weight andspleen/body weight ratios were 4-fold greater in leukemic rats(Table 1). There was a 13-fold increase in WBC and 30-50%

decreases in RBC, PCV, and HB concentration in spontaneouslyleukemic rats compared to the 26-month-old controls.

Serum Chemistry. There were no clinical chemistry determinations made on animals with spontaneous leukemia. However,the typical clinical pathology profile of hypoglycemia, hyperbili-

rubinemia, and elevated serum enzyme responses in spontaneously leukemic F344 rats were reported previously (9), andthe responses detailed later in this report for transplanted leukemic cases were identical.

Pathology. The spontaneous MNC leukemia has been adequately described in the Fischer 344 rat (1, 3, 9) and presentedno differently in this study. Large indented acentric nuclei, decreased cytoplasmic/nuclear ratios, and numerous red-stained,cytoplasmic granules are typical features of these leukemic cells(Figs. 1 and 2). The spleen and the liver were infiltrated byleukemic MNC in all of the cases and the lung, lymph nodes,adrenals, kidneys, and bone marrow were usually infiltrated aswell.

Cellular Biochemistry. The activities of six enzymes in the

Tabte!Body weight, spleen weight, and hematology in F344 rats with spontaneousleukemia(mean ±SE, N = 6)

ControlsLeukemicChangeSignificanceBody(g)470

±19433 ±13

0.9XNS"Spleen

(g)1

.70 ±0.286.81 ±1.71

4x<0.01Spleen/body

x1000.36

±0.061.63 + 0.52

4.5X<0.05WBC

(1r^/mm3)9.9

±1.9133 ±48

13x<0.05RBC(10"/mm3)8.1

±0.94.0 ±0.9

0.5X<0.01PCV(%)44.0

±4.126.1 ±4.0

0.6x<0.01HB(g/di)16.5

±1.511.3 ±0.9

0.7X<0.01

a NS, not significant.

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ENZYME ACTIVITY IN LEUKEMIC MONONUCLEAR CELLS

Table 2Enzyme activity in mononuclear cells from F344 rats with spontaneous leukemia (mean ±SE, N = 6-8)

Specific enzyme activity (milliunits/mg protein)

HMS Embden-Meyerhof TCA

G6PD 6PGD PK" LDH ICDH MDH

Spleen mononuclearcellsNormalLeukemicChanges0Blood

mononuclearcellsNormalLeukemicChanges32

±10127±164.0X112±21119±

171.1X8±454

+66.8X47

±841±70.9x2±120

±310.0X5±25±20374±831629±1714.4x560

±1021295±4412.3X•f21

±410.5X4±111

±52.8X1181

±2102721±2162.3X696

±1181481±3692.1X

" PK, pyruvate kinase; LDH, láclate dehydrogenase.b Only one value above detection level.c All changes statistically significant, at P < 0.01.

MNC from spleen and peripheral blood are shown in Table 2.The specific activities of all enzymes were elevated from 2- to11-fold in leukemic spleen MNC compared to normal spleno-

cytes. By comparison there were variable and smaller increases(2-3-fold) in leukemic MNC from blood that were restricted toenzymes from the Embden-Meyerhof pathway and the tricarbox-

ylic acid cycle.

Transplanted Leukemia

Body Weight and Organ Weight. In the first serial transplantation of leukemic mononuclear spleen cells recipients were killedat 112 days posttransplantation. There was a 6% decrease inbody weight, a 15-fold increase in absolute spleen weight, anda 16-fold increase in spleen/body weight ratio (Table 3). In the

second serial transplantation recipients were killed 103 daysafter cell transfer. These rats weighed 34% less than controlsand there were 20- and 30-fold increases in absolute spleen

weight and spleen/body weight ratio, respectively.Hematology. Table 4 illustrates hematograms from the serial

transplantation experiments. The WBC in leukemic rats increased 8-27-fold while the RBC decreased more than 60%

below controls. The PCV and HB concentration exhibited comparable decreases.

Serum Chemistry. Table 5 depicts clinical pathology in theleukemic MNC recipients, characterized by hypoglycemia andmarked hyperbilirubinemia, with total bilirubin concentrations 6-16-fold greater than controls. There were 3-6-fold increases inalanine aminotransferase, 2-3-fold increases in alkaline phos-phatase and sorbitol dehydrogenase, and a 2-fold increase in

lactate dehydrogenase activity. There were no detectablechanges in the serum enzyme activities of a-hydroxybutyric

dehydrogenase and creatine kinase or in the concentrations ofserum urea nitrogen or serum protein (data not included).

Pathology. The transplanted cases of MNC leukemia werefundamentally identical to the spontaneous cases and it wasimpossible to delineate between them by histopathological examination. In all transplanted cases the spleen and liver wereextensively involved. In addition all lymph nodes, and particularlythe mesenteric lymph nodes, were moderately to severely infiltrated by leukemic MNC. All other tissues known to be affectedin spontaneous leukemia were similarly infiltrated by leukemicMNC in the transplantation cases.

Cellular Biochemistry. Enzyme activities in spleen MNC fromrats with transplanted leukemia were lower than controls (trans-

Table 3

Body and spleen weight in F344 rats with transplanted leukemia (mean ±SE,N= 12)

Serial transplantation 1"

ControlsLeukemicChangeBody

(g)332

+ 4311 + 11

0.9XSpleen

(g)0.63

±0.019.6 ±0.42

15xSpteen/body

x1000.19

±0.013.1 ±0.14

16x

Serial transplantation 2Controls 340 ±9 0.63 ±0.02 0.19 + 0.01Leukemic 225 ±8 12.6 ±0.49 5.6 ±0.19Change 0.7x 20x 30x

* Animais in transplantation 1 sacrificed at 112 days and in transplantation 2 at

103 days postinjection with leukemic mononuclear spleen cells. All changes exceptbody weight in transplantation 1 significant, at P < 0.01.

Table 4Hematology in F344 rats with transplanted leukemia (mean ±SE, N = 12)

Serial transplantation1sControlsLeukemicChangeSerial

transplantation2ControlsLeukemicChangeWBC

(Itf/mm3)6.4

±0.3852.9±3.68x7.9

±0.3213±2027xRBC

(KP/mm3)9.0

±0.073.2±0.540.4x9.3

±0.21.8±0.20.2XPCV

(%)44.7

±0.419.1±2.90.6x45.9

±0.914.4±1.10.3xHB(g/di)15.1

±0.16.3±0.90.4X15.7

±0.34.5±0.40.3x

8 Animals in transplantation 1 sacrificed at 112 days and in transplantation 2 at

103 days postinjection with leukemic mononuclear spleen cells. All changes significant, at P < 0.01.

plant cases 1 and 2 in Table 6) in direct contrast to the 2.3-10.5-fold increases in the same cellular enzymes from spontaneously leukemic rats (Table 2). These decreases were 50-70%

below controls in the serial transplantation experiments (P <0.01) with respect to glucose-6-phosphate dehydrogenase, 6-

phosphogluconic dehydrogenase, and malate dehydrogenase.The pattern of enzyme responses in blood MNC from rats with

transplanted leukemia were not as uniform. The decreases belowcontrols (P < 0.05) in glucose 6-phosphate and 6-phosphoglu-

conate dehydrogenases and in isocitrate dehydrogenase enzymeactivities were not reproduced in blood MNC in the second serialtransplantation, but the 2-3-fold significant increases in malate

dehydrogenase activity were present in blood MNC in bothtransplantation experiments (Table 6). The latter increase wasthe only cellular enzyme response in rats with induced leukemia

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ENZYME ACTIVITY IN LEUKEMIC MONONUCLEAR CELLS

Table 5Clinical chemistry in F344 rats with transplanted leukemia (mean ±SE, N = 12)

Señaltransplantationl"ControlsLeukemicChangeSerial

transplantation2ControlsLeukemicChangeGlucose

(mg/dl)198

±6141±50.7X190

±1298±90.5XBilirubin

(mg/dl)0.34

±0.072.70±0.466x0.26

±0.054.10±0.8116xUnits/literLOH"216

±29383±472x302

±45602±1222xALAT69

±6232±563x68

±9441±746xALK-P127

±3242±102x128

±5398±143XSDH28

±582±113x26

±955±82x

" LDH, lactate dehydrogenase; ALAT, alanine aminotransferase; ALK-P, alkaline phosphatase; SDH, sorbitol dehydrogenase.'' Animals in transplantation 1 sacrificed at 112 days and in transplantation 2 at 103 days postinjection with leukemicmononuclearspleencells. All changes significant,

at P < 0.05.

TaWe6Enzymatic activity in mononuclear cells from F344 rats with spontaneous or transplanted leukemia

Mean specific enzyme activity (milliunits/mg protein ±SE).

Spleen MNCSerial

transplantcases6G6PD

ControlLeukemic6PGDControl

LeukemicPK"Control

LeukemicLDH

ControlLeukemicICDH

ControlLeukemicMDH

ControlLeukemicSpontaneous

cases0

(N =6-8)32

±10127±16C8±4

54±6C2±120

±3°374

±831629±171C2e

21±4C1181

±2102721 ±216CExperiment

1(N=10)56

±628±2°18±4

14±15±1

4±0.7800

±33737 ±452

±0.52±0.62299

±981032±48CExperiment

2(N =10)56

±916±1C43

±414±1C5

±0.74±0.6963

±95845 ±771e

1±0.2491

5 ±4392138 ±294CSpontaneous

cases8(N =6-8)112±21119±1747

±841±75±2

5±2560

±1021295±4414±

111±5696

±1181481 ±369CBlood

MNCSerial

transplantcases"Experiment

1(N =10)51

±634±5C19±3

10±2C5±2

4±1765

±101854 ±524

±0.91±0.1C406

±901075 ±144CExperiment

2(N =10)24

±422±115±

117±13

±0.36 ±0.4C808

±96996 ±591

±0.31±0.4688

±942250 ±107*

a Rats were 104 weeks old.b Rats were 22-24 weeks old.0 Significantlydifferent from controls, at P < 0.05." PK, pyruvate kinase; LDH, lactate dehydrogenase.* One sample only.

similar to the cellular enzyme responses in spontaneously leukemic rats.

The specific enzyme activity of acetylcholinesterase in ratswith transplanted leukemia (Table 7) decreased 34% belowcontrols in spleen MNC (P < 0.05) and 85% below controls inblood MNC (P < 0.01). In the second serial transplantation MNCacetylcholinesterase activity was 87% below control values inspleen and 57% below control values in blood (P < 0.01).

Mononuclear Cell Protein Concentration

The total protein concentration in the enriched MNC preparations of blood were lower in leukemic than in control rats andthe 50% decrease was comparable in both the spontaneous andtransplanted cases of leukemia (data not presented). By contrastthe total protein concentration in enriched MNC preparations

from spleen was consistently greater in leukemic than in controlrats and the 2-fold increase was comparable in both the spon

taneous and transplanted cases of leukemia (data not presented).

DISCUSSION

The clinical and morphological presentations of spontaneousand transplanted leukemia in F344 rats were indistinguishable.Leukemic animals lost weight, were pale, and had palpablespleens that were as much as 20-fold larger than normal. WBC

were grossly elevated to up to 27 times normal and RBC andPCV and HB concentrations were markedly reduced.

The pattern of MNC enzyme responses from spontaneouslyleukemic rats describes an elevation of enzymes in glycolysisand the hexose monophosphate shunt pathway similar to that

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Table 7Acetylcholínesterase activity in mononuclear cells from F344 rats withtransplanted* leukemia (mean ±SE, N = 12)

Specific activity (milliunits/mg protein)

Spleen Blood

Sehal transplantation1NormalLeukemicChangeSignificanceSerial

transplantation2NormalLeukemicChangeSignificance85

±1156±30.7XP

<0.05107

±1214±10.1XP

< 0.0144±56.4

±0.70.1XP

<0.0146

±520±20.4xP

< 0.01"Animals were given s.c. injections of 2 x 10' spleen mononuclear cells from

F344 rats with spontaneous leukemia; animals from transplantations 1 and 2sacrificed at 112 and 103 days postinjection, respectively.

reported by Weber eíal. (25-28) for neoplasms of the liver,

kidney, and colon. Lengle ef a/. (29) examined mixed thymocytepreparations from spontaneously leukemic thymuses of AKRmice and also reported 2-3-fold elevations above controls in

specific activities of four glycolytic enzymes, including pyruvatekinase and láclate dehydrogenase. They found that energy production was generally proportional to metabolic activity of eachcell preparation, as indicated by thymidine uptake, and that thehigher rates of energy production in thymic lymphoma cells wereassociated with increases in glycolysis, fatty acid breakdown,the Krebs cycle, and the pentose phosphate pathway. However,Weber ef al. (30) has described differences in the biochemicalstrategy of neoplasms related to variations in their cellular origin.For example they described a decrease in pyruvate kinaseactivity in muscle sarcomas that was in direct contrast with theelevations of pyruvate kinase observed in the other tumors thathad been studied previously.

Here we present evidence in spleen mononuclear leukemiccells from rats with the transplanted tumor for reversal of theexpression of various enzyme activities. This includes selectedenzymes from the HMS, glycolysis, and the TCA cycle. Themitochondria! and cytoplasmic components of the TCA enzymesICDH and MDH were assayed together; therefore discriminationof these enzyme responses between their cellular compartmentswas not possible.

These were unexpected results and a second serial transplantation experiment was performed confirming the original resultsand also demonstrating that the extent of the decrease in severalenzyme activities (Table 6) was associated with the severity ofthe leukemia, as defined by morphological and clinical determinations (cf. Tables 3 to 5). As in many in vivo biological modelsthere was variance between serial transplantations 1 and 2exemplified by the HMS enzymes and MDH activities in controlpreparations (Table 6), as well as the expected variations inenzyme activities associated with age (31-33) which necessitated the use of age-matched controls for each comparison.

There is no satisfactory explanation for the difference betweenthe two transplant cases in control 6-PGD and MDH activity in

spleen MNC and control G6PD enzyme activity in blood MNC(Table 6). Although methodological error cannot be ruled out theconsistency of the HMS and MDH responses in the two transplant cases argues for their biological validity.

In subsequent transplantation experiments to be reported indetail in a separate paper, morphological and biochemical responses were measured at monthly intervals. Decreases inglucose-metabolizing enzymes from spleen MNC were not lim

ited to the terminal phases of the disease, but in some enzymesthe decreases in activity preceded other diagnostic indices ofleukemia, when active tumor proliferation was presumably underway.

There appear to be only a limited number of hypotheses thatcould explain this dramatic change in expression of enzymaticactivity from the leukemic spleen mononuclear cells. The originalleukemic MNC from spontaneously leukemic rats may havebecome gradually altered after serial transplantation throughsyngeneic hosts, culminating in the enzymatic expression described here.

It is also possible that the preponderant type of MNC isolatedfrom the spleens of rats with induced leukemia was functionallyand therefore biochemically different than the preponderant celltype isolated from spleen of rats with spontaneous leukemia.Szelenyi ef al. (34) found that acetylcholinesterase activity ofnormal human peripheral blood lymphocytes was entirely confined to the lower density T-lymphocytes and was deficient in B-

lymphocytes. Later the same investigators (35) separated theperipheral blood lymphocytes from normal donors and frompatients with chronic lymphocytic leukemia and found a 60-95%

decrease in acetylcholinesterase activity in leukemic cells correlated with an increase in cell counts. Functional membranemarker analyses by erythrocyte-forming rosette technique indi

cated that the decrease in acetylcholinesterase activity was dueto decreased T-cell content in the mixed lymphocyte population

and was supported by the finding that purification and concentration of the T-cells led to restoration of normal acetylcholines

terase activity.We have demonstrated 34-87% decreases in acetylcholines

terase activity in the spleen MNC from rats with transplantedleukemia, strongly suggesting that there was a specific decreasein the percentage of T-cells in the mixed mononuclear spleen cell

culture. Stromberg ef al. (11) previously used identical procedures to isolate mixed mononuclear spleen cells from the samesource of animals as studied here. They reported a 69% decrease in T-cells in 2-year-old Fischer 344 rats with spontaneous

leukemia. We were not previously assaying acetylcholinesteraseactivity and thus cannot directly compare MNC acetylcholinesterase activity between rats with spontaneous and transplantedleukemia.

It is more difficult to adequately explain the decrease in HMSand glycolytic enzyme activities in leukemic cells by a change incellular composition. Oridinarily neoplastic cells display an integrated pattern of biochemical responses characterized by increases in these glucose metabolizing enzymes for energy production, NADPH production for biosynthetic processes, andprovision of ribose 5-phosphate for RNA and DNA syntheses(25-28, 36-38).

There are, however, data to indicate that certain biochemicalresponses vary inversely with the rate of growth of tumor transplants. Dentón ef al. (38) examined human colon tumors andtumor xenografts in nude mice and reported that HMS andglycolytic enzyme activities were much lower in slowly growing,well differentiated colon tumor xenografts than in rapidly growing, poorly differentiated ones, with primary human colon carci-

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ENZYME ACTIVITY IN LEUKEMIC MONONUCLEAR CELLS

nomas showing intermediate activities. For example there were400% greater increases in pyruvate kinase and 500% greaterincreases in G6PD activities in the rapidly growing line of humancolon carcinoma xenografts carried in nude mice than in theslowly growing tumor cell line, compared to normal colon mucosaenzyme activities. In another comparison there were 150% increases in HMS enzyme responses observed in spontaneoushuman colon carcinomas, whereas in chemically induced mousecolon carcinomas there were no changes in G6PD and transal-dolase activities and a 40% decrease in the 6-PGD enzyme level

(39) as compared to normal colon mucosa enzyme activities.An analogous situation may exist in the rat leukemia transplant

model used here since the growth rate of the splenic leukemiccells should be unchecked and faster in immunologically deficientolder rats (40) with spontaneous leukemia compared to thegrowth rate of the splenic leukemic cells in young recipient hostswith vigorous immune surveillance mechanisms.

We conclude that enhanced mononuclear cell enzymatic activity is not essential for the phenotypic expression of Fischer ratleukemia since the enzymatic responses between the spontaneous and transplanted leukemia cases were diametrically opposed. Elevated glycolysis and HMS enzyme activities mayreflect a neoplastic transformation necessary for de novo nucleicacid syntheses in spontaneous cases whereas the neoplasticMNC from transplanted cases may depend more on salvagepathways for their nucleotide requirements. Regardless of thereasons for these metabolic differences, these transplantable ratMNC provide abundant tumorigenic material with novel biochemical expressions that may be useful in the study of chemothera-

peutic intervention.

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ENZYME ACTIVITY IN LEUKEMIC MONONUCLEAR CELLS

o

•O0OnOFig. 1. Morphology of F344 rat mononuclear leukemic cell. Wright-Giemsa-stained peripheral blood smear. The acentric nucleus and numerous cytoplasmic granules

of the large leukemic cell are evident, x 2,000.

I

Tit

Fig. 2. Ultrastructural morphology of a F344 rat mononuclear leukemic cell. The large indented acentric nucleus, golgi apparatus, mitochondria, and electron-densegranules (red granules of Wright-Giemsa-stained cells) are evident, x 13,600.

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1985;45:4301-4307. Cancer Res   Michael P. Dieter, Robert R. Maronpot and John E. French  Spontaneous or Transplanted LeukemiaMononuclear Cells from Fischer 344 Rats with Either Comparison of the Morphology and Enzyme Activity of

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