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A and B blood-group antigens in continuous cell lines
A and B blood-group
antigens
in continuous cell lines
205
9. Hsu, T. C. and LIU, T. T., Evolution 2, 49 (1948). KEYL, H. G., Chromosoma 8, 739 (1957). LEWIS, E. B., Cold Spring Harbor Symp. Quanf. Biol. 16, 159 (1951). LEWONTIN, R. C. and HUBBY, J. L., Genetics 54, 595 (1966). MCCARTHY, M. D., Am. Nat. 79, 228 (1945). METZ, C. W., J. Heredity 26, 177 (1935). ___ Genetics 22, 543 (1937). PANITZ, R., Nafurwissenschaffen 47, 359 (1960). __ Chromosoma 17, 199 (1965). PAVAN, C., Brookhaven Symp. Biol. 18, 222 (1965). RITOSSA, F. M., ATWOOD, K. C., LINDSLEY, D. L. and SPIEGELMAN, S., Natl Cancer Inst. Monograph 23, 449 (1966). 20. SCHREIBER, G. and GUEDES, A. S., Rev. Brasil. Malarial. Doencas Trop. 11, 97 (1959) abstract. 21. WELSHONS, W. J., Science 150, 1122 (1965).
10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
A AND B BLOOD-GROUP D. FRANKS,’ Department
ANTIGENS
ROSEMARY
IN CONTINUOUS
LISKE
CELL LINES
and B. W. GURNER
of Pathology, University of Cambridge,
Cambridge, England
Received May 8, 1967
THEA
and
B blood-group
distinguishing
between
tions on several
human
several
other
strated
on human
species.
agglutination however,
when
(rabbit);
swine kidney; Although were made possibility
from
cell lines
from
alleged.
ATCC
tests
were
Consequently
cells
and on cell lines from
sera known (Table
can be demon-
to give strong
I). A and
of animal CRP
cells were
BHK
rabbit
can,
II), using
found
21 (Syrian
(cottontail
mixed
B antigens
of other species (Table
These included
hamster);
without
grown
in this laboratory,
immediately
after
of a cell line with on cell lines
confirmed
of the widespread it is essential
A nor B antigens
had not been cultured
the cultures
always
cell strains,
to aid in
of investiga-
to be
hamster); papilloma);
(bovine).
cells which
Similarly,
diploid
cultures
clone A (Chinese
of contamination
In view
sera. Several
for markers the results
neither
or B-positive
ovary
were
describes
and anti-B
for A and B antigens.
Collection
candidates
on tissue cells of a variety
and anti-B
routinely.
[l, 5, lo],
A-positive
and LCLN
with
that
tested
some
confirmed
received
cell lines and human
cell lines, using anti-A
with
anti-A
negative
are obvious
cell lines. This paper
It will be shown
be demonstrated
the same RK7
antigens
human
by
they
any growth
the results
receipt.
from
the
cells taken
were
Stocks
were received,
one of the other
obtained
testing
cells
to eliminate
cell lines being
American
directly
always
of frozen
Type
from
the
grown Culture
an ampoule
in this laboratory.
contamination
in this type
after
of study
of cell lines with of prove
all of the cells were tested
cells from
other
species
the cells do come from the species
by mixed
agglutination
with
specific
anti-mouse and anti-human sera, and also with a specific antiserum against the species from which the cell line was alleged to be derived, if this was other than mouse 1 Beit Memorial Research Fellow. Experimental
Cell Research 48
D. Franks,
Rosemary
Liske and B. W. Gurner
or human. A cell line was studied further only if it gave positive reactions with the antiserum against the species from which the line was derived, and a negative reaction with all other antisera with which it was tested. In recent reports it has been claimed by Hagiwara [6] and by Chessin, Bramson, Kuhns and Hirschhorn [2] that a human cell line, the FL line, contains B-positive cells. In the present study, FL cells were obtained from the American Type Culture Collection, and were tested with a battery of anti-B sera known to give strong mixed agglutination. The FL cells did not give mixed -agglutination with any of these antisera. Neither Hagiwara nor Chessin et al. gave details of any tests to confirm that
TABLE
I. Human cell lines and strains Cell
line
Human
diploid
cell strains
P. Jacobs, M.R.C., Holly London 14 15 17 17
Hill,
G. Christofinis, Glaxo Laboratories
cell lines
AV, (CCL 21) Detroit-6 (CCL 3) Detroit-6, clone 12 (CCL Linaker MS Detroit 98 (CCL 18) Detroit 532 (CCL 54) Hela-NCTC 3950 Hela-NCTC 3952 Hep 2 (CCL 23) Hela (CCL 2) Minnesota EE (CCL 4) Hela 229 (CCL 2.1) Intestine 407 (CCL 6) Burkitt lymphoma EBl Chang livier (CCL 13)
Experimental
were not found.
Source
s44 MRC-I Emerson-I Hill II P58 II WI-38 WI-26 Glaxo HEL Glaxo HEL Glaxo HEL Glaxo HEK Aneuploid
in which A and B antigens
Cell Research
48
3.1)
ATCC ATCC ATCC A. P. Wyatt D. Hobson; K. G. Brand; ATCC ATCC Nat1 Cancer Inst. Nat1 Cancer Inst. ATCC ATCC ATCC ATCC ATCC M. A. Epstein ATCC
Beale
A and B blood-group
antigens
in continuous
207
cell lines
the FL cells which they were using were human cells. However, tests with specific antisera were subsequently done by Chessin et al. (personal communication) and it is claimed that these showed that the “FL” cellswere human cells. Since the “FL” cell line used by Chessinet al. is no longer available, neither the presenceof B antigen on these cells nor their speciescan be confirmed. It was also claimed that the number of cells which were B-positive increased when the cells were grown in tissue-culture medium containing additional amounts of the constituent sugars of the blood-group substances.FL cells obtained from the ATCC were therefore grown in medium containing additional sugars in concentrations ranging from 0.05 to I ymol/l, and also in medium in which the galactose content was increased to $56 millimols/l and the glucose was omitted. None of these treatments led to the appearance of B-positive cells. TABLE
II.
Subculturenumber
Cellline RA/McCarthy RK ll/Christofinis RK 13/Christofinis RK 13/Christofinis RK 13/Christofinis RK 13/Kelus RK 3/Beale RK 7/Christofinis lGR/Daniel lGH/Daniel lGC/Daniel LM/Merchant C,LM/Merchant LMat,C,/Merchant C,LM/Merchant C3H-MS2/Daniel C3H-Sl/Daniel CJH-Tl/Daniel C57-SS/Daniel BSC-I/Christofinis Vervet VJ/Beale MDCK/Stulberg DK-Kenya/Beale YRGN/ EMK-lOl/Hayflick BSC-l/Hayflick - , Antigen
Cell lines in which either A or R anfigen
About
50 64 25 83 169
Unknown 44 Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown 64 &104 Unknown Unknown Unknown Unknown Unknown absent;
+ , Antigen
present
Species Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rat Rat Rat Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Vervet Vervet
has been found.
B
-
M + Mt Mt M + M+ M+ M+ -
M+ M+ M+ M+ M i monkey monkey
Dog Dog Chinese hamster Rhesus monkey Vervet monkey on all cells; M + , Antigen
Cell line references
A
M + M + + + + M + M + present Experimental
M+ M+ M+ + M + M+ M+ + M + M + M+ M+ M+ + + + M+ M+ on some cells. Cell Research
48
D. Franks,
208
Rosemary
Liske and B. W. Gurner
It has also been claimed by Moor-Jankowski [9] that the human A blood-group antigen was present on the AHF/adult human fibroblast, ERK, and KB cell lines. The AHF cell line is no longer available, but it has not been possible to confirm MoorJankowski’s findings with the ERK and KB cell lines. Moor-Jankowski did, however, show that the B antigen was present on cells of the MCN cell line, and that this was in TABLE
III.
Tests
for
the
presence
of
l3 antigen on Cell
FL
(CCL
62) cells.
lines
L-i Antisera
(humaFCCL62)
m+
-
m+
-
-
m+
(Waj.)
-
-
mf
anti-human
+
+
(Str.)
Anti-B
(Ha.)
Anti-B
(Hu.)
Anti-B Rabbit
Antigen present
-
(mouse)
-
Anti-B
-, Antigen
(bnE&)
absent; +, Antigen on some cells.
present
on
all
cells;
m +,
fact a mouse cell line, and not a human line. The presence of B antigen has also been demonstrated or rhesus-monkey cell lines [4, 81. The absence of A and B antigens from established human cell lines might be expected from the observations 13, 71 that blood-group antigen disappears at an early stage during the development of cultures from human tissues of known ABO blood group. It is interesting to find that A and B antigens are not found on cells of human diploid cell strains, and this suggests that it is not simply the change to aneuploid which causes the disappearance of A or B antigen. We gratefully acknowledge the receipt of grants from the National Health, U.S. Public Health Service.
Institutes
of
REFERENCES 1. BRAND, K. J. and SYVERTON, J. T., J. Nat2 Cancer Inst. 28, 147 (1964). 2. CHESSIN, L. N., BRAMSON, S., KUHNS, W. J. and HIRSCHHORN, K., Blood 25, 944 (1965). 3. DE CARLI, L., MODIANO, G., Nuzzo, F., DE ANDREIS, M. and AULISA, B., Atti ASS. Genef. ItaIicmo 6, 99 (1961). 4. FRANKS, D., COOMSS, R. R. A., BESWICK, T. S. L. and WINTER, M. M., Immunology 6, 64 (1963). 5. FRANKS, D., GURNER, B. W., COOMBS, R. R. A. and STEVENSON, R., Expfl Cell Res. 28, 609 (1962). 6. HAGIWARA, A., Expfl Cell Res. 28, 615 (1962). 7. HBGMAN, C. F., ExptZ CeZI Res. 21, 137 (1960). 8. KANO, K., Int. Arch. Allergy 30, 281 (1966). 9. MOOR-JANKOWSKI, J., J. genet. hum. 12, 88 (1963). 10. STULBERG, C. S., SIMPSON, W. F. and BERMAN, L., Proc. Sot. expfl Biol. Med. 108,434 (1961).
Experimental
Cell Research
48
antigens
in continuous cell lines
205
9. Hsu, T. C. and LIU, T. T., Evolution 2, 49 (1948). KEYL, H. G., Chromosoma 8, 739 (1957). LEWIS, E. B., Cold Spring Harbor Symp. Quanf. Biol. 16, 159 (1951). LEWONTIN, R. C. and HUBBY, J. L., Genetics 54, 595 (1966). MCCARTHY, M. D., Am. Nat. 79, 228 (1945). METZ, C. W., J. Heredity 26, 177 (1935). ___ Genetics 22, 543 (1937). PANITZ, R., Nafurwissenschaffen 47, 359 (1960). __ Chromosoma 17, 199 (1965). PAVAN, C., Brookhaven Symp. Biol. 18, 222 (1965). RITOSSA, F. M., ATWOOD, K. C., LINDSLEY, D. L. and SPIEGELMAN, S., Natl Cancer Inst. Monograph 23, 449 (1966). 20. SCHREIBER, G. and GUEDES, A. S., Rev. Brasil. Malarial. Doencas Trop. 11, 97 (1959) abstract. 21. WELSHONS, W. J., Science 150, 1122 (1965).
10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
A AND B BLOOD-GROUP D. FRANKS,’ Department
ANTIGENS
ROSEMARY
IN CONTINUOUS
LISKE
CELL LINES
and B. W. GURNER
of Pathology, University of Cambridge,
Cambridge, England
Received May 8, 1967
THEA
and
B blood-group
distinguishing
between
tions on several
human
several
other
strated
on human
species.
agglutination however,
when
(rabbit);
swine kidney; Although were made possibility
from
cell lines
from
alleged.
ATCC
tests
were
Consequently
cells
and on cell lines from
sera known (Table
can be demon-
to give strong
I). A and
of animal CRP
cells were
BHK
rabbit
can,
II), using
found
21 (Syrian
(cottontail
mixed
B antigens
of other species (Table
These included
hamster);
without
grown
in this laboratory,
immediately
after
of a cell line with on cell lines
confirmed
of the widespread it is essential
A nor B antigens
had not been cultured
the cultures
always
cell strains,
to aid in
of investiga-
to be
hamster); papilloma);
(bovine).
cells which
Similarly,
diploid
cultures
clone A (Chinese
of contamination
In view
sera. Several
for markers the results
neither
or B-positive
ovary
were
describes
and anti-B
for A and B antigens.
Collection
candidates
on tissue cells of a variety
and anti-B
routinely.
[l, 5, lo],
A-positive
and LCLN
with
that
tested
some
confirmed
received
cell lines and human
cell lines, using anti-A
with
anti-A
negative
are obvious
cell lines. This paper
It will be shown
be demonstrated
the same RK7
antigens
human
by
they
any growth
the results
receipt.
from
the
cells taken
were
Stocks
were received,
one of the other
obtained
testing
cells
to eliminate
cell lines being
American
directly
always
of frozen
Type
from
the
grown Culture
an ampoule
in this laboratory.
contamination
in this type
after
of study
of cell lines with of prove
all of the cells were tested
cells from
other
species
the cells do come from the species
by mixed
agglutination
with
specific
anti-mouse and anti-human sera, and also with a specific antiserum against the species from which the cell line was alleged to be derived, if this was other than mouse 1 Beit Memorial Research Fellow. Experimental
Cell Research 48
D. Franks,
Rosemary
Liske and B. W. Gurner
or human. A cell line was studied further only if it gave positive reactions with the antiserum against the species from which the line was derived, and a negative reaction with all other antisera with which it was tested. In recent reports it has been claimed by Hagiwara [6] and by Chessin, Bramson, Kuhns and Hirschhorn [2] that a human cell line, the FL line, contains B-positive cells. In the present study, FL cells were obtained from the American Type Culture Collection, and were tested with a battery of anti-B sera known to give strong mixed agglutination. The FL cells did not give mixed -agglutination with any of these antisera. Neither Hagiwara nor Chessin et al. gave details of any tests to confirm that
TABLE
I. Human cell lines and strains Cell
line
Human
diploid
cell strains
P. Jacobs, M.R.C., Holly London 14 15 17 17
Hill,
G. Christofinis, Glaxo Laboratories
cell lines
AV, (CCL 21) Detroit-6 (CCL 3) Detroit-6, clone 12 (CCL Linaker MS Detroit 98 (CCL 18) Detroit 532 (CCL 54) Hela-NCTC 3950 Hela-NCTC 3952 Hep 2 (CCL 23) Hela (CCL 2) Minnesota EE (CCL 4) Hela 229 (CCL 2.1) Intestine 407 (CCL 6) Burkitt lymphoma EBl Chang livier (CCL 13)
Experimental
were not found.
Source
s44 MRC-I Emerson-I Hill II P58 II WI-38 WI-26 Glaxo HEL Glaxo HEL Glaxo HEL Glaxo HEK Aneuploid
in which A and B antigens
Cell Research
48
3.1)
ATCC ATCC ATCC A. P. Wyatt D. Hobson; K. G. Brand; ATCC ATCC Nat1 Cancer Inst. Nat1 Cancer Inst. ATCC ATCC ATCC ATCC ATCC M. A. Epstein ATCC
Beale
A and B blood-group
antigens
in continuous
207
cell lines
the FL cells which they were using were human cells. However, tests with specific antisera were subsequently done by Chessin et al. (personal communication) and it is claimed that these showed that the “FL” cellswere human cells. Since the “FL” cell line used by Chessinet al. is no longer available, neither the presenceof B antigen on these cells nor their speciescan be confirmed. It was also claimed that the number of cells which were B-positive increased when the cells were grown in tissue-culture medium containing additional amounts of the constituent sugars of the blood-group substances.FL cells obtained from the ATCC were therefore grown in medium containing additional sugars in concentrations ranging from 0.05 to I ymol/l, and also in medium in which the galactose content was increased to $56 millimols/l and the glucose was omitted. None of these treatments led to the appearance of B-positive cells. TABLE
II.
Subculturenumber
Cellline RA/McCarthy RK ll/Christofinis RK 13/Christofinis RK 13/Christofinis RK 13/Christofinis RK 13/Kelus RK 3/Beale RK 7/Christofinis lGR/Daniel lGH/Daniel lGC/Daniel LM/Merchant C,LM/Merchant LMat,C,/Merchant C,LM/Merchant C3H-MS2/Daniel C3H-Sl/Daniel CJH-Tl/Daniel C57-SS/Daniel BSC-I/Christofinis Vervet VJ/Beale MDCK/Stulberg DK-Kenya/Beale YRGN/ EMK-lOl/Hayflick BSC-l/Hayflick - , Antigen
Cell lines in which either A or R anfigen
About
50 64 25 83 169
Unknown 44 Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown 64 &104 Unknown Unknown Unknown Unknown Unknown absent;
+ , Antigen
present
Species Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rat Rat Rat Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Vervet Vervet
has been found.
B
-
M + Mt Mt M + M+ M+ M+ -
M+ M+ M+ M+ M i monkey monkey
Dog Dog Chinese hamster Rhesus monkey Vervet monkey on all cells; M + , Antigen
Cell line references
A
M + M + + + + M + M + present Experimental
M+ M+ M+ + M + M+ M+ + M + M + M+ M+ M+ + + + M+ M+ on some cells. Cell Research
48
D. Franks,
208
Rosemary
Liske and B. W. Gurner
It has also been claimed by Moor-Jankowski [9] that the human A blood-group antigen was present on the AHF/adult human fibroblast, ERK, and KB cell lines. The AHF cell line is no longer available, but it has not been possible to confirm MoorJankowski’s findings with the ERK and KB cell lines. Moor-Jankowski did, however, show that the B antigen was present on cells of the MCN cell line, and that this was in TABLE
III.
Tests
for
the
presence
of
l3 antigen on Cell
FL
(CCL
62) cells.
lines
L-i Antisera
(humaFCCL62)
m+
-
m+
-
-
m+
(Waj.)
-
-
mf
anti-human
+
+
(Str.)
Anti-B
(Ha.)
Anti-B
(Hu.)
Anti-B Rabbit
Antigen present
-
(mouse)
-
Anti-B
-, Antigen
(bnE&)
absent; +, Antigen on some cells.
present
on
all
cells;
m +,
fact a mouse cell line, and not a human line. The presence of B antigen has also been demonstrated or rhesus-monkey cell lines [4, 81. The absence of A and B antigens from established human cell lines might be expected from the observations 13, 71 that blood-group antigen disappears at an early stage during the development of cultures from human tissues of known ABO blood group. It is interesting to find that A and B antigens are not found on cells of human diploid cell strains, and this suggests that it is not simply the change to aneuploid which causes the disappearance of A or B antigen. We gratefully acknowledge the receipt of grants from the National Health, U.S. Public Health Service.
Institutes
of
REFERENCES 1. BRAND, K. J. and SYVERTON, J. T., J. Nat2 Cancer Inst. 28, 147 (1964). 2. CHESSIN, L. N., BRAMSON, S., KUHNS, W. J. and HIRSCHHORN, K., Blood 25, 944 (1965). 3. DE CARLI, L., MODIANO, G., Nuzzo, F., DE ANDREIS, M. and AULISA, B., Atti ASS. Genef. ItaIicmo 6, 99 (1961). 4. FRANKS, D., COOMSS, R. R. A., BESWICK, T. S. L. and WINTER, M. M., Immunology 6, 64 (1963). 5. FRANKS, D., GURNER, B. W., COOMBS, R. R. A. and STEVENSON, R., Expfl Cell Res. 28, 609 (1962). 6. HAGIWARA, A., Expfl Cell Res. 28, 615 (1962). 7. HBGMAN, C. F., ExptZ CeZI Res. 21, 137 (1960). 8. KANO, K., Int. Arch. Allergy 30, 281 (1966). 9. MOOR-JANKOWSKI, J., J. genet. hum. 12, 88 (1963). 10. STULBERG, C. S., SIMPSON, W. F. and BERMAN, L., Proc. Sot. expfl Biol. Med. 108,434 (1961).
Experimental
Cell Research
48