ANATOMIC PATHOLOGY
Original Article
Serum Transferrin Receptor Level Is Not
Altered in Invasive Adenocarcinoma
of the Breast
HEATHER N. RAAF, M.D., DONALD W. JACOBSEN, PH.D., SUSAN SAVON, M.S.,
AND RALPH GREEN, M.B., B.CH., M.D.
trations was 2.60-7.34 mg/L (mean, 4.44 mg/L) compared
with 2.85-8.80 mg/L (mean, 5.49 mg/L) in the control group.
Nine patients with in situ adenocarcinoma of the breast had
transferrin receptor concentrations of 3.68-6.66 mg/L (mean,
4.94 mg/L). For both the invasive carcinoma group and the in
situ group, the means were not significantly different from
those of the control group (P = 0.06 and 0.32, respectively). It
was concluded that the differential expression of transferrin
receptor on the surface of malignant tumor cells in adenocarcinoma of the breast was not reflected by changes in circulating
transferrin receptor concentrations. (Key words: Breast carcinoma; Enzyme-linked immunosorbent assay; Tumor markers;
Serum transferrin receptor; Transferrin receptor) Am J Clin
Pathol 1993; 99:232-237
The transferrin receptor is expressed on the surface of rapidly
dividing cells that require iron as a co-factor for essential redox
reactions and deoxyribonucleotide synthesis. Transferrin receptors are expressed on the surface of breast carcinoma cells
but not on benign breast tumor cells. In this study, the authors
investigated whether transferrin receptor concentrations in the
serum were elevated in patients with invasive adenocarcinoma
of the breast. The transferrin receptor was isolated and purified
from human placenta by affinity chromatography. The serum
transferrin receptor concentration was determined using an enzyme-linked immunosorbent assay in 19 patients with invasive
breast adenocarcinoma, 12 of whom had involvement of axillary lymph nodes. These results were compared with those
from 16 normal age-matched female controls. In the invasive
breast cancer group, the range of transferrin receptor concen-
The expression of transferrin receptor, a cell surface glycoprotein, is associated closely with cell growth and proliferation. The receptor binds holotransferrin and internalizes the protein by receptor-mediated endocytosis.
Among its numerous and essential functions, iron is a
component of the mitochondrial electron transport
chain that carries out cellular respiration and is an essential co-factor for ribonucleotide diphosphate reductase,
the enzyme responsible for the balanced formation of
deoxyribonucleotides for DNA synthesis. Transferrin is
required for cell growth in serum-free cell culture media,12 most likely for its iron-scavenging ability. In vitro
deprivation of iron by picolinic acid, a chelating agent,
inhibits cell proliferation,3 even in the presence of apotransferrin.4 Cells express receptors as they move from
the G1 to the S phase, with surface density reaching a
maximum just before mitosis.5'6 Antibodies that block
the transferrin binding site on the receptor also prevent
7-9
Resting and terminally differentiated
From the Department of Laboratory Hematology, The Cleveland proliferation.
Clinic Foundation, Cleveland, Ohio. Dr. Raaf currently is affiliated cells have few or no transferrin receptors on their surwith the Cuyahoga County Coroner's Office, Cleveland, Ohio.
faces.10"13
Surface transferrin receptors are expressed on maligPresented in part at the Pathology Resident Awards competition at
nant but not benign breast tumor cells.14 Binding studies
the joint meeting of the American Society of Clinical Pathologists and
the College of American Pathologists, New Orleans, Louisiana, Ocof microsomal preparations of breast cancer tissue show
tober 1991. (Dr. Raaf was the winner in the clinical pathology cateincreased transferrin receptors over normal breast tisgory.)
sue.15 The transferrin receptor concentration in breast
Received January 27,1992; revised manuscript accepted for publication May 11, 1992.
cancer is correlated inversely with the estrogen receptor
Address reprint requests to Dr. Green: From the Department of Labconcentration.16 The recent discoveries that (1) soluble
oratory Hematology, The Cleveland Clinic Foundation, Cleveland,
transferrin receptors circulate in the serum17 and (2) they
Ohio 44195.
232
RAAF ET AL.
233
Breast Adenocarcinoma
Transferrin Receptor Level in nvasive
I)
may be elevated in malignancy 1819 have prompted the
current study to determine whether serum transferrin
receptor levels are increased in patients with breast adenocarcinoma.
MATERIALS AND METHODS
Patient Population
We identified the subjects in this study prospectively
before they underwent a biopsy or mastectomy for breast
tumors, including those undergoing a reoperation for residual tumors based on pathologic findings from a
biopsy, between August 1990 and January 1991. Pathologic reports were used to identify patients with invasive
and in situ adenocarcinoma and to identify those with
axillary lymph node involvement. Those with a diagnosis of invasive adenocarcinoma were included in the "invasive adenocarcinoma" group. Sera from these patients
were obtained from preoperative specimens submitted
for routine chemical analysis. Nineteen patients with invasive adenocarcinoma were identified, 12 of whom had
axillary lymph node involvement. No further attempt
was made to review the charts of these patients for their
stage of disease. Nine patients with in situ (intraductal)
carcinoma comprised the second study group. All sera
were frozen immediately and stored at - 7 0 °C for up to
6 months before they were tested. The third study group
consisted of sera from five women known to have stage
IV adenocarcinoma of the breast. These sera had been
stored at - 7 0 °C for up to 6 months. Sera from 16
healthy female volunteers, aged 40 years or older with
normal iron status and no history of breast cancer, were
used as normal controls.
Purification of Placental Transferrin Receptor
The transferrin receptor was isolated and purified
from human placental tissue using a combination of two
methods.20'21 Two fresh human placentas were transported from the delivery room in plastic bags on ice and
processed immediately at 4 °C unless otherwise specified. The placentas were stripped of their cords and
membranes and cut into approximately 20 g pieces. The
placental sections were homogenized at high speed in a
Waring blender for 1 minute in 1.5 volumes of 10 mmol/
L potassium phosphate buffer, pH 7.5, containing 150
mmol/L NaCl (KP r NaCl buffer). The homogenate then
was centrifuged at 25,000g for 90 minutes. The supernatant was aspirated and discarded. The crude membrane
pellet was resuspended in 1,500 mL of KPi-NaCl buffer
and frozen at - 2 0 °C. The transferrin receptors were
solubilized in Triton X-100 as follows. We added 500
mL of ICP-NaCl buffer containing 4% Triton X-100 to
the thawed crude membrane preparation in 1,500 mL of
KPi-NaCl buffer for a final Triton X-100 concentration
of 1%. The mixture was homogenized in a Waring
blender (twice for 30 seconds each at high speed) and
then placed in a beaker and subjected to sonication for
two 5-minute bursts using a 1-cm probe. The homogenate was stirred for 2 hours and then centrifuged at
25,000g for 30 minutes. The supernatant, containing the
solubilized receptor, was titrated to pH 5 with 1 N HC1.
Deferoxamine (381 mmol/L in 2.0 mL of distilled water)
was added to a final concentration of 0.2 mmol/L, and
the mixture was stirred for 15 minutes. The pH then was
adjusted to 8.0 with 1 N NaOH. Ammonium sulfate was
added slowly while stirring over 20 minutes to a final
concentration of 40% w/v. The solution was stirred for
an additional 30 minutes and then centrifuged at
20,000g. After removal of the supernatant by aspiration,
the pellet, which floated because of the presence of Triton X-100, was resuspended in a total volume of 300 mL
of K P r N a C l buffer and stirred overnight. The suspension was dialyzed overnight against 4 L of KPi-NaCl
buffer with buffer changes at 4 and 8 hours.
The dialysate was clarified by centrifugation at
17,000g for 2 hours and passed through a Sepharose CL4B column (3 x 6 cm [Pharmacia LKB Technology, Piscataway, NJ]) equilibrated with KPi-NaCl buffer. The
Sepharose CL-4B column effluent (150 mL) was applied
to a Sepharose-transferrin column (6-mL bed volume)
at a rate of 20 mL/hr. The column was washed rapidly
with 1 L of K P r N a C l buffer containing 0.2% Triton
X-100 and then slowly with 20 mL of KP-NaCl buffer
containing 10 mmol/L of 3-([3-cholamidopropyl]-dimethylamino)-l -propane sulfonate (CHAPS) to remove
the Triton X-100. Then, 20 mL of iron-removal buffer
(50 mmol/L sodium citrate, pH 4.9, containing 100
mmol/L NaCl, 10 mmol/L CHAPS, and 1.0 mmol/L
deferoxamine) was passed through the column at 2 mL/
min. The removal of iron from the column resulted in a
color change from orange to white. The column was
washed immediately with 20 mL of 50 mmol/L of N(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid
(HEPES), pH 7.5, containing 100 mmol/L NaCl and 10
mmol/L CHAPS at 20 mL/min. The transferrin receptor was eluted with two column volumes of 50 mmol/L
HEPES, pH 7.5, containing 500 mmol/L sodium thiocyanate and 10 mmol/L CHAPS at 2 mL/min. The eluate
was applied to a 3 X 12-cm Sephadex G-25 M column
(20-mL bed volume [Pharmacia LKB Biotechnology,
Piscataway, NJ]), previously equilibrated with KPiNaCl buffer. Fractions containing the transferrin receptor were pooled and concentrated by membrane
filtration. Final purification was achieved using highperformance liquid chromatography on a TSK 3000SW
Vol. 99 •No. 3
234
ANATOMIC PATHOLOGY
Original Article
gel permeation column (7.5 X 800 mm, Beckman Instruments, Fullerton, CA). The fractions were stored at
- 7 0 °C.
The binding capacity of the isolated receptor was assessed using an ammonium sulfate precipitation iodine
125-labeled transferrin binding assay.20 Samples containing solubilized transferrin receptor were incubated at
37 °C for 30 minutes with 0.5 pmol of iodine 125-labeled transferrin in the presence or absence of 500 pmol
of cold transferrin receptor in 10 mmol/L TRIS HC1, pH
8.0, containing 150 mmol/L NaCl (total volume, 600
JUL). After incubation, the mixture was cooled to 4 °C for
5 minutes in ice water. Cold saturated ammonium sulfate (400 juL) was added to a final concentration of 40%.
Then mixture was centrifuged at 4 °C for 30 minutes in a
microfuge at top speed. The supernatant was removed,
and the pellet and supernatant were counted separately.
Nonspecific binding was determined in tubes containing
a large excess of unlabeled transferrin. The purity of
the transferrin receptor preparation was assessed using
sodium dodecyl sulfate polyacrylamide gel electrophoresis.22
The serum transferrin receptor concentration was determined by an enzyme-linked immunosorbent assay.23
These authors reported that transferrin receptor levels in
sera stored at - 7 0 °C are stable for at least 1 year. Antibodies to the transferrin receptor were generously provided by James D. Cook, M.D., Kansas University Medical Center (Kansas City, KS). Antibody E2H10 was used
as the coating antibody, and horseradish peroxidase conjugated antibody A4A6 was used as the indicator. The
plates were read at 490 nM on a microtiter plate reader
(Molecular Devices Corp., Menlo Park, CA). The assays
were run in triplicate with high, normal, and low sera as
controls. There was good between-assay and within-assay reproducibility. For the high, normal, and low serum
controls, the between-assay coefficients of variation were
10.5%, 12.1%, and 11.6%, respectively. The overall
mean coefficient of variation for within-assay precision,
based on an assay of samples in triplicate, was 7.3%.
Other Methods
The Sepharose-transferrin column was prepared according to a published method 24 as follows. We combined 40 g of Sepharose CL-4B with 50 mL of water and
100 mL of 2.0 mol/L Na 2 C0 3 on ice with overhead stirring. We added 10 g of cyanogen bromide (in 5 mL of
acetonitrile) and stirred vigorously for 90 seconds. The
reaction was terminated by collecting the activated beads
on Whatman 4 paper (Whatman Inc., Clifton, NJ) in a
chilled Buchner funnel and rapidly washing with 2 L of
chilled 0.1 mol/L NaHC0 3 . The activated Sepharose
A
B
C
97,400
66,200
45,000
31,000
•x^PIF"
i
<r- 2 1 , 5 0 0
•
m
<-
14,400
FIG. I. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of
the transferrin receptor purified from human placenta. Lane A, 5.0 Mg
of protein; lane B, 5.1 Mg of protein; and lane C, molecular weight
markers: 97,400, phosphorylase b; 66,200, bovine serum albumin;
45,000, ovalbumin; 31,000, carbonic anhydrase; 21,500, soybean trypsin inhibitor; and 14,400, lysozyme.
then was combined with 30 mL of 0.4 mol/L N a H C 0 3
and 200 mg of holotransferrin in 40 mL of cold phosphate-buffered saline. The mixture was stirred overnight
at 4 °C. Glycine (100 mL of 1.0 mol/L, pH 9.0) was
added to the reaction mixture and allowed to warm to
room temperature. After stirring for 2 hours, the beads
were harvested by collection on Whatman 4 filter paper.
The beads were washed thoroughly with cold phosphatebuffered saline and stored in that buffer at 4 °C. We
prepared the iodine 125-labeled transferrin receptor by
the chloramine T method 25 and purified it by high-performance liquid chromatography on a TSK 3000 SW gel
permeation column. The protein concentration was determined by the bicinchoninic acid (BCA) protein assay
method (Pierce, Rockford, IL), according to the manufacturer's instructions. Statistical analysis was performed
using Students' /-test and Wilcoxon's rank test.
RESULTS
Highly purified placental transferrin receptor (5.4 mg)
was obtained from two fresh human placentas. The preparation migrated as a single major band during sodium
dodecyl sulfate polyacrylamide gel electrophoresis with
A.J.C.P. • March 1993
235
RAAF ET AL.
Transferrin Receptor Level in Invasive Breast Adenocarcinoma
TABLE
SERUM TRANSFERRIN RECEPTOR LEVELS BY
DISEASE GROUP
Disease Group
Controls
Breast cancer
Invasive
Significance
In situ
Significance
Stage IV
Significance
N
Mean + SD Median
16 5.49 ±1.44
5.34
Minimum Maximum
2.85
8.83
19 4.60 ± 1.20
4.55
2.60
/-test (vs. Control), P value = 0.06
9 4.94 ±0.92
4.72
3.68
/-test (v.v. Control), P value = 0.32
5 7.53 ±3.72
5.29
4.16
Wilcoxon test, P value = 0.54
7.34
6.66
11.83
Values arc given as mg/L.
an apparent molecular weight of 94,000 Daltons (Fig. 1).
On the basis of protein concentration and the determination of the transferrin receptor concentration by enzyme-linked immunosorbent assay (with the standards
provided by Dr. Cook), the purity of this transferrin receptor preparation was estimated to be greater than 98%.
The serum transferrin receptor concentrations from
16 normal female controls, aged 40 years or older,
ranged from 2.85 to 8.80 mg/L (mean, 5.49 mg/L). This
agreed well with the mean of 5.56 mg/L reported for a
group of 45 healthy women.23 In 19 patients with invasive adenocarcinoma of the breast, serum transferrin receptor concentrations ranged from 2.60 to 7.34 mg/L
(mean, 4.44 mg/L). These levels were not significantly
different from those of the controls (P = 0.06). Nine
patients with in situ adenocarcinoma of the breast had
transferrin receptor concentrations ranging from 3.68 to
6.66 mg/L (mean, 4.94 mg/L). These values were not
significantly different from those of the controls (P
= 0.32). In five patients with known stage IV breast
cancer, the concentrations were normal in three (5.29,
5.06, and 4.15 mg/L) and elevated in two (11.83 and
11.33 mg/L). The patient with a concentration of 11.83
mg/L had a serum iron concentration of 310 iig/L (normal, 350-1,700 Mg/L), and her serum ferritin level was
104 Mg/L (normal, 150-4,050 Mg/L). There was insufficient serum from this patient to determine her total ironbinding capacity and transferrin saturation. In the second patient, with a serum transferrin receptor concentration of 11.33 mg/L, her iron level was 310 Mg/L; total
iron-binding capacity, 1,850 Mg/L (normal, 2,200-4,800
Mg/L); and transferrin saturation, 17% (normal, 2055%). The serum ferritin concentration was 4,059 Mg/L.
The transferrin receptor concentrations in the patients in
the stage IV group did not differ significantly from those
of controls (P = 0.54, Wilcoxon test, Table 1 and Fig. 2).
tain malignancies.1819,23 It is of potential clinical interest
to know which tumors have elevated levels and whether
serum transferrin receptor concentrations might be helpful in identifying occult malignancy or the recurrence of
previously identified malignancy. Adenocarcinoma of
the breast was selected to test this possibility because earlier studies had shown a clear difference between the
transferrin receptor content of benign and malignant
breast tumor cells.14
Using the antibodies provided by Cook and the assay
described by his group23 and the transferrin receptor purified in our own laboratory, we were able to achieve excellent interlaboratory correlation in the measurement of
serum transferrin receptor concentrations. The serum
transferrin receptor concentrations in the two control
groups did not differ significantly (P > 0.50). Our
method for isolating the transferrin receptor was similar
to the one used by Flowers and associates.23 These methods appear to yield a large quantity of pure transferrin
receptor and a reproducible assay for the measurement
of its concentration in serum.
No significant change was identified between agematched normal control volunteers and patients with
invasive adenocarcinoma of the breast, identified by the
pathologic reports. However, the clinical extent of invasion could not be assessed from these reports.
To test the hypothesis that our patients with invasive
cancer might not have had sufficient tumor bulk to produce a positive result, we tested sera from patients with
known advanced-stage breast cancer. Two of five of
these patients had elevations of the transferrin receptor
level that could not be explained by an iron deficiency.
OT
12.0
,
o
11.0
B)10.0
E
9.0 "
o 8.0 a.
a>
o 7.0 "
w
CC 6.0 "
c
5.0 4.0 V)
2
OT
o
3.0
2.0
1.0
0
Control
Invasive
Insitu
Stage IV
Disease Group
DISCUSSION
Serum transferrin receptor concentrations are elevated in iron deficiency, conditions associated with increased erythropoiesis (including hemolysis), and in cer-
FIG. 2. Concentrations of transferrin receptor in sera from patients
with adenocarcinoma of the breast. The patients were divided into
three groups according to the stage of disease: invasive (n = 19), in situ
(n = 9), and stage IV (n = 5). Levels from age- and sex-matched controls (n = 16) also are shown.
Vol. 99 • No. 3
236
ANATOMIC PATHOLOGY
Original Article
This may indicate that, with sufficient tumor bulk, the
transferrin receptor concentration will rise. A large test
population would be necessary to provide statistically
significant data. Even so, such an association would have
no advantage with respect to the detection of recurrence
or early invasion, in which instances monitoring with a
simple serum test would be most helpful.
Why serum transferrin receptor concentrations are
not elevated in adenocarcinoma of the breast is not
known, but there are several possible explanations. Although present in high density on the surface of cells in
invasive breast adenocarcinoma, the receptors may not
reach the circulation until a late stage of the disease. A
certain threshold level of tumor neovascularization may
be required for sufficient numbers of receptors from the
breast tumors to reach the circulation. Alternatively,
there may be less inherent interaction of the breast
cancer cells with the vasculature than is the case in other
tumors, such as hepatocellular carcinoma,18 in which the
cells have an intimate association with the circulation
and malignant cells therefore may have better access.
The potential for circulation of transferrin receptors
was reported first by Pan and Johnstone,26 who found
that sheep reticulocytes shed multivesicular endosomes
containing transferrin receptor during erythropoiesis.
Transferrin receptors may not be shed as readily in adenocarcinoma of the breast as in erythroid or other cell
types. Alternatively, there may be faster clearance of
serum transferrin receptors in adenocarcinoma of the
breast. In our study, the transferrin receptor concentration actually tended to be slightly lower in patients with
in situ adenocarcinoma, and even more so, almost to a
statistically significant level, in invasive adenocarcinoma. Although we believe that these differences probably would disappear in a larger study, these results also
were consistent with a more rapid clearance of tumor-related transferrin receptors.
Finally, transferrin receptor from adenocarcinoma of
the breast may be immunologically distinct and no
longer detected by our monoclonal antibodies. One
group found elevated serum transferrin receptor concentrations in lymphoma and myeloma,18 and another
found essentially normal concentrations.19 Although
such a difference may be only a reflection of differing
tumor bulk in the study groups, it might indicate that the
antibody combination used by the first group detected
an antigenically altered tumor-related serum transferrin
receptor, although that from the second group did not.
There is evidence supporting mutation of the transferrin
receptor,27 and altered transferrin receptors have been
found with defective glycosylation in acute T-cell
leukemia.28
Although there appears to be no clinical utility in measuring serum transferrin receptor levels in patients with
adenocarcinoma of the breast, the data from the laboratory that detected elevations of serum transferrin receptor concentrations in other solid tumors, such as gastric,
colonic, and esophageal cancer, should be verified using
other antibody systems.18 Perhaps differences in transferrin receptor structure and processing may be uncovered that will lead to a better understanding of the membrane-associated steps involved in cell proliferation and
malignant transformation.
Acknowledgments. The authors thank James D. Cook, M.D., Kansas
University Medical Center, who provided the antibodies to the transferrin receptor; Ina Hardesty, R.N., The Cleveland Clinic Foundation,
who helped in the prospective identification of patients with breast
tumors and collection of preoperative sera; Dr. Robert Kiwi, Chief of
Obstetrics, University Hospitals of Cleveland, Cleveland, Ohio, who
helped obtain the fresh placentas; Dr. Manjula Gupta, Department of
Immunopathology, The Cleveland Clinic Foundation, who provided
stored sera from women known to have stage IV adenocarcinoma of
the breast from a bank of stored sera; and Sharon Medendorp, M.P.H.,
Department of Biostatistics and Epidemiology, Research Institute, The
Cleveland Clinic Foundation, who performed the statistical analysis.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
REFERENCES
Barnes D, Sato G. Serum-free cell culture: A unifying approach.
Cell 1980;22:649-655.
Breitman TR, Collins SJ, Keine BR. Replacement of serum by
insulin and transferrin supports growth and differentiation of
the human promyelocyte cell line, HL60. Exp Cell Res
1980;126:494-498.
Fernandez-Pol JA, Bono VH Jr., Johnson GS. Control of growth
by picolinic acid: Differential response of normal and transformed cells. Proc Natl Acad Sci U S A 1977;74:2889-2893.
Robbins E, Pederson T. Iron: Its intracellular localization and possible role in cell division. Proc Natl Acad Sci U S A
1970;66:1244-1251.
Chitamber CR, Massey EJ, Seligman PA. Regulation of transferrin
receptor expression in human leukemia cells during proliferation and induction of differentiation. J Clin Invest
1983;72:1314-1325.
Musgrove E, Rugg C, Taylor I, Hedley D. Transferrin receptor
expression during exponential and plateau phase growth of human tumor cells in culture. J Cell Physiol 1984; 118:6-12.
Trowbridge IS, Domingo DL. Anti-transferrin receptor monoclonal antibody and toxin antibody conjugates affect growth of
human tumour cells. Nature 1981;294:171-173.
Trowbridge IS, Lopez F. Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits human tumor cell
growth in vitro. Proc Natl Acad Sci U S A 1982; 79:1175-1179.
White S, Taetle R, Seligman PA, et al. Combinations of anti-transferrin receptor monoclonal antibodies inhibit human tumor cell
growth in vitro and in vivo: Evidence for synergistic antiproliferative effects. Cancer Res 1990;50:6295-6301.
Larrick JW, Creswell P. Modulation of cell surface iron transferrin
receptors by cellular density and state of activation. J Supramol
Struct 1979;11:579-586.
Trowbridge IS, Omary MB. Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin. Proc
Natl Acad Sci U S A 1981;78:3039-3043.
Gatter KC, Brown G, Trowbridge IS, et al. Transferrin receptors in
human tissues: Their distribution and possible clinical relevance. J Clin Pathol 1983;36:539-545.
A.J.C.P. • March 1993
RAAF ET AL.
Transferrin Receptor Level in Invasive Breast Adenocarcinoma
13. Petraki H, loannides CG, Filli S, Papmichail M. Loss and reappearance of transferrin receptors in human leukemic cell lines.
Exp Cell Biol 1986;54:80-88.
14. Faulk SP, Hsi B-L, Stevens PJ. Transferrin and transferrin receptors in carcinoma of the breast. Lancet 1980;2:390-392.
15. Shindelman JE, Ortmeyer AE, Sussman HH. Demonstration of
the transferrin receptor in human breast cancer tissue: Potential
marker for identifying dividing cells. IntJCancer 1981 ;27:329—
334.
16. Tonik SE, Shindelman JE, Sussman HH. Transferrin receptor is
inversely correlated with estrogen receptor in breast cancer.
Breast Cancer Res Treat 1986;7:71-76.
17. Kohgo Y, Nishisato T, Kondo H, et al. Circulating transferrin
receptor in human serum. Br J Haematol 1986;64:277-281.
18. Kohgo Y, Niitsu Y, Nishisato T, et al. Transferrin receptors of
tumor cells: Potential tools for diagnosis and treatment of malignancies. In: Spik G, Montreuil J, Crichton RR, Mazurier,
eds. Proteins of Iron Storage and Transport. New York: Elsevier, 1985, pp 155-169.
19. Klemow D, Einsphar D, Brown TA, et al. Serum transferrin receptor measurements in hematologic malignancies. Am J Hematol
1990;34:193-198.
20. Seligman PA, Allen RH. Isolation of transferrin receptor from human placenta. Methods Enzymol 1987; 147:239-247.
237
21. Turkewitz AP, Amatruda JF, Borhani D, et al. A high yield purification of the human transferrin receptor and properties of its
major extracellular fragment. J Biol Chem I988;263:83188325.
22. Laemmli UK. Cleavage of structural proteins during the assembly
of the head of bacteriophage T„. Nature 1970;227:680-685.
23. Flowers CH, Skikne BS, Covell AM, Cook JD. The clinical measurement of serum transferrin receptor. J Lab Clin Med
1989;114:368-77.
24. March SC, Parikh I, Cuatrecasas P. A simplified method for cyanogen bromide activation of agarose for affinity chromatography.
Anal Biochem 1974;60:149-152.
25. McConahey PJ, Dixon FJ. A method of trace iodination of proteins for immunologic studies. Int Arch Allergy Appl Immunol
1966;29:185-189.
26. Pan B. Johnstone RM. Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: Selective externalization
of the receptor. Cell 1983;33:969-977.
27. Lesley JF, Schulte RJ. Selection of cell lines resistant to anti-transferrin receptor antibody: Evidence for a mutation in transferrin
receptor. Mol Cell Biol 1984;4:1675-1681.
28. Petrini M, Pelosi-Testa E, Sposi NM, et al. Constitutive expression
and abnormal glycosylation of transferrin receptor in acute Tcell leukemia. Cancer Res 1989;49:6989-6996.
Vol. 99 • No. 3