Lactoferrin binding by leukemia cell lines

1987 70: 264-270

Y Yamada, T Amagasaki, DW Jacobsen and R Green Lactoferrin binding by leukemia cell lines

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264 Blood. Vol 70. No 1 (July). 1987: pp 264-270

Lactoferrin Binding by Leukemia Cell Lines

By Yasuaki Yamada, Tatsuhiko Amagasaki, Donald W. Jacobsen, and Ralph Green

Monocytes and macrophages have receptors for the iron-

binding protein lactoferrin. Lactoferrin acts as a potentinhibitor of granulocyte-macrophage colony stimulating

factor production when it binds to these cells. Using a

rosette assay and immunofluorescence. we have shown

that cultured leukemia cells, including the human erythroid

leukemia cell line K562, also have lactoferrin binding sites.

The number of binding sites on K562 cells was estimated

using soluble �Fe-lactoferrin. Inhibition studies demon-

strate that lactoferrin binding sites are distinct and unre-

Iated to receptors for transferrin or the Fc portion of IgG.

which are present on K562 cells. However, electrostatic

L ACTOFERRIN,* a cationic glycoprotein that binds two

atoms of ferric iron per molecule,’ is present in high

concentration in milk ( I mg/mL),23 other body secretions,45

and in secondary granules of myeloid cells (3 pg/ I 06 neutro-

phils),�8 but is scarcely detectable in the serum (less than I

pg/mL).9 It has many biochemical similarities to trans-

ferrin,’#{176}�’2which is present in plasma (2 to 4 mg/mL)’3 and

other body fluids. A major biological role for transferrin as

an iron transport and delivery protein has been defined.

Immature erythroid cells, activated lymphocytes, and neo-

plastic cells, which require iron for hemoglobin synthesis or

replication, take up iron efficiently through the specific

receptor for transferrin.’4’8 On the other hand, the biological

significance of lactoferrin remains obscure. One of its roles is

in the regulation of normal myebopoiesis. Lactoferrin binding

to the surface of monocytes through a putative specific

receptor results in suppression of the release of granulocyte-

macrophage colony stimulating factor.’9 Another suggested

role is as a bacteriostatic or bactericidal agent.4�#{176} Apo-

lactoferrin depresses the multiplication of iron-dependent

microbial strains. Little is known about the relation between

lactoferrin and neoplastic cells. By applying a rosette-

5Lactoferrin and transferrin refer to the diferric forms, unless

otherwise stated.

From the Section of Investigative Hematology. Department of

Laboratory Hematology. and Department of Brain and Vascular

Research, Cleveland Clinic Foundation.Submitted May 12. /986; accepted March 9. 1987.

Supported in part by a research grant from the Cleveland Clinic

Foundation (RPC No. I 749). This is publication no. 03-87from the

Department of Laboratory Hematology. Cleveland Clinic Founda-

tion.

Presented in Part at the American Society of Hematology. NewOrleans. December 1985. YY and TA are special fellows in the

Department of Laboratory Hematology at The Cleveland Clinic

Foundation.

Address reprint requests to Ralph Green. MD. Department of

Laboratory Hematology. Cleveland Clinic Foundation. 9500 Euclid

Aye, Cleveland. OH 44106.The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. §1734 solely to

indicate this fact.

C I 987 by Grune & Stratton. Inc.

0006-4971/87/7001-0041$3.00/0

forces may be important for lactoferrin binding. since other

polycationic proteins (eg, protamine) inhibit lactoferrin

binding. Prior treatment of K562 cells with trypsin nearly

abolishes lactoferrin binding. However, these cells recover

their ability to bind lactoferrin when trypsin is removed.

Unlike transferrin receptors. the expression of lactoferrin

binding sites is not regulated by cellular iron status. Cyto-

sine arabinoside arrests the proliferation of K562 cells and

simultaneously leads to a reduction in lactoferrin surface

binding, suggesting that lactoferrin binding may be depen-

dent on cell proliferation.

S 1987 by Grune & Stratton, Inc.

forming assay that was developed in our laboratory,2’ we

have found that neoplastic cells also express lactoferrin

binding sites similar to those reported for monocytes or

macrophages. We report here the nature of lactoferrin

binding to cultured leukemia cells.

MATERIALS AND METHODS

Cell preparation. K562 cells, a human erythroid leukemia cell

line, were used to analyze the kinetics of lactoferrin binding. Cells

were grown in RPMI 1640 medium supplemented with 5% heat-

inactivated fetal bovine serum, 100 U/mI penicillin, 100 �tg/mL

streptomycin, and 0.25 pg/mI fungizone (Irvine Scientific, Santa

Ana, CA). MOLT-4 cells and CCRF-CEM cells (human T lympho-blastic leukemia cell lines), U937 cells (human histiocytic lym-

phoma cell line), HL-60 cells (human acute promyelocytic leukemia

cell line), CCF-YI cells (a human B cell line established in our

laboratory from a patient with Ph’ negative juvenile chronic myelo-

cytic leukemia), Raji cells (human Burkitt lymphoma cell line), and

L1210 cells (mouse B lymphocytic leukemia cell line) were also used

and were grown under similar conditions but with 10% fetal bovine

serum medium. All experiments were conducted using cells atgrowth densities of 3 to 5 x l0� cells/mI (logarithmic phase). Cellswere washed once with RPMI 1640 + 0.5% bovine serum albumin

(fraction V; Sigma Chemical Co. St Louis), resuspended in the same

medium at a cell density of I x 106/mL, and used for rosette

formation.

Mononuclear cells were separated from heparinized normal blood

by a density-gradient centrifugation technique using Ficoll-

Hypaque (FH; Pharmacia, Piscataway, NJ). Cells were then passed

through a nylon-wool column22 to enrich T lymphocytes. The resul-

tant T lymphocytes were activated by stimulation with concanavalin

A (Con A; Sigma) for 96 hours and evaluated for lactoferrin

binding.

Lactoferrin and transferrin. Purified human apo-lactoferrin

from colostrum (Calbiochem-Behringer, La Jolla, CA) and human

apo-transferrin (Sigma) were saturated with iron according to the

method described by Bates and Schlabach.2”23 Iron saturation was

confirmed spectrophotometrically by an increase in absorbance at

460 nm (A460/A280 = 0.045 to 0.048 for fully saturated trans-

ferrin).24 Monoclonal antibodies (MoAbs) against the human trans-

ferrin receptor (42/6 and B3/25)25 were generously supplied by Dr

Ian Trowbridge (Salk Institute, La Jolla, CA). OKT9 antihuman

transferrin receptor antibody was purchased from Ortho Diagnostic

Systems (Raritan, NJ).Lactoferrin coating of bovine red blood cells and rosetting

procedure. Lactoferrin was coated on bovine red blood cells (B-

RBC) using CrC13 according to the method described originally for

the protein A hemolytic plaque assay26 and modified subsequently by




co

80

60

40

20

z0

4

0

I-

an0

LACTOFERRIN BINDING BY LEUKEMIA CELLS 265

this laboratory for transferrin-coated B-RBC.2’ Successful coupling

of lactoferrin on B-RBC was confirmed with fluorescein isothiocya-

nate (FITC)-labeled antilactoferrin antibody (Cappel Worthington

Biochemicals, Malvern, PA). Apo-lactoferrin, transferrin, or albu-

mm-coated B-RBC were prepared similarly, using the same concen-

trations of protein. Cultured neoplastic cells or T lymphocytes

(1 x 106/mL in 0.25 ml) were mixed with 0.1 mL of lactoferrin,

transferrin, or albumin-coated B-RBC (0.5% packed cell volume) in

U-bottom 12 x 75-mm tubes (Falcon #2054, Oxnard, CA). The

mixtures were centrifuged at 30 x g for three minutes and then

incubated at 37#{176}Cfor 45 minutes (except for the time-course study).

The pellets were carefully resuspended, and rosette formation was

estimated visually using a hemocytometer. More than 200 cells were

counted, and those cells binding more than three B-RBC on their

surface were considered positive for rosette formation. All experi-

ments were performed in triplicate.

Immunofluorescence assay. Lactoferrin binding was also evalu-

ated by an immunofluorescence assay using soluble lactoferrin.

After washing three times with PBS, 1 x 106 pelleted cells were

incubated with 100 zL of lactoferrin solution (50 pg/mI in PBS) at

4#{176}Cfor 60 minutes. The treated cells were washed three times with

PBS, and then incubated with 50 �L of a 1/20 dilution of FITC-

conjugated rabbit antihuman lactoferrin antibody (F(ab’)2 fraction,

Cappel Labs). After washing three times with PBS, the labeled cells

were examined using a fluorescence-activated cell sorter.

59Fe-lactoferrin binding assay. Apo-lactoferrin was saturated

with 59Fe (59FeC13, New England Nuclear, Boston) by the methodused for preparing nonradiolabeled diferric-lactoferrin.2”23 K562

cells (1 x 106) suspended in Hank’s balanced salt solution (HBSS)

were incubated at 4#{176}Cfor 60 minutes with various concentrations of

59Fe-lactoferrin. After incubation cells were washed three times with

HBSS, and radioactivity was measured in a gamma spectrometer.

Benzidine staining. K562 cells were cultured in the presence of3.6 x iO� mol/L cytosine arabinoside (Sigma) to induce erythroid

differentiation27 for assessment of differentiation-associated change

in lactoferrin binding ability. Hemoglobin synthesis in these cells

was estimated by benzidine staining.27 The proportion of cells that

synthesized hemoglobin (stained blue) was assessed by light micros-

copy.

Other chemicals. Deferoxamine mesylate (Desferal, CIBA

Pharmaceutical Co, Summit, NJ) or ferric ammonium citrate(Sigma) were dissolved in distilled water and added to culture media

at final concentrations of 1 x l0� mol/L and 10 pg/mI respec-

tively to determine the effect of iron content of the culture solution

on the expression of transferrin receptor and lactoferrin binding

sites. Cells treated with 0.25% trypsin solution (Irvine Scientific) for

one minute at 37#{176}C,cells treated with 2 mg/mL of deoxyribonu-

clease I or ribonuclease A (DNase or RNase, Sigma) for one hour at

room temperature, and cells treated with 0.1 U/mI of neuramini-

dase (Calbiochem-Boehringer) for 20 minutes at 37#{176}Cwere used to

assess the susceptibility of lactoferrin binding sites to digestion by

these enzymes. Human IgG (Sigma) was incubated for 30 minutes

at 60#{176}Cto prepare heat-aggregated IgG for use in experiments to

study inhibition of rosette formation. Human milk lysozyme andsalmon protamine (Sigma) were also used to study the specificity of

lactoferrin binding.

RESULTS

Time-course study of rosette formation. Mixtures of

K562 cells and lactoferrin-, apo-lactoferrin- or albumin-

coated B-RBC were incubated for up to 90 minutes at 37#{176}C.

Rosette formation with lactoferrin-coated B-RBC increased

rapidly and reached a plateau after 45 minutes incubation

(Fig 1). Rosette formation was lower with apo-lactoferrin-

0 l5 30 45 60 90

INCUBATION TIME (mm)

Fig i . Time course of rosette formation. K562 cells were

incubated with Iactoferrin-(---*). apo-lactoferrin (O-O). or

aIbumin-I�---i�) coated B-RBC. Each point is the mean of three

assays; vertical bars indicate standard deviations.

coated B-RBC. Control albumin-coated B-RBCs did not

form rosettes throughout the incubation time.

Determination of lactoferrin binding sites by Scatchard

analysis. 59Fe-radiolabeled lactoferrin showed saturable

binding to K562 cells (Fig 2). A Scatchard plot analysis of

these data showed maximum binding of 4.9 x 1O�’ molecules

per cell; the dissociation constant was 7.4 x I0_6 mol/L. A

competition assay using a tenfold excess of unlabeled lacto-

ferrin was also attempted but was unsuccessful due to the

technical problem caused by cell aggregation at lactoferrin

concentrations greater than 2.5 mg/mL.

Specificity ofrosette-forming assayfor detection of lact 0-

ferrin binding sites. To test the specificity of the rosette-

forming assay, K562 cells were preincubated with either

soluble lactoferrin, transferrin, or MoAbs against transferrin

receptor for 30 minutes at room temperature before addition

of lactoferrin-coated B-RBC. Rosette formation was inhib-

ited by prior treatment with soluble lactoferrin in a dose-

dependent manner (Fig 3). Soluble apo-lactoferrin also

inhibited rosette formation almost to the same extent as did

soluble lactoferrin (Table 1 ). However, neither transferrin

nor three types of MoAbs against transferrin receptor inhib-

ited rosette formation with lactoferrin-coated B-RBC (Table

1); all inhibited rosette formation with transferrin-coated

B-RBC at the same concentrations).2’ Heat-aggregated IgG

at a concentration of 64 �g/mL, which is sufficient to fully

saturate all Fc receptors on K562 cells,28 did not inhibit

rosette formation (Table 1 ). Since lactoferrin is a cationic

protein (p1 = 8.7),29 nonspecific electrostatic binding to the

cell surface membrane was considered. To investigate this

possibility, we used two kinds of cationic proteins in competi-

tion studies: human milk lysozyme and salmon protamine.

Lysozyme caused no inhibition, but protamine substantially

inhibited rosette formation. Furthermore, neuraminidase, an

enzyme that cleaves membrane surface sialic acid resulting

in decreased cell surface negative charge, also partially

inhibited rosette formation.

Trypsin sensitivity of lactoferrin binding sites. K562

cells were examined for rosette formation with lactoferrin-

coated B-RBC before and after digestion with trypsin. In

three experiments, rosette formation decreased markedly




MOLECULES/CELL z I0�

0

l00

Positivity: 4.7%

0 4 8 16 32 64 128

266 YAMADA ET AL

C

C

0ILF CONCENTRATION (pg/mI)

Fig 2. Dose-response binding of �‘Fe-radiolabeIed lactoferrinto K562 cells. The mean of three experiments is shown. AScatchard plot analysis (inset). indicates that 4.9 x i0� moleculesare bound to each K562 cell at saturation. The dissociationconstant (K,,) derived from the concentration of free Iigand at halfsaturation of the binding sites is 7.4 x i0’ mol/L.

following trypsin digestion (from 82.9% ± 0.6% to

1 1 .8% ± 3.9%) but recovered almost to pretreatment levels

following culture of the digested cells for five hours at 37#{176}C

in culture medium without trypsin (74.6% ± 2.0%). Trypsin

sensitivity of lactoferrin binding by MOLT-4 cells was also

shown using the immunofluorescence assay (Fig 4). Binding

z0

Iz

IF (pg/mi)

Fig 3. Inhibition of rosette formation by soluble lactoferrin.K562 cells were pretreated with soluble lactoferrin before rosetteformation. Each point is the mean of three assays; vertical barsindicate standard deviations.

Table 1 . Specificity of Rosette- Forming Assay

Treatment of K562 Cells

Inhibition ofRosette Formation 1%)

Mean ± SD (n - 3)

Lactoferrin (64 �g/mL)

Apo-lactoferrin (64 �zg/mL)

Transferrin (64 �zg/mL)

Apo-transferrin (64 �g/mL)

MoAbs against transferrin receptor

42/6 (1 zg/mL)

B3/25 (1 �.tg/mL)

OKT9 (1 �tg/mL)

Heat-ag�egated human lgG (64 �g/mL)

Cationic proteins

Human milk Iysozyme (64 jcg/mL)

Salmon protamine (64 �zg/mL)

Neuraminidase (0.1 U/mL)

74.0 ± 1.8

77.9 ± 3.6

- 1 .9 ± 6.8

- 3. 1 ± 9.0

9. 1 ± 3.9

3.4 ± 5.9

5.4 ± 8.7

- 1 . 5 ± 3.3

- 1 .9 ± 1.0

44.6 ± 8.9

31.5 ± 4.0

9(562 cells were pretreated with these agents before rosette forma-

tion for 30 minutes at room temperature except for neuraminidase

treatment, which was performed at 37#{176}C.

A

B

LU

DCz...aLUU

D

Positiv,ty:673%

Positivity 47.7%

RELATIVE FLUORESCENCE INTENSITY

Fig 4. Trypsin sensitivity of lactoferrin binding sites. LabeledMOLT-4 cells (-) were processed on a fluorescence-activated cell sorter and compared with control cells (----) treatedwith FITC-conjugated antibody only. Nontreated cells (A). trypsin-digested cells (B). DNase-treated cells (C). and RNase-treated cells(D) were analyzed for lactoferrin binding.




z0I-

4

0

U.

LU

LUan0

lOO

8O�

60�

40

20

0

A0

a-z0Ua-anz 120

4

C, 110�l0O

0 24 48 72

CULTURE TIME (HRS)

96

Fig 5. Influence of iron content ofculture media on the expression oftransferrin receptor (A) and lactoferrinbinding sites (B). K562 cells were cul-tured with deferoxamine (A-t�) or

ferric ammonium citrate (-) for

varying times up to 96 hours and com-

pared with control cells (O-O) for

their transferrin and lactoferrin binding

ability. Each point is the mean of threeassays; vertical bars indicate standard

deviations.


was almost completely abrogated by trypsin digestion (Fig

4B). DNase did not remove but rather enhanced lactoferrin

binding (Fig 4C). Interestingly, RNase caused slight inhibi-

tion of lactoferrin binding (Fig 4D).

Influence of iron availability on the expression of lacto-

ferrin binding. K562 cells were cultured up to 96 hours in

the presence of the iron chelator deferoxamine ( I x I O�

mol/L) or 10 pg/mL ferric ammonium citrate. At various

times lactoferrin and transferrin binding by these cells was

assessed with the rosette-forming assay. Proliferation status

of K562 cells was not affected by either agent at these

concentrations, and the doubling times of these cells were

similar to untreated control cells. Rosette formation with

transferrin-coated B-RBC increased two to three times fol-

lowing deferoxamine treatment but decreased to less than

one-half following addition of ferric ammonium citrate (Fig

5A). However, rosette formation with lactoferrin-coated

B-RBC was not influenced by these agents (Fig 5B).

Influence of cell proliferation status on expression of

lactoferrin binding. K562 cells were cultured with cytosine

arabinoside (C-ara) and examined for rosette formation with

lactoferrin-coated B-RBC at 24-hour intervals. The culture

solution was replaced by fresh media containing C-ara every

other day. Cell proliferation ceased after 24 hours in C-ara

medium, but the viability remained greater than 90%. The

proportion of benzidine-positive cells increased daily and

reached 77.7% at 144 hours, indicating maturation to hemo-

globin-producing cells. There was a corresponding inverse

decline in rosette formation rate (Fig 6). This decrease in

lactoferrin binding could be curtailed by removing C-ara

from the culture media.

Lactoferrin binding sites on various cells. Expression of

lactoferrin binding sites was examined on other neoplastic

cell lines as well as on normal blood lymphocytes before and

after activation with Con A (Table 2). The immunofluores-

cence assay was always more sensitive than the rosette-

forming assay. Four (K562, MOLT-4, L I 2 10, and CCF-Y I)

of eight cell lines examined showed abundant lactoferrin

binding sites. The rosette-forming assay failed to detect

lactoferrin binding sites on activated T lymphocytes, but the

immunofluorescence assay showed that approximately one

half of activated T lymphocytes display lactoferrin binding.

0 24 48 72 144

CULTURE TIME (HRS)

Fig 6. Effects of C-ara on the expression of lactoferrin bindingsites. Rosette formation was performed on K562 cells cultured in

the presence of C-ara (i-). For some cells C-ara was removedat 48 hours. and culture was continued without C-ara (----).

Each point is the mean of three assays; vertical bars indicatestandard deviations.

DISCUSSION

Although more than 25 years have passed since the

iron-binding protein lactoferrin was found in milk,3 its bio-

logical functions are still poorly understood. Receptor-like

binding of lactoferrin to human peripheral monocytes has

been described.30’3’ Furthermore, it has been reported that

lactoferrin has an inhibitory effect on the release of granulo-

cyte-macrophage colony stimulating factor (GM-CSF) from

monocytes and macrophages.’9 Few studies have been done

on the relation between lactoferrin and neoplastic cells. Two

recent articles have shown that lactoferrin is an essential

nutrient for the growth of neoplastic cell lines in serum-free

medium. Growth stimulation of Bri 7 cells, a human B

lymphocytic leukemia cell line, was greater with lactoferrin

than with transferrin.32 Similarly, growth of HT29, a human

colon adenocarcinoma cell line, was also stimulated by

350

� 300

0

I-. 250z0U 200

� ISO4C,4 100anLU

D..a4

>0 I I I I

0 24 48 �T2 96

CULTURE TIME (HRS)




268 YAMADA ET AL

Table 2. Lactoferrin Binding by Neoplastic Cell Lines

and Normal T Lymphocytes

Cells

Rosette Formation %

Mean ± SD In - 3)Immunofluorescence

Assay (%)

K562 90.0 ± 3.8 97.7

MOLT-4 21.3 ± 5.2 67.3

L1210 28.9 ± 3.3 ND

CCF-Y1 13.0 ± 1.4 91.8

CCRF-CEM 2.9 ± 0.5 ND

HL-60 0.7 ± 0.7 9.7

U937 0 14.0

Raji 0 4.9

Resting T lymphocytes 0 2.8

Mitogen-stimulated

T lymphocytes 0 55.4

lactoferrin.33 The effect was more pronounced in the pres-

ence of iron. These reports led us to study lactoferrin binding

sites on neoplastic cells.

In the present studies we used a rosette-forming assay to

detect lactoferrin binding. Rosette formation rate was

greater with lactoferrin-coated than with apo-lactoferrin-

coated B-RBC, suggesting a higher binding affinity of

iron-saturated lactoferrin. Although differences in the quan-

tity of protein coating cannot completely be excluded, it is

unlikely that this is the cause of the discrepancy, since the

same concentration of lactoferrin or apo-lactoferrin was used

for coating. By analogy, iron-saturated transferrin has

higher binding affinity to its receptor than apo-trans-

ferrin.3�’36 The transferrin molecule becomes more compact

and spherical after it has bound iron,34’”’35 and this may be

advantageous for receptor binding. On the other hand,

rosette formation with lactoferrin-coated B-RBC was inhib-

ited by soluble lactoferrin irrespective of its iron content,

suggesting that low affinity binding is sufficient to suppress

rosette formation. However, lactoferrin-coated B-RBC

rosette formation was not inhibited by transferrin, MoAbs

against the transferrin receptor, or heat-aggregated human

IgG. This indicates that lactoferrin occupies binding sites

different from those for either the transferrin receptor or the

Fc receptor for IgG, which are both present on K562 cells.28

The number of lactoferrin binding sites on K562 cells was

estimated by Scatchard analysis using 59Fe-lactoferrin. Bind-

ing of 59Fe-lactoferrin was saturable, and the number of

binding sites per cell (4.9 x lO�), though high, agrees with

figures reported for adherent mononuclear cells (3.3 x IO�

per cell) and nonrosetting lymphocytes (2.5 x lO� per cell)

prepared from normal human blood.30 Because of problems

with cell aggregation that we encountered at high lactoferrin

concentrations, it was not possible to eliminate nonspecific

binding, so that our figure is likely an overestimate of the

number of binding sites.

Since lactoferrin is a cationic protein (p1 = 8.7),29 we

considered that binding to the negatively charged cell surface

might be of a nonspecific electrostatic nature. We therefore

pretreated K562 cells with two other cationic proteins to

block surface-negative charges. The more cationic lysozyme

(p1 = 10.5 to I l.O)�� did not inhibit lactoferrin-B-RBC

rosette formation. However, protamine caused considerable

inhibition, although the inhibitory activity was less than that

of soluble lactoferrin. Sialic acid, one of the major sources of

surface negative charge, was removed by neuraminidase

treatment’#{176} of K562 cells and resulted in a decrease of rosette

formation. Taken together these findings suggest that elec-

trostatic factors are intimately involved in lactoferrin bind-

ing to cells. Similar electrostatic factors have been impli-

cated in the binding of lactoferrin to macrophages and liver

reticuloendothelial cells. Lactoferrin binds to alveolar

macrophages in competition with the other cationic neutro-

phil granule glycoproteins, elastase and cathepsin G.4’

Removal of sialic acid from lactoferrin increases its binding

affinity to mouse peritoneal macrophages.42 Moreover, based

on a study of lactoferrin uptake by the liver, the possible

existence of common binding sites for certain cationic pro-

teins on liver reticuloendothelial cells has been postulated.43

The lactoferrin binding sites we have demonstrated on

neoplastic cell lines are similar in some respects to the

lactoferrin binding sites described on macrophages and liver

reticuloendothelial cells. However, there are also differences,

such as the sensitivity to trypsin digestion. We found marked

sensitivity to trypsin digestion of lactoferrin binding by

neoplastic cells. In contrast, lactoferrin binding by human

peripheral blood monocytes is somewhat resistant to trypsin

digestion.” A further difference is the sensitivity to DNase

and RNase. Lactoferrin binding sites on neoplastic cells were

resistant to DNase but were partially sensitive to RNase,

suggesting that lactoferrin binding sites may be RNase-

susceptible acidic groups present on the cell surface.�#{176} In

contrast to neoplastic cells, lactoferrin binding sites on

normal human monocytes are sensitive to DNase treat-

ment.”

Transferrin receptor expression of cultured cells is regu-

lated by the iron concentration of the culture medium. An

iron chelator, deferoxamine, increases the number of trans-

ferrin receptors, whereas excess iron decreases transferrin

receptor expression.24’45’�’47”'8 We confirmed this observation

by finding a good inverse correlation between iron supply and

transferrin-receptor expression using our rosette-forming

assay. Lactoferrin shows close structural homology to trans-

ferrin at the iron binding site, but unlike transferrin, lactofer-

rin binding to cells was not influenced by cellular iron status.

This finding supports the view that K562 cells cannot take up

iron from lactoferrin even though cells can bind and internal-

ize lactoferrin (unpublished observation).

Four of eight leukemic cell lines as well as activated T

lymphocytes showed lactoferrin binding, but resting T lym-

phocytes did not. Similarly, receptor-like binding of lactofer-

rin to fresh leukemia cells, but not to normal lymphocytes or

platelets has been reported.49 Furthermore, we were able to

demonstrate diminished expression of lactoferrin binding by

C-ara treatment of K562 cells. These results seem to indicate

that there is a close relationship between lactoferrin binding

and cell proliferative status. Although monocytes and acti-

vated lymphocytes also have lactoferrin binding sites, analy-

sis in conjunction with other cell markers may prove useful

for the identification of leukemic cells in blood or neoplastic

cells in body fluids suspected to contain metastatic tumor.





REFERENCES

I. Masson PL, Heremans JF: Metal-combining properties of

human lactoferrin (red milk protein) I . The involvement of bicar-

bonate in the reaction. Eur J Biochem 6:579, 1968

2. Masson PL, Heremans JF: Lactoferrin in milk from different

species. Comp Biochem Physiol 39: 1 19, 1971

3. Groves ML: The isolation of a red protein from milk. J Am

Chem Soc 82:3345, 19604. Masson PL, Heremans JF, Prignot JJ, Wauters G: Immuno-

histochemical localization and bacteriostatic properties of an iron-

binding protein from bronchial mucus. Thorax 21:538, 1966

5. Masson PL, Heremans JF: Studies on lactoferrin, the iron-

binding protein of secretions. Protides Biol Fluids Proc Colloq

14:115, 1966

6. Masson PL, Heremans JF, Schonne E: Lactoferrin, an iron-

binding protein in neutrophilic leukocytes. J Exp Med 130:643,

1969

7. Baggiolini M, Duve C, Masson PL, Heremans JF: Association

of lactoferrin with specific granules in rabbit heterophil leukocytes. J

Exp Med 131:559, 1970

8. Leffell MS, Spitznagel JK: Fate of human lactoferrin and

myeloperoxidase in phagocytizing human neutrophils: Effects of

immunoglobulin G subclasses and immune complexes coated on

latex beads. Infect Immun 12:813, 1975

9. Broxmeyer HE, Gentile P, Bognacki J, Ralph P: Lactoferrin,

transferrin and acidic isoferritins: Regulatory molecules with poten-

tial therapeutic value in leukemia. Blood Cells 9:83, 1983

10. Aisen P, Leibman A: Lactoferrin and transferrin: A compara-

tive study. Biochim Biophys Acta 257:314, 1972

I I . Metz-Boutigue MH, Jolles J, Mazurier J, Spik G, Montreuil

J, Jolles P: Structural studies concerning human lactoferrin: Its

relatedness with human serum transferrin and evidence for internal

homology. Biochimie 60:38, 1978

12. Metz-Boutigue MH, Jolles J, Mazurier J, Schoentgen F,

Legrand D, Spik G, Montreuil J, Jolles P: Human lactoferrin:

Amino acid sequence and structural comparisons with other trans-

ferrins. Eur J Biochem 145:659, 1984

13. Olesen H, Terp B: Transferrin determination by Laurell

electrophoresis in antibody containing agarose gel. Scand J Clin Lab

Invest2l:14, 1968

14. Jandl JH, Katz JH: The plasma-to-cell cycle oftransferrin. J

Clin Invest 42:314, 1963

I 5. Larrick JW, Cresswell P: Transferrin receptors on human B

and T lymphoblastoid cell lines. Biochim Biophys Acta 583:483,

I 979

16. Galbraith GMP, Galbraith RM, Faulk WP: Transferrin

binding by human lymphoblastoid cell lines and other transformed

cells. Cell Immunol 49:215, 1980

17. Galbraith RM, Werner P. Arnaud P, Galbraith GMP: Trans-

ferrin binding to peripheral blood lymphocytes activated by phyto-

hemagglutinin involves a specific receptor. J Clin Invest 66:1135,

1980

18. Van Renswoude J, Bridges KR, Harford JB, Klausner RD:

Receptor-mediated endocytosis of transferrin and the uptake of Fe

in K562 cells: Identification of a nonlysosomal acidic compartment.

Proc NatI Acad Sci USA 79:6186, 1982

19. Broxmeyer HE, Smithyman A, Eger RR, Meyers PA,

DeSousa M: Identification of lactoferrin as the granulocyte-derived

inhibitor of colony-stimulating activity production. J Exp Med

148:1052, 1978

20. BuIlen JJ, Armstrong JA: The role of lactoferrin in the

bactericidal function of polymorphonuclear leukocytes. Immunology

36:781, 1979

21. Yamada Y, Jacobsen DW, Green R: Visualization of the

receptor for transferrin on K562 cells by a rosette-forming assay. J

Immunol Methods 86:95, 1986

22. Danilovs J, Ayoub G, Terasaki P1: B-lymphocyte isolation by

thrombin-nylon wool, in Terasaki P1 (ed): Histocompatibility Test-

ing. Los Angeles, UCLA Tissue Typing Laboratory, 1980, p 28723. Bates GW, Schlabach MR: The reaction of ferric salts with

transferrin. J Biol Chem 248:3228, 1973

24. Bottomley 55, Wolfe LC, Bridges KR: Iron metabolism in

K562 erythroleukemic cells. J Biol Chem 260:681 1, 1985

25. Mendelsohn J, Trowbridge I, Castagnola J: Inhibition of

human lymphocyte proliferation by monoclonal antibody to trans-

ferrin receptor. Blood 62:821, 1983

26. Gronowicz E, Coutinho A, Melchers F: A plaque assay for all

cells secreting Ig of a given type or class. Eur J Immunol 6:588,

1976

27. Hicks DG, Ohlsson-Wilhelm BM, Farley BA, Kosciolek BA,

Rowley PT: K562 cell erythroid differentiation: Requirement for a

factor in fetal bovine serum. Exp Hematol 13:273, 1985

28. Ichiki AT, Wust CJ, Lozzio CB: Characterization of the Fc

(IgG) receptor on the pluripotential leukemia cell K562. Clin Exp

Immunol 59:64, 1985

29. Moguilevsky N, Retegui LA, Masson PL: Comparison of

human lactoferrins from milk and neutrophilic leukocytes. Relative

molecular mass, isoelectric point, iron-binding properties and uptake

by the liver. Biochem J 229:353, 1985

30. Bennett RM, Davis J: Lactoferrin binding to human periph-eral blood cells: An interaction with a B-enriched population of

lymphocytes and a subpopulation of adherent mononuclear cells. J

Immunol 127:1211, 1981

31. Birgens HS, Hansen NE, Karle H, Kristensen LO: Receptor

binding of lactoferrin by human monocytes. Br J Haematol 54:383,

1983

32. Hashizume 5, Kuroda K, Murakami H: Identification oflactoferrin as an essential growth factor for human lymphocytic cell

lines in serum-free medium. Biochim Biophys Acta 763:377, 1983

33. Amouric M, Marvaldi J, Pichon J, Bellot F, Figarella C:

Effect of lactoferrin on the growth of a human: Adenocarcinoma cell

line-Comparison with transferrin. In Vitro 20:543, 1984

34. Kornfeld 5: The effect of metal attachment to human apo-

transferrin on its binding to reticulocytes. Biochim Biophys Acta

194:25, 1969

35. Tsunoo H, Sussman HH: Characterization of transferrin

binding and specificity of the placental transferrin receptor. Arch

Biochem Biophys 225:42, 1983

36. Klausner RD. Ashwell G, Van Renswoude J, Harford JB,

Bridges KR: Binding ofapotransferrin to K562 cells: Explanation of

the transferrin cycle. Proc Natl Acad Sci USA 80:223, 1983

37. Fuller RA, Briggs DR: Some physical properties of hen’s egg

conalbumin. J Am Chem Soc 78:5253, 1956

38. Azari PR, Feeney RE: Resistance of metal complexes ofconalbumin and transferrin to proteolysis and to thermal denatur-

ation. J Biol Chem 232:293, 1958

39. Alderton G, Ward WH, Fevold HL: Isolation of lysozyme

from egg white. J Biol Chem 157:43, 1945

40. Weiss L: The cell periphery. Int Rev Cytol 26:63, 1969

41. Campbell EJ: Human leukocyte elastase, cathepsin G, and

lactoferrin: Family of neutrophil granule glycoproteins that bind to

an alveolar macrophage receptor. Proc Natl Acad Sci USA 79:6941,

1982

42. Van Snick JL, Masson PL: The binding of human lactoferrin

to mouse peritoneal cells. J Exp Med 144:1568, 1976




270 YAMADA ET AL

43. Moguilevsky N, Retegui LA, Courtoy PJ, Castracane CE, 46. Bridges KR, Cudkowicz A: Effect of iron chelators on the

Masson PL: Uptakeoflactoferrin by the liver III. Critical roleofthe transferrin receptor in K562 cells. J Biol Chem 259:12970, 1984protein moiety. Lab Invest 50:335, 1984 47. Louache F, Testa U, Pelicci P. Thomopoulos P. Titeux M,

Rochant H: Regulation of transferrin receptors in human hemato-44. Bennett RM, Davis J, Campbell 5, Portnoff 5: Lactoferrin

binds to cell membrane DNA. Association of surface DNA with an poietic cell lines. J Biol Chem 259:1 1576, 198448. Mattia E, Rao K, Shapiro DS, Sussman HH, Klausner RD:

enriched population of B cells and monocytes. J Clin Invest 71:61 1,I 983 Biosynthetic regulation of the human transferrin receptor by des-

ferrioxamine in K562 cells. J Biol Chem 259:2689, 1984

45. Cudkowicz A, Klausner RD, Bridges KR: Regulation of the 49. Birgens HS, Karle H, Hansen NE, Kristensen LO: Lactofer-

transferrin receptor in K562 erythroleukemia cells. Prog Clin Biol rin receptors in normal and leukemic human blood cells. Scand J

Res 165:509, 1984 Haematol 33:275, 1984




Lactoferrin binding by leukemia cell lines

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