[CANCER
RESEARCH
35, 2609-2612,
September
1975]
Brief Communication
Expression by Human Neuroblastoma Cells of an Antigen
Recognized by Naturally Occurring Mouse Anti-Brain Autoantibody
Sue Ellen Martin1 and W. John Martin
Laboratory of Biochemical Genetics, National Heart and Lung Institute [S.E.M.],
Institute [W. I. M.], NIH, Bethesda, Maryland 200/4
and Viral Leukemia and Lymphoma Branch, National Cancer
MATERIALS
SUMMARY
AND METHODS
Mouse Sera. For the experiments reported in this study,
sera of normal 6- to 9-month-old male C3Hf/HeN
mice
were used.
Murine Cell Lines. The N B 1 cell line was established
adrenal metastasis of a primary
These experimentsindicatethat mousebrain antigen2 is from a neuroblastoma
ovarian
teratoma
of
a
C3H/Helcrf
mouse. The contact
present on four human neuroblastoma
cell lines but not on
inhibitable
epithelial
cell
line
Ter-A
is
1 of several cell lines
other human tumor cell lines tested, including four glial cell
derived
from
the
ovarian
teratoma.
N-T16
is a cloned cell
lines and a retinoblastoma.
These findings demonstrate the
(2).
value of naturally occurring antibodies in normal mouse line derived from the C 1300 murine neuroblastoma
Human
Cell
Lines.
The
human
cell
lines
assayed
for
sera for the detection and serological classification
of
the
expression
of
the
M
BA-2
antigen
are
listed
in
Table
I.
human tumor antigens and indicate that considerable
Absorption of Mouse Sera. Aliquots of undiluted mouse
caution should be used in attempts to distinguish tumor
sera were absorbed with a 1:3 packed volume of either
specific and tissue-specific antigen expression on human
tumor cells at 4°for 40 mm or homogenized normal tissues
neuroblastoma
cells.
at 25°for 40 mm. The suspensions were centrifuged at 8000
A number of human tumor cell lines of both neural and
nonneural origin have been assayed for the expression of an
interspecies brain antigen (mouse brain antigen 2), detected
by naturally occurring antibodies in normal mouse sera.
x g for 1.5mm in a microcentrifuge (Beckman Instruments,
INTRODUCTION
Table
I
Human cell lines tested for expression of MBA -2 antigen
Normal sera of mice of a wide variety of strains including
germ-free and congenitally athymic (nude) mice are cyto
origin of
Designation
toxic, in the presence of rabbit complement,
for the linePassage―Source―SK-N-MCNeuroblastoma54ASK-N-SHNeuroblastoma28ALANEVNeuroblastoma8B
cell
of cell lineTissue
teratoma-derived
murine neuroblastoma
cell line NB 1
(6—8).The cytotoxicity of normal serum for NBI cells is
due to naturally occurring 1gM antibodies (6-8). Anti-NB1
cytotoxic antibodies in normal mouse sera can be specifi
+ 77C
+
cally absorbed by homogenized brain and kidney tissue of D118MGAstrocytoma215CHS0683Glioma13CHA207Glioblastoma29DY-79Retinoblastoma>
mice and man and by homogenized brain tissue of rats,
guinea pigs, and chickens (6). The interspecies brain antigen
recognized by NOA2 in mouse sera has been designated
100EFLNormal
MBA-2. It is of interest to know whether the expression of amnion19BMTOsteogenic
MBA-2 is restricted to a subpopulation of neural cells. We sarcoma102BT
carcinoma28B
24Bladder
have therefore tested a variety of human tumor cell lines of
both neuronal and glial origin for the ability to absorb
a The
passage
number
of
the
cell
lines
when
first
received
in
our
specifically anti-MBA-2
antibody activity from normal
laboratory. The cell lines were tested within 5 subsequent passages.
mouse serum.
b The
cell
lines
were
received
from
the
following
sources:
A,
Dr.
June
1 Graduate
2 The
student
at
abbreviations
University
used
are:
College
NOA,
London.
naturally
occurring
antibodies;
MBA-2, mouse brain antigen 2; BSS-lO, balanced salt solution containing
10% fetal calf serum; RC', rabbit complement.
Received April 25, 1975; accepted June 17, 1975.
SEPTEMBER
1975
Biedler, Memorial Sloan Kettering Research Institute ( I); B, Dr. Robert
Seeger, Department
of Pediatrics
and Department
of Immunology,
University of California, Los Angeles, Los Angeles, Calif.; C, Cell Cul
ture Laboratory,
Naval Biomedical
Research
Laboratory,
Oakland,
Calif.; D, Dr. Stuart
Aaronson,
National
Cancer Institute,
NIH.
Bethesda, Md.; E, Dr. Marshall Nirenberg, National Heart and Lung
Institute, NIH, Bethesda, Md.
2609
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer Research.
S. E. Martin and W. I. Martin
@
60
Inc., Rockville, Md.), and the absorbed sera were tested for
residual cytotoxic activity. Tissue absorption
does not
release anticomplementary
factors (6).
50
51Cr Release Cytotoxicity Assay. A 2-step complement
dependent 51Cr release cytotoxicity assay was performed as
40
previously described (5—7).Briefly, 20 @l
of a suspension of
U)
6 x l0@ ‘Cr-labeled tumor cells in BSS-lO were mixed with
U)
20 sl of unabsorbed or absorbed mouse serum appropriately
30
U
diluted. The mixture was incubated at 37°for 60 mm. Cells
U
were washed with 4 ml of BSS-lO and resuspended in 30 @l
w
20
of a 1:4 dilution of mouse tissue-absorbed
RC'. The mix
ture was incubated at 37°for 60 mm, 2 ml of BSS-lO were
added, and the tubes were centrifuged at 800 x g for 10 mm.
10
The radioactivity in I ml of supernatant (a) and that in the
remaining 1 ml of supernatant plus the cell pellet (b) were
measured. The absolute percentage of lysis of target cells is
1:1
1:2
1:4
1.8
defined by the formula [(2 x a)/(a + b)] x 100. Four
SERUM DILUTION
groups of controls were included in each experiment. The
Chart I. Anti-NBI cytotoxicity of normal mouse serum either unab
control groups were as follows: medium control (Med),
initial incubation with BSS-l0 and 2nd incubation with sorbed (•); absorbed with the human neuroblastoma cell lines LANTB
BSS-lO; antibody control (Ab), initial incubation with (0- - -0), SK-N-SH (V)@ SK-N-MC (•),and LANEV (@- - -s); or
absorbed with the cell lines FL ((A), MT (D—D), Y-79 (0), and T 24
mouse serum and 2nd incubation with BSS- 10; complement
(@—@).
control (Comp), initial incubation with BSS-10 and 2nd
incubation with RC'; and maximum release (FT), initial
60
incubation with BSS-lO followed by 3 cycles of freezing and
thawing. The specific (%) lysis was calculated by the
formula
50
>.
-4
0.
U)
(Absolute % lysis due to serum + RC')
— (Ab
— Mcd)
— (Comp)
x
40
100
FT-Comp
U)
U)
The observed values for Ab and Med controls did not differ
by more than 1% lysis. The Comp value for NB1 cells
averages 8.9%, for N-Tl6 cells it averages 13.7%, and for
Ter-A cells the average is 10.8%. These values were only
slightly higher than the corresponding values observed with
the Med controls, i.e., the RC' used was essentially nontoxic
for the cell lines tested. In each assay, duplicate determina
tions were performed.
>.
-J
0
30
U.
0
LU
U)
20
10
0
1:1
1:2
RESULTS
Aliquots
1:4
SERUM
of normal
mouse
serum
were absorbed
with
1:8
DILUTION
Chart 2. Anti-NB1 cytotoxicity of normal mouse serum either unab
sorbed (•),absorbed with the human glial tumor cell lines I 18 MG (Es),
eitherhumanneuroblastomaor nonneuralhumancell lines HS0683 (A), and A172 (0); or absorbed with the human neuroblastoma
and tested for residual cytotoxic activity. Chart 1 illustrates
that absorption of normal mouse serum with each of 4
human neuroblastoma
cell lines significantly reduced anti
NB1 cytotoxic activity. Included in this experiment were
aliquots of the same pool of serum absorbed with either a
retinoblastoma,
an osteogenic sarcoma, a bladder carci
noma, or a normal amnion-derived
cell line. None of the
latter cell lines significantly absorbed anti-NB1 cytotoxic
activity from normal sera. Chart 2 illustrates the results of
an experiment in which the human neuroblastoma cell line,
SK-N-MC, and 3 human glial tumor cell lines, Al72, 118
MG, and HS0683, were tested for their ability to reduce
anti-NB1 cytotoxic activity of mouse serum. Again, 5K-NMC cells significantly reduced anti-NB1 cytotoxic activity.
2610
cell line SK-N-MC
(U).
None of the glial cell lines tested, however, absorbed
anti-NB1 cytotoxic NOA. In similar experiments, the glial
tumor cell line HA207 was found not to absorb anti-NBI
cytotoxic activity in normal mouse serum. When sera
absorbed with the neuroblastoma
and glial cell lines were
tested for cytotoxic activity against the murine neuroblas
toma cell line N-T16, no significant reduction of cytotoxic
ity was observed. Chart 3 depicts results obtained in an
experiment in which sera absorbed with the neuroblastoma
cell lines SK-N-MC and SK-N-SH, and the glial cell lines
HSO683 and Al72 were tested against N-Tl6 target cells.
CANCER RESEARCH
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer Research.
VOL. 35
Neurob!astoma
90
A ntigen
Table 2
Expression of NB/-associated antigen MBA-2 on normal brain and
kidney tissue
80
ofTissue
Residual cytotoxicity
serumaNBI
70
usedfor
Ter-AMousebrain9.0
absorptionabsorbed
60
107.7Mousekidney―12.1
106.5Mouseliver105.2
112.0Human
U)
(%)
97.7Human
brain36.7
98.2Human
kidney54.8
liver104.2
>.
U
@4O
96.7
LU
Q.
U)
a Aliquots
30
of
normal
mouse
serum
were
absorbed
with
a
1:3
volume
of
homogenized tissues, and the absorbed serum was tested for cytotoxic
activity against NBI and Ter-A target cells. The results are expressed as
the percentage of the specific lysis achieved against these target cells by
unabsorbed serum.
20
b It
has
yet
to
be determined
whether
the
MBA-2
antigen
present
in
the
kidney is in the form of antigen:antibody
complexes and reflects the
occurrence in normal mouse sera of autoantibodies
directed against this
antigen.
10
and kidney tissue of both mice and man and is, therefore, a
normal tissue, rather than a tumor-specific, antigen. These
findings are relevant to the assumption that the common
Chart 3. Anti-N-T16
cytotoxicity
of normal mouse serum either
neuroblastoma
antigen recognized by lymphoid cells and
unabsorbed
(•); absorbed with the human neuroblastoma
cell lines
sera of neuroblastoma-bearing
patients is tumor specific (3,
SK-N-MC (0) and SK-N-SH (s); or absorbed with the human glial cell
4). Thus, a lack of cross-reactivity
with other tumor cell
lines HS0683 (A) and Al72 (0).
lines, even including cells of glial origin, would not necssar
ily exclude that the anti-neuroblastoma
reactivity seen in
These absorbed sera were also tested for residual cytotoxic
patients might be directed against the brain-associated
ity against the murine teratoma-derived
cell line, Ter-A. No
normal tissue antigen MBA-2.
reduction in anti-Ter-A cytotoxicity was apparent (data not
MBA-2 is not detectable on the N-Tl6 neuroblastoma,
shown). As reported in detail elsewhere, of several normal
and it is quite possible that some human neuroblastoma cell
mouse and human tissues tested, only brain and kidney
lines will also lack MBA-2. The nature of MBA-2 and its
tissue are capable of specifically absorbing anti-NB1 cyto
significance in terms of the biological behavior of neuroblas
toxicity from normal mouse sera. The results of I such
toma cells are presently obscure. By testing for the expres
experiment are depicted in Table 2. Data indicating the 1gM
sion of MBA-2 on a wide variety ofhuman neuroblastomas,
nature of the anti-NB1 RC'-dependent
cytotoxic activity of
it may be possible to correlate M BA-2 expression with a
normal sera of C3Hf/HeN
mice have been presented
particular clinical characteristic.
It is also possible that
previously. Briefly, the cytotoxic activity is (a) present only
MBA-2 expression may unexpectedly
occur on certain
in the excluded fraction (1gM peak) of mouse serum
tumors that are not neuroblastomas.
Thus, for example, an
chromatographed
on a Sephadex G-200 column; (b) abol
antigen recognized by murine NOA on the N-T16 cell line is
ished by exposure of mouse serum to 0. 1 M 2-mercaptoetha
also present on the murine glioblastoma G26 and on I of 2
nol; and (c) inactivated by preincubation of mouse serum
murine lung tumor cell lines tested (unpublished data).
with a specific goat anti-mouse
1gM immunoglobulin
Again, however, the detection of aberrant expression of cell
antibody preparation (6—8).In addition, genetic studies have
surface antigens by malignant cells could be important in
indicated
that mice genetically
defective in thymus
understanding the diverse clinical behavior of tumors of the
independent
1gM antibdy production have little or no
same tissue origin. Finally, a wide variety of cell surface
detectable serum anti-NBI cytotoxic activity (8).
antigens can be detected on murine tumors using NOA
(7—9).Detailed studies on antigenic cross-reactivity detected
by murine NOA between murine and human tumor cell
DISCUSSION
lines may provide a valuable approach to the serological
classification and detection of human tumors.
The results reported in this paper indicate that an antigen
detected by mouse NOA on the murine neuroblastoma cell
line NB! is present on the 4 human neuroblastoma cell lines
ACKNOWLEDGMENTS
tested but not on a variety of other human tumor cell lines
tested, including a retinoblastoma
and 4 glial tumor cell
We wish to thank Dr. Robert Seeger of the Department of Pediatrics
lines. The antigen can readily be detected on normal brain and the Department of Immunology, University of California, Los
1:1
1:2.
1:4
1:8
SERUM DILUTION
SEPTEMBER
1975
2611
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer Research.
S. E. Martin and W. J. Martin
Angeles; Dr. June Biedler of Memorial Sloan Kettering Research Insti
tute;
and
Dr.
Marshall
Nircnberg
of
the
Laboratory
of
Biochemical
1968.
4. Jose, D. G., and Seshadri, R. Circulating Immune Complexes in
Genetics, National Heart and Lung Institute, NIH, for generously
providing neuroblastoma cell line for use in this study.
Human Neuroblastoma:
Direct Assay and Role in Blocking Specific
Cellular Immunity. Intern. J. Cancer, /3: 824-838, 1974.
5. Martin, S. E. Mouse Brain Antigen Detected by Rat Anti-C1300
Antiserum. Nature, 249: 71-73, 1974.
REFERENCES
6. Martin, S. E., and Martin, W. J. Interspecies Brain Antigen Detected
by Naturally
1. Biedler, J. L., Helson, L., and Spengler, B. A. Morphology and Growth,
Tumorigenicity,
and Cytogenetics of Human Neuroblastoma
Cells in
Continuous Culture. Cancer Res., 33: 2643—2652, 1973.
2. Breakefield, X. 0., and Nirenberg, M. Selection for Neuroblastoma
Cells That Synthesize Certain Transmitters. Proc. Natl. Acad. Sd.
U.S.; 7/: 2530—2533,1974.
3. Hellstriim,
I., Hellström, K. E., Pierce,
Demonstration
of Cell Bound and Humoral
G. E., and Bill, A. H.
Immunity against Human
Neuroblastoma Cells. Proc. NatI. Acad. Sci. U. S., 60: 1231-1238,
2612
Occurring
Mouse Anti-Brain
Autoantibody.
Proc. NatI.
Acad. Sci. U. S., 72: 1036-1040, 1975.
7. Martin,
S. E., and Martin,
W. J. Anti-Tumor
Antibodies
in Normal
Mouse Sera. Intern. J. Cancer, /5: 658-664, 1975.
8. Martin,
S. E., and Martin,
CBA/HN
W. J. X Chromosome
Linked Defect of
Mice in Production of Tumor Reactive Naturally Occur
ring 1gM Antibodies. J. Immunol., //5: 502-508. 1975.
9. Martin, W. J., and Martin, S. E. Naturally
Occurring
Cytotoxic
Anti-Tumour Antibodies in Sera of Congenitally Athymic (Nude)
Mice. Nature New Biol., 249: 564-565, 1974.
CANCER RESEARCH
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer Research.
VOL. 35
Expression by Human Neuroblastoma Cells of an Antigen
Recognized by Naturally Occurring Mouse Anti-Brain
Autoantibody
Sue Ellen Martin and W. John Martin
Cancer Res 1975;35:2609-2612.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/35/9/2609
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1975 American Association for Cancer Research.