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1501
A Novel Tumor-Associated Antigen Expressed in
Human Uterine and Ovarian Carcinomas
Kenzo Sonoda, M.D.’
Manabu Nakashima, M.D.’
Tsunehisa Kaku, M.D.’
Toshiharu Kamura, M.D.‘
Hitoo Nakano, M.D?
Takeshi Watanabe, M.D.’
’ Department of Molecular Immunology, Medical Institute of Bioregulation, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Department of Gynecology and Obstetrics,
Faculty of Medicine, Kyiishu University, Fukuoka. Japan.
The authors thank Dr. Masazumi Tsuneyoshi for
providing paraffin embedded tissue blocks; Dr.
Zenon Steplewski for thi: GA 733 MoAb; Dr.
Akira Yachi for the YH 206 MoAb; Dr. Keigo
Endo for the CA .I25 MoAb; Dr. Osamu Hayakawa for the CAC-1 cell; Dr. Masaru Sakamoto
for the TMCC cell; Dr. Yoshihiro Kikuchi for the
MH, KF, HRA, and KK cells; Dr. Masato Nishida
for the HAG-2 cell; and the National Institute of
Hygienic Sciences for the MCAS cell, Kuramochi, PA-I, TYK-nu, and BeWo cells.
Address for reprints: Takisshi Watanabe, M.D.,
Department of Molecular Immunology, Medical
institute of Bioregulation, Kyushu University 60,
Maidashi 3-1 -1, Higashi-ltu, Fukuoka, 812-82,
Japan.
Received November 28,1995: accepted January
2, 1996.
0 1996 American Cancer Society
BACKGROUND. A large number of monoclonal antibodies (MoAhs) against human
tumor cells have been generated and it has been shown that these MoAbs are
useful tools in the diagnosis and treatment of cancer patients, as well as in the
basic investigation of the oncogenesis and characterization of cancer cells.
METHODS. The 22-1-1 MoAb was established by cell fusion between mouse rnyeloma cells and spleen cells derived from mice immunized with the human uterine
cervical adenocarcinoma cell line, SiSo. The tissue distribution and biologic characteristics of the 22-1-1 antigen (Ag) were examined.
RESULTS. The 22-1-1 Ag was distinct from the known tumor-associated antigens
such as YH 206, GA 733, CA 125, carcinoembryonic antigen, and sialyl Le”molecules
in an expression pattern in human tumor cell lines. An immunohistochemical
study revealed that 22-1-1 Ag was expressed in 87.5% of uterine cervical adenocarcinomas, 66% of uterine endometrial adenocarcinomas, and 58.8% of ovarian carcinomas. Moreover, 22-1-1 Ag was detected in 87.7% of uterine cervical squamous
cell carcinomas; however, it was not detected in normal uterine cervical or ovarian
tissues, except in uterine endometrial glands, in which its expression was observed
at low levels. The 22-1-1 Ag was secreted into cell culture supernatant fluids and
was also detected in the vaginal discharges of uterine cervical carcinoma patients.
The antigenic epitope of 22-1-1 Ag was shown to be a protein with a molecular
weight of 78 kilodaltons using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.
CONCLUSIONS. The 22-1-1 MoAb reactive to a novel tumor-associated antigen was
generated. This Ag was expressed in cancer cells derived mainly from the uterus
and ovary. Moreover, 22-1-1 Ag was secreted in the vaginal discharges of uterine
cervical carcinoma patients. 22-1-1 MoAb is a potential tool for the study of oncogenesis and the management of cancer patients. Cancer 1996; 721501-9.
0 1996 American Cancer Society.
KEYWORDS monoclonal antibody, tumor-associated antigen, uterine carcinoma,
ovarian carcinoma, flow cytometric analysis, irnmunohistochemical examination.
T
o date, many monoclonal antibodies (MoAbs) have been generated in
the field of cancer research. Some MoAbs have been used to evaluatle
the therapeutic effects of various treatments on cancer patient^.'-^ Other
MoAbs have been applied to basic research to examine the growth characterization, immunogenicity, and physiology of tumor
These investigations have shown that MoAbs are useful tools and the analysis of
antigens detected by such MoAbs is important in the research of oncogenesis and the characteristics of cancer cells. Adenocarcinoma of the uterine
cervix accounts for 5-20% of cervical cancers, and its incidence has recently been reported to be
However, the carcinogenesis
and biologic characteristics of this adenocarcinoma still remain unclear.
Moreover, this type of cancer shows low sensitivity against radiation and
1502
CANCER April 15,1996 I Volume 77 / Number 8
chemotherapy in comparison with squamous cell carcinoma of the uterine c e ~ ~ i x . ’ Previously,
”’~
we established
a new human cell line, SiSo, from a patient afflicted with
uterine cervical adenocarcinoma to study the biologic
characteristics of this d i ~ o r d e r .In
’ ~this report, we present
a new MoAb, 22-1-1, against a novel tumor-associated
antigen by immunizing mice with SiSo cell line. We investigated the tissue distribution and biologic property of
22-1-1 Ag and compared its characteristics with other
tumor-associated antigens such as CA 125, CA 19-9, and
carcinoembryonic antigen (CEA) using immunohistochemical analysis.
MATERIALS AND METHODS
Cell Lines
The human tumor cell lines used in this study (Table
1) and a mouse myeloma cell line, X63. Ag8. 653, were
maintained in a complete culture medium, RPMI- 1640,
with fetal calf serum (Whittaker Bioproduct, Walkersville,
MD) at 10% volume (of solute) per volume (of solvent)
containing L-glutamine (1 rnM; Flow Laboratory, Irvine,
Scotland), ,f?-mercaptoethanol (5 x
Gibco Laboratories, Grand Island, NY), N-2-hydroxyethylpiperazineN’-2-ethanesulfonic acid (Hepes) (pH 7.2) (10 mM;
Gibco), nonessential amino acids (0.1 mM; Gibco), and
sodium pyruvate (1 mM; Gibco).
Immunization and Cell Fusion
We previously established the SiSo cell line from a uterine
cervical adenocarcinoma ~ a t i e n t . Balb/c
’~
mice (6 weeks
old and female) were immunized subcutaneously with 1
x lo6 SiSo cells immersed with 0.5 mL of a complete
adjuvant, Titer Max (CytRx, Norcross, GA). After 2 weeks,
1 x lo6SiSo cells mixed with incomplete Freund adjuvant
(Organon Teknika, Turnhout, Belgium) were injected intraperitoneally for the second immunization. For a boost,
sonicated 1 x lo6 SiSo cells in 1 mL phosphate-buffered
saline (PBS) (pH 7) were used. Four days after the boost,
spleen cells were fused with X63. Ag8. 653, at the ratio of
5:l (spleen cells:X63), using polyethylene glycol 4000. For
monoclonal antibody screening, SiSo and other human
tumor cell lines were stained by indirect immunofluorescence with the culture supernatant fluids of growing
hybridoma colonies. Positive hybridomas were subcloned
by limiting dilution. Supernatant fluids from cloned hybridomas were screened by indirect immunofluorescence
for binding to uterine or ovarian carcinoma but not to
other organ tumor cell lines. One positive clone was obtained and termed as 22-1-1. 22-1-1 MoAb was defined
as an IgM class antibody.
lrnmunofluorescence and Flow Cytometry
The 22- 1-1Ag distribution on tumor cell lines was investigated by flow cytometric analysis. Briefly, each tumor cell
TABLE 1
Expression of 22-1-1 Antigen on Human Tumor Cell Lines
Cell line
Pathologic diagnosis
SiSo
TMCC
CAC-1
HeLa
MH
Kuramochi
MCAS
KF
HRA
KK
HAC-2
PA-1
TK-nu
BeWo
K562
Daudi
CCRF CEM
MM-8
MM-28
MM-33.1
KB
HEp-2
PC-10
GoTo
ZR-75- 1
PC-9
A-549
MKN-45
Kato 111
Panc-1
Chang liver
H1
CoLo 320DM
CoLo 699
LOVO
HT-1197
Uterine cervical adenocarcinonia
Uterine cervical adenocarcinoma
Uterine cervical adenocarcinoma
Uterine cervical adenocarcinoma
Ovarian mucinous adenocarcinoma
Ovarian mucinous adenocarcinoma
Ovarian mucinous adenocarcinonia
Ovarian serous adenocarcinoma
Ovarian serous adenocarcinoma
Ovarian clear cell carcinoma
Ovarian clear cell carcinoma
Ovarian undifferentiated carcinoma
Ovarian undifferentiated carcinoma
Choriocarcinoma
Erythroid leukemia
Burkitt lymphoma
Lymphoblastic leukemia
Malignant melanoma
Malignant melanoma
Malignant melanoma
Oral epidermoid carcinoma
Laryngeal epidermoid carcinoma
Lung squamous cell carcinoma
Adrenal neuroblastorna
Breast cancer
Lung adenocarrinoma
Lung adenocarcinonia
Gastric cancer
Gastric cancer
Pancreatic cancer
Liver cell
Cholangioma
Colon cancer
Colon cancer
Colon cancer
Bladder cancer
22-1-1 A$
t:positive, -: negative.
“The 2-1-1 Ag expression on tumor cell lines was detected by flow cylornetric analysis
was harvested upon mild trypsinization, treated by 0.25%
tqpsin with 0.5 mM ethylene diamine tetra acetic acid
(EDTA),and 1 x lo5 cells were suspended in a cell-staining buffer PBS containing 0.5% bovine serum albumin
and 0.05% NaN, with 10% goat serum for 20 minutes.
The cells were then incubated with 100 yL of 22-1-1 hybridoma culture supernatant fluid on ice for 45 minutes.
After washing 3 times, the cells were incubated with fluorescein isothiocyanate-conjugated goat antimouse IgM
(Cappel Laboratories, West Chester, PA) antibody on ice
for 45 minutes. The cells were washed 3 times with the
staining buffer and a flow cytometric analysis was performed. At the same time, the reactivities of YH 206
MoAb,“ GA 733 M o A ~ , ‘ ~and
, ’ ~CA 125 MoAblg against
the cell lines were examined. Stained cells were analyzed
A Novel Human Tumor-Associated Antigen/Sonoda et al.
1503
TABLE 2
22-1-1 Monoclonal Antibody Reactivity against Neoplasms in Female
Genital Organs
Pathologic diagnosis
No. of cases
IJterine cervixh
Squamous dysplasia
Squamous cell carcinoma in situ
Microinvasive squamous cell carcinoma
Invasive squamous cell carcinoma
lnvasive adenocarcinoma
10
4
2
57
Uterine corpus
Endometrial adenocarcinoma
FIGURE 1. Adenocarciiioma of the uterine cervix. The 22-1-1 Ag existed
both in the cytoplasm and on the membrane of cancer cells. Moreover,
22-1-1 Ag was detected in the glandular lumen of the tumor cells (magnification XIOO).
t)y the Coulter Epics flowcytometry Epics Elite (Coulter,
Iiialeah, FL). MoAb YH 206, GA 733, and CL4 125 were
provided by Dr. Akira Yachi (Sapporo IJniversity School
o f Medicine, Sapporo, Japan), Dr. Tenon Steplewski (The
Wistar Institute. Philadelphia, PA), and Dr. Keigo Endo
( ( ;unnia Ilniversity School of Medicine, Gunma, Japan),
respt:ctively.
lrnrnunohistochemical Examination
For immunohistochemical analysis, streptoavidin-biotin
inethodology (IIistofine SAB-PO Kit; Nichirei, Tokyo, Japan) was used for formalin fixed and paraffin embedded
~pecin~ens.2"'The
22-1-1 Ag was localized with a 1 2 0 dilution of hybridoma culture supernatant fluid. Immunohistoclicniical analysis of other tumor-associated antigens,
iricluding CA 125, C A 19-9 and CEA, was also performed.
(:A 19-9 and CEA MoAbs were purchased from Nichirei
(Tokyo, Japan). Paraffin embedded tissue blocks of nongynecologic cancer were provided by Dr. Masazumi Tsuneyoshi (Kyushu University, Fukuoka, Japan). Tissue sections with less than 5% reactive tumor cells were considcrcd negative, and those with more than 5% reactive cells
was defined as positive.
Chernilurninescent Western Dot Blotting
I o tlctcct secreted 22-1-1 antigen in cell culture supernatant fluid, chemiluminescent Western dot blotting was
performed. Briefly, each sample was applied to a nitroceliulosc~filter by a blotting apparatus (Rio-Rad, Richmond,
(;A). The sample was fixed on the filter by air-drying. After
3% hydrogen peroxide (H,O,) treatment to remove a n
endogenous peroxidase activity (10 minutes at room temperature). the filter was soaked in 5% nonfat milk in TrisI~
Ovaryh
Mucinous cyst adenocarcinoma
Serous cyst adenocarcinorna
Clear cell carcinoma
Endometrioid adenocarcinonia
Total of ova:ian carcinomas
No. of
positive caw'
0 10%)'
64
2 (50%)
2 (100%)
50 (87.7%)
56 (87.5%)
103
64! (66%)
7
5
1
3
4
4
4
17
1
10 (58.8%)
For immunohistochemiral analysis, streploavidin-biotinmerhodology was tised for formalin tixed
and paraffin embedded specimens.Tissue sections with greater than 5% reaclive cells were defined
as positive.
"Nonrancrrous legions of uterine cenir or ovary \ w e negative for 22-1-1 ~ g .
' Kuniber in parenrhesisis he percenrage of positive c a w
buffered saline-Tween 20 (TBS-T) containing 5% normal
goat serum (45 minutes at room temperature). The filter
was incubated with 22-1-1 MoAh in TBS-I' containing 5%
normal goat serum for one hour at room temperature.
After washing 3 times with TBS-T, the filter was incubated
for 1 hour with peroxidase conjugated goat antimouse
IgM antibody (Cappel Laboratories, West Chester, PA).
After washing 3 times with TBS-T, the filter was soaked
with enhanced chemiluminescence kit substances (Ametsham, Buckinghamshire, United Kingdom) and exposed
for 15 minutes.
Radioimrnunoprecipitation
To investigate the molecular weight of 22-1-1 Ag, metabolic-radioactive labeling method was performed. Briefly,
SiSo, Panc- 1, and Co1.0 699 were collected by mild trypsinization. After washing twice with PBS, each tumor cell
was resuspended at a concentration of 5 x 10"cells/mL in
3 mL of methionine-free HPMI 1640 (Gibco I.aboratories,
Grand Island, NY) containing 10% fetal calf serum dialyzed against PBS. After adding 100 pCi of I"'SJmethionine, the cells were incubated for 16 hours at a
humidified 37 "C, 5% C 0 2 incubator. Labeled cells were
collected and washed twice with ice-cold PRS. Each labeled cell pellet was incubated with a cell lysis buffer (15
mM nonanoyl-n-methyl-glucamide [Mega-g], 150 mbl
NaCI, 5 mM EDTA, SO mM l'ris-HC1 IpH 7.51, 0.5% noni-
1504
CANCER April 15,1996 / Volume 77 / Number 8
TABLE 3
22-1-1 Monoclonal Antibody Reactivity against Genital Organ Tissues
of Healthy Females
Tissue
No. of cases
No. of positive cases
Uterine cervix
Uterine endometrium
Proliferative phase
Secretory phase
Atrophic endometrium
Ovary
5
0
3
3
4
5
la
3a
Id
0
Only endometrial gland cells were weakly stained.
FIGURE 2. lmmunohistochemical analysis of the uterine cervix by 221-1 MoAb. Normal uterine cervical tissue is negative for 22-1-1 Ag A:
squamous epithelium, B: cervical gland cells. (Original magnification
X200).
det P-40 [NP-401,and 2 mM phenyl methyl sulfonyl fluoride [PMSFI) for 15 minutes at 4 "C. The cell lysate was
incubated with 22- 1- 1 MoAb, after which goat antimouse
IgM antibody (Cappel Laboratories West Chester, PA)
and, finally, protein A beads (Pierce, Rockford, IL) were
added (1 hour at 4 "C, respectively). The eluate was electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced
and nonreduced conditions.
Enzyme Treatment
Partial purified 22-1-1 Ag from culture supernatant fluid
by ammonium precipitation was treated by trypsin (Difco
Laboratories, Detroit, MI), neuraminidase (Sigma, St.
Louis, MO), and 0-glycanase (Genzyme, Cambridge, MA).
Briefly, 22-1-1 Ag was incubated with trypsin at concentrations from 0.01 to 10 mglmL (20 hours at room temperature). After incubation, 22- 1- 1 Ag was transferred onto
nitrocellulose filters for Western blot analysis. 22- 1- 1 Ag
was then treated by 0-glycanase (4 mUl100 pg protein
of sample) in 10 mM calcium acetate, and 20 mM sodium
cacodylate solution (pH 6.8) (20 hours at 37 "C). After,
22-1-1 Ag was treated by neuraminidase (20 mUlmL) in
0.1 M sodium acetate buffer (pH 5) (4 hours at 37 "C).
SiSo cells were treated by neuraminidase (Sigma, St.
Louis, MO), hyaluronidase (Sigma),tunicamycin (Sigma),
and N-acetyl a - D galactosaminidase (Sigma), after which
the expression of 22- 1-1Ag was analyzed by flow cytometry. Briefly, SiSo cells were treated by 10-1000 UlmL of
hyaluronidase with 500 UlmL of aprotinin in HEPESbuffer saline (30 minutes at 37 "C). SiSo cells were then
cultured with tunicamycin (0- 10 pg/mL, for 24 hours and
96 hours). After SiSo cells were incubated with 5 mM of
N-acetyl a-D galactosaminidase in culture medium (36
hours at 37 "C). Then, 1 x 10' SiSo cells were treated by
neuraminidase (100 mU/mL) in HEPES-buffer saline (20
mM of HEPES, and 140 mM of NaCl [pH 71) at 37 "C for
30 minutes.
RESULTS
22-1-1 Ag Distribution on Human Tumor Cell lines
The 22-1-1 MoAb reacted with tumor cell lines derived
from uterine cervical adenocarcinoma and ovarian adenocarcinoma, such as SiSo, TMCC, MH, Kuramochi, KF,
and HRA. However, it did not react with any tested tumor
cell lines that were derived from other organs (Table 1).
In comparison with MoAbs reactive to other tumor-associated antigens, such as YH 206, GA 733, and CA 125, the
distribution pattern of 22-1-1 Ag was significantly different (data not shown). Additionally, Kato 111, which expressed CEA and sialyl Le" molecules, was negative for
22-1-1 Ag. Therefore, the 22-1-1 Ag was distinct from YH
206, GA 733, CA 125, CEA, and sialyl Le" molecules.
Tissue Distribution of 22-1 -1 Antigen
Immunohistochemical analysis with 22-1- 1 MoAb was
performed using paraffin embedded specimens. 22-1- 1
Ag existed both in the cytoplasm and on the membrane of
the adenocarcinoma cells derived from the uterine cervix.
Moreover, 22-1-1 Ag was detected in the glandular lumen
of the cancer cells, indicating that 22-1-1 Ag was also
secreted (Fig. 1).
The 22-1-1 MoAb reacted with 87.5% of the cases
with uterine cervical adenocarcinomas, 66% of the cases
with uterine endometrial adenocarcinoma, and 58.8% of
the cases with ovarian carcinoma (Table 2). The 22-11 MoAb also reacted with adenoma malignum (highly
differentiated adenocarcinoma) of the uterine cervix.
With regard to ovarian carcinomas, 22-1-1 MoAb stained
mucinous adenocarcinomas stronger than serous or endometrioid adenocarcinomas. 22- 1-1 Ag was detected not
A Novel Human Tumor-Associated Antigen/Sonoda et al.
1505
TABLE 4
Reactivities of Various Monoclonal Antibodies against Uterine
Carcinomas
MoAb
22-1-1
0 125
CA 19-9
CFA
Uterine cervical squamous
cell carcinoma
Uterine cervical Uterine endometrial
adenocarcinoma adenocarcinoma
50/57 (87.7%)a
56/64 (87.5%)
27/40 (67.5%)
29/40 (72.5%)
36/40 (90%)
4/10 (40'%)
5/10 (50%)
6/10 (8O'?h)
681103 (66%)
30140 (75%)
19/40 (47.5%)
10/40 (25%)
C U : carcinoembrponic antigen.
Numbers of positive cases of numbers of total cases. Nuinbers in parentheses are percent positivity.
a
FIGURE 3. The secretion of 22-1-1 Ag to the cell culture supernatant
fluid. The supernatant fluid of each cell line at the indicated initial cell
number and culture period was applied to a nitrocellulose filter. Filters
were incubated with 22-1-1 MoAb followed by peroxidase conjugate goat
antimouse IgM. Detection of 22-1-1 Ag was performed with a chemiluminescent substance.
only in adenocarcinomas of the female genital tract, but
also in 87.7% of cases with uterine cervical squamous cell
carcinoma. With regard to uterine cervical squamous cell
neoplasms, 22-1-1 MoAb did not react with squamous
dysplasia but it strongly stained the cytoplasm of microinvasive arid invasive carcinoma cells (Table 2). 22-11 MoAb reacted weakly with normal uterine endometrial
gland cells. However, no reactivity was observed in the
normal uterine cervical squamous epithelium, cervical
gland cells (Fig. 2), or ovarian tissues (Table 3).
The 22-1-1 MoAb reactivity was compared with that
of CA 125, CA 19-9, and CEA MoAbs in uterine cancers
(Table 4). 22-1-1 Ag was expressed in a comparatively
broader and stronger manner in uterine cancers than
were other tumor-associated antigens.
The Secretion of 22-1-1 Ag to Culture Supernatant Fluid
The culture supernatant fluids of SiSo, HeLa, and CAC-1
were examined in the secretions of 22-1-1 Ag (Fig. 3). 221-1 Ag was detected in the culture supernatant fluids of
FIGURE 4. The 22-1-1 Ag was detected in the vaginal discharges iof
uterine cervical cancer patients. The vaginal discharges were collected
from healthy donors and cancer patients. Protein concentration of samples
was determined by optical density at 280 nm and was adjusted to O.!%
100 p,L of each indicated diluted sample was used for Western blot anal!/sis. The vaginal discharge samples were collected from healthy donors
(lanes 1 to 8), a uterine cervical squamous cell carcinoma patient (lane
9), and uterine cervical adenocarcinoma patients (lanes 10 and 11). Lanes
12 and 13 contain the culture supernatant fluids of SiSo cells and phosphate-buffered saline, respectively.
FIGURE 5. The molecular size of 22-1-1 Ag was estimated. Each cell
line was labeled metabolically with [35S]-methionine.22-1-1 Ag was immunoprecipitated and analyzed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under reduced and nonreduced conditions. Cell line 1:SiSo; 2:Panc-1; 3:CoLo 699.
SiSo cells that expressed 22-1-1 Ag and the amount of
secreted 22-1-1 Ag was found to increase depending on
the culture period and cell number. However, 22-1-1 Ag
was not found in the supernatant fluids of HeLa and CAC.1 in which 22-1-1 Ag was negative.
Moreover, 22- 1- 1 Ag was also detected in the vaginal
discharges of uterine cervical cancer patients, but not in
normal healthy donors (Fig. 4).
Molecular Size of 22-1-1 Ag
The molecular size of 22-1-1 Ag was assessed using SiSo
cell lysates after [%I -metabolic labeling. Molecules of apparently 78 kilodaltons in weight were detected in SiSo
cell lysates under both reduced and nonreduced conditions of sodium dodecyl sulfate-polyacrylamide gel elec-
1506
CANCER April 15,1996 / Volume 77 / Number 8
TABLE 5
Sensitivity of 22-1-1 Antigen to the Treatment by Various Enzymes
Enzyme treatment
Condition for reaction
Substrates
Sensitivity of 22-1-1 antigen
Trypsid
Hyaluronidaseb
Tunicamycinb
O-glycanasea
N-acetyl IY-Dgalactosaminidaseb
Neuraminidase'
0.01-10 mglmL, 20 hr, RT
10-1000 UlmL, 30 min, 37 'C
10 mglmL, 24-96 hr, 37 'C
4 mu,20 hr, 37 'C
5 mM,36 hr,37 'C
20-100 mUlmL, 37 'C
Protein
N-linked glycan
K-linked glycan
O-linked glycan
O-linked glycan
Sialic acid
Sensitive
Resistant
Resistant
Resistant
Resistant
Resistant
hr: hour; min: minute; R T room temperature.
Partially purified 22-1-1 antigen from culture supernatant fluid of SiSo cells was treated with each enzyme and Western dot blotting was performed using 22-1-1 MoAb.
SiSo cells were incubated with each enzyme and the cell surface expression of 22-1-1 antigen was then analyzed by flow cytomey.
'The partially purified 22-1-1 Ag was treated with neuraminidase for 4 hours and analyzed by Western dot blotong. SiSo cells were also treated with neuraminidase for 30 minutes and the evpression of 22-1-1
Ag on the cell surface was analyzed by flow cytornetiy.
a
trophoresis; however, they were not seen in lysates of
Panc-1 and CoLo 699 cells that were not positive for 221-1 Ag (Fig. 5). Moreover, affinity purified 22-1-1 Ag from
SiSo cell culture supernatant fluids was also shown to
have the same molecular weight of 78 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(data not shown).
Sensitivity of 22-1-1 Ag Against Enzyme Treatment
The sensitivity of 22-1-1 Ag against various enzymes was
investigated (Table 5). 22-1-1 Ag was shown to be sensitive to trypsin treatment, because 22-1-1 antigenicity was
scarcely detected after trypsin digestion in a concentration-dependent manner (data not shown). Conversely,
22-1-1 Ag was resistant to treatment with hyaluronidase,
tunicamycin, O-glycanase, N-acetyl a - D galactosaminidase, and neuraminidase.
DISCUSSION
To date, many MoAbs have been generated in the field
of cancer research and the utility of these MoAbs has
been extensively investigated. MoAbs such as CA 125, CA
19-9, CEA, and alpha-fetoprotein have been used to detect tumor-associated antigens in the serum of cancer
patients after treatment. Radioisotope-conjugated MoAbs
have been employed to detect tumor tissues of primary
sites and m e t a s t a s e ~ . * ~
~~
A-number
of cancer therapies
using MoAb specific for tumor-associated antigens have
been attempted. In addition to the direct effects of MoAbs
by antibody-dependent cellular cytotoxicity and complement-dependent cellular c y t o t o x i ~ i t y , ~anticancer
~-~~
drugs- or radionuclides- conjugated MoAbs have been
applied in the treatment of ~ a n c e r . ~Moreover,
*-~~
several
MoAbs have been manufactured for immunohistologic
discrimination of benign versus malignant cells in histologic and cytologic preparation^.^^-^' These MoAbs also
have been used to examine the origin, growth profile, and
function of tumor
In the case of gynecologic neoplasms, several MoAbs
have been reported to be useful. B72.3 and Ca 1 MoAbs
were reactive with both endometrial hyperplasias and
carcinoma^.^'^^^ CA 1, HMFG 1, and HMFG 2 MoAbs were
found to be most reactive with immature metaplasia as
opposed to squamous cell carcinoma^.^^ CEA MoAb has
been shown to be of value both in the discrimination of
ovarian primary adenocarcinomas from metastatic adenocarcinomas of colonic origin and in the differentiation
of endocervical versus endometrial adenocarcinoOC 125 MoAb was reported to recognize a majority of serous, clear cell, endometrioid, and undifferentiated ovarian tumors, but not mucinous, sex cord, or germ
cell t ~ m o r s .MSN-1
~ ~ . ~MoAb
~
has been shown to be useful
in confirming endometrioid or clear cell differentiation
in ovarian and endometrial tumors and in distinguishing
between atypical endometrial hyperplasias and those
without a t ~ p i a . ~1C5
' MoAb, which reacted with various
adenocarcinomas derived from the female genital tract
but not with squamous cell carcinomas, was reported to
be an improved tool for the detection of adenocarcinomas of the uterine cervix through tissue imprints and
cervical smears.48
We produced 22-1-1 MoAb from mice immunized
with SiSo cells derived from uterine cervical adenocarcinoma. Although 22- 1- 1 MoAb reacted weakly with normal
glandular cells of the uterine endometrium, it was not
detected in the normal uterine cervix or ovarian tissues.
On the contrary, 22-1-1 MoAb strongly stained uterine
cervical adenocarcinomas (87.5%), uterine endometrial
adenocarcinomas (66%), and ovarian adenocarcinomas
(58.8%).Moreover, 22-1-1 MoAb was shown to react with
squamous cell carcinomas of the uterine cervix (87.7%).
In addition, there is a possibility that 22-1-1 Ag expression
A Novel Human Tumor-Associated Antigen/Sonoda et al.
1507
TABLE 6
Reativities of the Combination of 22-1-1 MoAb with Any One of Either CA 125, CA 19-9, or CFA MoAbs
-
MOW
Uterine cervical squamous
cell carcinoma
Uterine cervical
adenocarcinoma
Uterine endometrial
adenocarcinoma
22-1-1 \nth CA 125
22-1-1 mth CA 19-9
22-L-1 wth CM
9\10
9/10 (90%)
lO/lO (100%)
38140 (95%)
39/40 (97.5%)
38/40 (95%)
35/40 (87 5%)
28/40 (70%)
23/40 (57 5%)
-
-
CEA carcinoenibryonic antigen
Numbers of positive cases of numbers of total cases Numbers in parenthesis are percent posltivlty
is related to a neoplastic progression, because 22-1-1 Ag
is detectable in rnicroinvasive and invasive squamous cell
carcinomas but not in dysplasia of the uterine cervix (Table 2). In nongynecologic cancers, 22-1-1 Ag was detected
in esophageal squamous cell carcinomas (two positive
cases of two total cases), gastric adenocarcinomas (three
positive cases of three total cases), colon adenocarcinomas (three positive cases of three total cases), and pancreatic adenocarciriomas (two positive cases of two total
cases), indicatiq that 22-1-1 Ag exists widely in various
cancer tissues.
We compared the tissue distribution of 22-1-1, CA
125, CA 19-9, and CEA Ag in uterine cancers. As reported
previo~sly,"".~~~'
CA 125 MoAb showed a significant reaction with uterine adenocarcinomas (67.5-75%), but
hardly any with squamous cell carcinomas (as shown in
Table 4). CA 19-9 MoAb was reported to react with 80.392.5% of uterine endometrial adenocarcinomas."~5' However, in OUT study, CA 19-9 Ag was detected in only 47.5%
of endometrial adenocarcinomas, whereas 22- 1- 1 Ag existed in 87% of uterine cervical carcinomas and in 66% of
endometrial adenocarcinomas. Therefore, 22- 1-1 MoAb
appears to react with uterine cancer cells more broadly
than CA 1;!5 and CA 19-9 MoAbs. CEA MoAb has been
shown to he of use in the differentiation of endocervical
versus endomei rial adenocarcinomas, because CEA
MoAb reacted with uterine cervical adenocarcinomas
more strongly 1 han with endometrial adenocarcinoma^.^^-^^ Mle found this to be true in our study as well.
(Table 4). With regard to squamous cell carcinomas, CEA
Ag was detected in 80% of tumor tissues, with this reactivity being slightly higher than that of a previous report.53
In addition, the combination of 22-1-1 MoAb with any
one of either CA, 125, CA 19-9, or CEA MoAb detected
greater than 90% of uterine cervical squamous cell carcinomas (Table 6). The combination of 22-1-1 MoAb with
CA 19-9 MoAb stained 97.5% of the cervical adenocarcinomas, and 22-1-1 MoAb combined with CA 125 MoAb
reacted with 87 5% of endometrial adenocarcinomas.
This data indicates that when 22-1-1 MoAb is combined
with one of the other MoAbs, it can detect cancer cells
more efficiently than any other MoAb alone. This might
be of value in diagnosing uterine cancer tissues.
In preliminary experiments, 22-1-1 Ag was detected
in the vaginal discharges of uterine cervical cancer patients, but not in that of normal healthy donors (Fig. 41,
indicating that 22-1-1 MoAb can be used in the diagnosis
of uterine cancer with vaginal discharge. This clinical relevance of 22-1-1 MoAb is now under investigation.
From flow cytometric analisis data, 22-1-1 Ag was
shown to be distinct from YH 206, GA 733, CA 125, CEA,
and sialyl Le" molecules. The tissue distribution of 221-1Ag was also different from those of CA 125, CA 199, and CEA antigens. Moreover, there is a possibility
that 22-1-1 Ag expression is related to a neoplastic progression. There have been no previous reports of a 78kilodalton tumor-associated antigen secreted in the
vaginal discharges of uterine cancer patients that dlemonstrates these characteristics. This evidence indic,ates
that 22-1-1 Ag is a novel tumor-associated antigen. Further investigations, including gene cloning and detection of 22-1-1 Ag in the serum of cancer patients, are
now in progress.
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