(CANCER RESEARCH 52, 3506-3509. June 15, 1992]
Advances in Brief
Molecular Analysis of the Protein Tyrosine Phosphatase 7 Gene in Human Lung
Cancer Cell Lines1
Tetsuya Tsukamoto, Takashi Takahashi,2 Ryuzo Ueda, Kenji Hibi, Haruo Saito, and Toshitada Takahashi
Laboratories of Immunology [T. Ts., To. T.] and Chemotherapy [Ta. T., R. U.], Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan; Second
Dépannent of Surgery, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan [K. H.]; and Department of Biological Chemistry and Molecular
Pharmacology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 0211S [H. S.J
Abstract
The protein tyrosine phosphatase y (PTPy) gene has recently been
suggested as a candidate tumor suppressor gene involved in the oncogenesis of human lung and renal cancers, although no direct evidence for
PTPy mutations has been demonstrated thus far. We explored the status
of PTPy in 31 human lung cancer cell lines as well as in various other
types of human tumor cell lines. Northern blot analysis revealed that two
independent cell lines expressed PTPy mR.VVs with sizes distinct from
those in human fetal and adult normal lung. However, our extensive
search for mutations in the PTPy gene failed to identify any abnormalities
in the cytoplasmic region, which contains two protein tyrosine phosphatase-like domains. These results warrant further examination of genetic
alterations in the extracellular and transmembrane domains of PTPy,
which had not been cloned at the time of the present study.
Introduction
Allelic losses are hallmarks of chromosome regions harboring
tumor suppressor genes. Restriction fragment-length polymor
phism analyses have revealed frequent allelic loss on chromo
somes 3p, 13q, and 17p in lung cancer and have provided the
foundation for discovery of the inactivation of the retinoblastoma and p53 genes on 13q and 17p, respectively (1, 2). Al
though the 3p deletion was first reported in 1982 by a conven
tional cytogenetic study (3) and later confirmed by restriction
fragment-length polymorphism analysis by us and others (49), no solid candidate for the tumor suppressor gene(s) on 3p
has been identified yet.
The human PTPy3 gene, PTPRG, is a member of the transmembrane protein tyrosine phosphatases (PTPases), which may
play a key role in signal transduction by reversing the effects of
protein tyrosine kinases, many of which are protooncogenes
(10). Based on a reduction of the PTPy gene dosage in lung
cancer and its chromosomal assignment to 3p21, which coin
cides with the commonly deleted region on 3p in lung cancer
(8, 9). LaForgia et al. (11) have recently suggested that PTPy
might be a tumor suppressor gene and a possible target for 3p
deletion in lung cancer. However, no direct evidence for muta
tions in the PTPy gene has been demonstrated, and even PTPy
mRNA expression in human fetal and adult normal tissues has
not been clearly demonstrated thus far. Therefore, we examined
tissue distribution of PTPy expression and explored the status
of the PTPy gene in human lung cancer.
Materials
and Methods
Southern and Northern Blot Analyses. Fetal tissue at 13 weeks of
gestation was obtained from an elective abortion, and adult normal
tissue was collected from postmortem and/or surgical specimens.
Twenty-one small cell lung cancer (SCLC) and four non-small cell lung
cancer (NSCLC) cell lines with prefix (ACC-LC-) were established in
our laboratories at Aichi Cancer Center in Japan. In addition, 6 NSCLC
cell lines as well as 30 cell lines from other human malignancies which
had been contributed by various investigators were also analyzed.
Derivations and culture conditions of these cell lines have been reported
previously (12, 13). Extraction of DNA and RNA from cell lines and
tissue samples were performed as described previously (12). Using the
2.8-kilobase £coRIfragment of the HPTP723S cDNA as a probe (14),
Southern and Northern blot analyses were performed with 10 ag of
genomic DNAs and total cellular RNAs, respectively.
RNase Protection Assay. The 32P-labeled antisense RNA probes
prepared from PTPy cDNA were hybridized with 10 ng of total cellular
RNA as described previously (2). The 3 antisense RNA probes used
were: HPTP-^Dl
(EcoRl-Xhol);
HPTPTD2
(Xho\-Acc\)\ and
HPTP7UT (Accl-EcoRl).
Polymerase Chain Reaction-Single Strand Conformation Polymor
phism (PCR-SSCP) Analysis. Random-primed cDNAs were made as
described previously (15). PCR-SSCP analysis was performed as de
scribed by Orila et al. (16) with slight modification. PCR products
labeled with [32P]dCTP were digested with appropriate restriction en
zymes to yield a higher sensitivity due to their smaller size and were
then electrophoretically separated on a 6% nondenaturing polyacrylamide gel at 5°C.The sense primers used were: SI, nt 79-99; S2, nt
457-474; S3, nt 986-1006; and S4, nt 1344-1361. The antisense
primers were: ASI, nt 508-525; AS2, nt 877-897; AS3, nt 1418-1435;
and AS4, nt 1812-1832. The nucleotide coordinates were obtained
from the sequence of Krueger et al. (14).
Results and Discussion
Northern blot analysis was first performed with total RNAs
extracted from human fetal and adult normal lungs as well as
from various other tissues, since expression of the PTPy gene
Received 4/10/92; accepted 5/12/92.
should be observed in a normal counterpart of possibly affected
The costs of publication of this article were defrayed in part by the payment
tumor types. Using the 2.8-kilobase £coRI fragment from a
of page charges. This article must therefore be hereby marked advertisement in
PTPy
cDNA clone (HPTP723S) as a probe (14), 9.6- and 6.2accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by a Grant-in-Aid for the Comprehensive Ten-Year Strat
kilobase
mRNAs were detected in all tissues examined (Fig. 1).
egy for Cancer Research from the Ministry of Health and Welfare, Japan, by
Interestingly, both fetal and adult normal lungs expressed PTPy
Grants-in-Aid for Cancer Research from the Ministry of Education, Science, and
Culture and the Ministry of Health and Welfare, Japan; and by a grant from the
mRNA most abundantly among the tissues studied, suggesting
Cancer Research Institute, Inc., New York.
the possibility that inactivation of PTPy might contribute to
2To whom requests for reprints should be addressed.
3 The abbreviations used are: PTPy, protein tyrosine phosphatase y; PTPase,
the oncogenesis of lung cancer.
protein tyrosine phosphatase; SCLC, small cell lung cancer; NSCLC. non-small
We next examined mRNA expression of PTPy in 31 lung
cell lung cancer; PCR-SSCP, polymerase chain reaction-single strand conforma
cancer cell lines by Northern blot analysis (Fig. 2a). Because
tion polymorphism; cDNA, complementary DNA; nt, nucleotide.
3506
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PTP> IN HUMAN LUNG CANCER
•¿
Fetus
militili
normal lung. Furthermore, neither ACC-LC-171 nor U-2 OS,
both of which exhibited abnormal-sized mRNAs by Northern
blot analysis, showed any abnormalities, suggesting that abnor
mal splicing or deletion may be located outside the cytoplasmic
domain.
Subtle mutations in the PTPy gene were also studied by
means of PCR-SSCP analysis, because we were concerned
about the possibility that some mutations might have been
Adult •¿
mili!
-9.6
-6.2,
SCLC —¿
28SO)
c
3
18S-
en co rco co •¿Ã“ÒÓÓÓÓÓÒ
•¿NSCLC
CD o
h- co
O
O o
-f
T
-j-j-j-j-J-J-J-Jo^CJO
ÒÒÒÒÓÒÓÒV^CC-r'
ggggggggy«^*
cn tn
ß-Actin
Fig. I. Detection of PTPy transcripts by Northern blot analysis in human fetal
and adult normal tissues. Transcripts of 9.6 and 6.2 kilobases were detected in
all tissues examined, while both fetal and adult normal lungs expressed the most
abundant transcripts of the /'//'-, gene. *, shorter exposures of fetal lung and
28S-
kidney as well as adult normal lung.
18Sour previous studies on p53 inactivation in lung cancer indi
cated that mutations found in cell lines faithfully reflect those
in the corresponding primary tumors (2, 13, 15), we used cell
lines instead of primary tumor samples for this analysis to
exclude inevitable contamination of normal cells such as
stremai cells. Eight-one % (17 of 21) of the SCLC and 50% (5
of 10) of the NSCLC cell lines expressed readily detectable
mRNA 9.6 and 6.2 kilobases in size which were identical to
those in normal lung, while the remaining cell lines exhibited
greatly reduced or undetectable levels of PTPy transcripts by
Northern blot analysis. Notably, ACC-LC-171 (SCLC-SM in
our previous report) (12) expressed two distinctly sized mRNAs
(6.8 and 4.5 kilobases), suggesting either a deletion of a part of
PTPy gene or the occurrence of abnormal splicing which has
been reported as a mechanism for retinoblastoma gene and p53
inactivation (15, 17). We also examined PTPy expression in
30 cell lines from other human malignancies (Fig. 2b) and
identified an additional cell line (U-2 OS, osteosarcoma) ex
pressing transcripts (10.8 and 7.6 kilobases) distinct from the
PTPy mRNAs in either normal lung or ACC-LC-171. In the
U-2 OS cell line, expression of normal-sized transcripts was
also observed. The remaining solid tumor cell lines examined
in this study (3 glioblastoma, 2 neuroblastoma, 3 melanoma, 7
gastrointestinal, 3 hepatoma, 1 breast, and 2 renal cancer cell
lines) expressed detectable levels of normal-sized mRNAs ex
cept for the HT29 colon cancer cell line. In contrast, only 2 of
the 8 hematopoietic tumor cell lines expressed detectable
mRNA (data not shown), which is in agreement with a previous
report by LaForgia et al. (Il ).
RNase protection assay was then performed to search for
subtle mutations in lung cancer, because no gross abnormalities
could be found by Southern blot analysis using the cDNA probe
and £coRI digests of genomic DNAs (data not shown). Con
sequently, 31 lung cancer cell lines as well as the U-2 OS
osteosarcoma cell line were examined using three antisense
RNA probes which can cover almost the entire cytoplasmic
domain of PTPy. Representative results of RNase protection
assay are shown in Fig. 3. We found no abnormal RNase
cleavage in any of the lung cancer cell lines when compared to
ß-Actin
JL
^»
A fli^Lflft M
28S-
18S-
ß-Actin
Fig. 2. Detection of PTPy transcripts by Northern blot analysis in lung cancer
cell lines (a) as well as in other types of human tumor cell lines (e). In a. SCLC
and NSCLC cell lines expressed varying degrees of PTPy transcripts, although
several cell lines such as ACC-LC-76 and VMRC-LCD
showed greatly reduced
or no detectable mRNA. Note that ACC-LC-171 exhibited 6.8- and 4.5-kilobase
transcripts which are distinct from those in normal lung (9.6 and 6.2 kilobases).
In A. other types of human solid tumors expressed 9.6- and 6.2-kilobase transcripts
identical to those of normal lung, whereas the U-2 OS osteosarcoma cell line was
found to express distinctly sized mRNAs (10.8 and 7.6 kilobases) in addition to
normal-sized transcripts (neuroblastoma. SK-N-SH; glioblastoma, MG-178 and
U-343; colon cancer, HT29, SW1116, and ColoS71; hepatoma, HepG2 and SkHepl; breast cancer, MCF-7; osteosarcoma, U-2 OS; renal cancer, SK-RC-9 and
AM-RC-6). The *, shorter exposure of U-2 OS.
3507
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IN HUMAN LUNG CANCER
O)
c
II
<D
Q. Z
co
z
oc
rce o o o o
9
o o o o ir.
o o o o v
<
<
<
<
£t
O
HPTPyDI
SCLC
i-
NSCLC
P.C.
SCLC
HPTPYD2
co
0)
HPTPyUT
NSCLC
fo
f~
f^ o r—co
»-i/i r~ co GÌ
r**
Ó 0 6 6 Ó 0
o o o o
< < < <
ot
D
tr
I«—¿I
Fig. 3. RNase protection assay of RNAs
extracted from adult normal lung, lung cancer
cell lines, and U-2 OS osteosarcoma cell line
(a-c). All cell lines including ACC-LC-171 and
U-2 OS cell lines exhibited RNase cleavage
patterns identical to those of adult normal
lung. ¡i.schematic diagram of the I'll'mRNA and the location of the antisense RNA
probes. In the coding region (boxed), the
darkly shaded box (TM) and the lightly shaded
boxes (DI and D2) represent the transmem
brane domain and the PTPase-like domains.
The transmembrane and extracellular domains
have not been completely characterized (10).
DI and D2, PTPase-like domains 1 and 2,
respectively; EC, extracellular domain.
o
_ O O O U
co _i _i _i _i
E O Ü O O
»-
^f^o^co
T-ifj^-cooiN"
g>
c
»-CNÕC33
SCLC
NSCLC
»o
•¿^
^ o f** to
*-u"ï»-cpOïr^
T
—¿
--
cicli,
S1-AS1MOCI
E
z
ÒÒÒÙÓÓi—'
< < < < < ° * ^
ÛOO
Ë.I.Ë.
§
J
ÒÒÒÒÒÒi—'
u °°°u ° * ^
S2-AS2//V» I
S3-AS3/ Ato I
EC
S4-AS4/ec/1
DI
TM
S1»-
-4AS1
S3»-
-»AS3
»AS2 S4»-
-4AS4
Fig. 4. SSCP analysis of PCR-amplified cDNA derived from normal lung and lung cancer cell lines (a-d) and schematic diagram of the PTPy mRNA as well as
location of the PCR primers used in this study (<•).
All lung cancer cell lines exhibited identical electrophoretic mobility when compared to adult normal lung in both
5' and 3' of the PTPase-like domain 1 (a and b) as well as in both 5' and 3' of the PTPase-like domain 2 (c and d). Vertical arrowheads, restriction sites used to
digest the cDNA/PCR products. P.C., independent plasmid clones used as positive controls which carry a misincorporation of nucleotides created by Taq polymerase.
3508
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PTPy IN HUMAN
LUNG CANCER
missed due to the intrinsic limitation of RNase protection assay,
i.e., less than 50% of the possible single base pair mismatches
can be detected by RNase protection assay (18). In contrast,
nearly 90% of single base changes could be detected by the
PCR-SSCP method (19). Since two PTPase-like domains have
been identified in PTPy and are thought to play key roles as
catalytic and regulatory domains (10), we examined both
PTPase-like domains by PCR-SSCP analysis using cDNAs
prepared from 31 lung cancer cell lines as well as from U-2 OS
cell lines. Again, we found no mutations in either of these
PTPase-like domains (Fig. 4). Altogether, we concluded that
the cytoplasmic domain of the PTPy gene appears to carry
mutations quite infrequently even if they exist.
The current study showed for the first time that the PTPy
gene is expressed ubiquitously in human fetal and adult normal
tissues and that fetal and adult normal lungs express PTPy
mRNA most abundantly among tissues tested. The presence in
two independent cell lines of PTPy mRNAs distinct from those
of normal lung is highly suggestive of the authenticity of PTPy
as a tumor suppressor gene at 3p21. However, our exhaustive
search for mutations in the PTPy gene failed to identify any
such abnormalities in lung cancer. The region we studied is the
cytoplasmic region of PTPy, which contains two PTPase-like
domains thought to be important for their activities (10). There
fore, a lack of evidence for mutations in the PTPy gene suggests
the following possibilities:
(a) Mutational hot spot(s) may be located outside of the
region studied, suggesting the need for a further search for
genetic alterations in the extracellular and transmembrane do
mains. It is possible that mutations in those regions may serve
as a preferential mechanism for PTPy inactivation, resulting
from alterations in binding with the unidentified ligand.
(b) PTPy may not be a tumor suppressor gene involved in
the oncogenesis of lung cancer, and the distinctly sized mRNAs
in two cell lines merely represent normal isoforms of PTPy
which are generated by alternative splicings. In this regard, it
should be noted that at least five isoforms of the human
leukocyte common antigen (CD45, a member of the transmem
brane PTPases) are known to be generated by alternative splic
ing of three exons encoding NHj-terminal segments of the
extracellular domain (20).
To elucidate these issues, further molecular characterization
of the PTPy gene including cloning of the entire cDNA as well
as an extensive search for possible mutations in the remaining
region are warranted.
2. Takahashi, T., Ñau, M. M., Chiba. I., Birrer, M. J., Rosenberg, R. K.,
Vinocour, M., Levitt, M., Pass, H., Gazdar, A. F., and Minna, J. D. p53: a
frequent target for genetic abnormalities in lung cancer. Science (Washington,
DC), 240:491-494, 1989.
3. Whang-Peng, J., Kao-Shan, C. S., Lee, E. C, Bunn, P. A., Carney, D. N.,
Gazdar, A. F., and Minna. J. D. Specific chromosome defect associated with
human small-cell lung cancer: deletion 3p( 14-23). Science (Washington DC),
215: 181-182, 1982.
4. Naylor, S. L., Johnson, B. E., Minna, J. D., and Sakaguchi, A. Y. Loss of
heterozygosity of chromosome 3p markers in small-cell lung cancer. Nature
(Land.), 329: 451-454, 1987.
5. Brauch, H., Johnson, B., Hovis, J., Yano, T., Gazdar, A., Pettengill, O. S.,
Graziano, S., Sorenson, G. O., Poiesz, B. J., Minna, J., Linehan, M., and
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Postmus, P. E., Poppema, S., and Buys, C. H. C. M. Deletion of a DNA
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Nature (Lond.), 330: 578-581. 1987.
7. Yokota, J., Wada, M., Shimosato, Y., Terada, M., and Sugimura, T. Loss of
heterozygosity on chromosome 3, 13 and 17 in small-cell lung carcinoma
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USA, 84: 9252-9256, 1987.
8. Hibi, K., Takahashi, T., Yamakawa, K., Ueda, R., Sekido, Y., Ariyoshi, Y.,
Suyama, M., Takagi, H., Nakamura, Y. and Takahashi, T. Three distinct
regions involved in 3p deletion in human lung cancer. Oncogene, 7: 445449, 1992.
9. Yokoyama, S., Yamakawa, K., Tsuchiya, E., Murata, M., Sakiyama, S., and
Nakamura, Y. Deletion mapping on the short arm of chromosome 3 in
squamous cell carcinoma and adenocarcinoma of the lung. Cancer Res., 52:
873-877, 1992.
10. Saito, H., and Streuli, M. Molecular characterization of protein tyrosine
phosphatases. Cell Growth Differ.. 2: 59-65, 1991.
11. LaForgia, S., Morse, B., Levy, J., Barnea, G., Cannizzaro, L. A., Li, F.,
Nowell, P. C., Boghosian-Sell, L., Click, J., Weston, A., Harris, C. C.,
Drabkin, H., Patterson, D., Croce, C. M., Schlessinger, J., and Huebner, K.
Receptor protein-tyrosine phosphatase 7 is a candidate tumor suppressor
gene at human chromosome region 3p21. Proc. Nati. Acad. Sci. USA, 88:
5036-5040, 1991.
12. Takahashi, T., Obata, Y., Sekido, Y.. Hida, T., Ueda, R., Watanabe, H.,
Ariyoshi, Y., Sugiura, T., and Takahashi, T. Expression and amplification
of myc gene family in small cell lung cancer and its relation to biological
characteristics. Cancer Res., 49: 2683-2688, 1989.
13. Takahashi. T., Takahashi. T., Suzuki, H., Hida, T., Sekido, Y., Ariyoshi, Y.,
and Ueda, R. The p53 gene is very frequently mutated in small-cell lung
cancer with distinct nucleotide substitution pattern. Oncogene, 6: 17751778, 1991.
14. Krueger, N. X., Streuli, M., and Saito, H. Structural diversity and evolution
of human receptor-like protein tyrosine phosphatases. EMBO J., 9: 32413252, 1990.
15. Takahashi, T., D'Amico. D., Chiba, I., Buchhagen, D. L., and Minna, J. D.
Acknowledgments
T., Buchhagen, D. L., Carbone, D., Piantadosi, S., Koga, H., Reissmann, P.
T., Slamon, D. J., Holmes, E. C., and Minna, J. D. Mutations in the p53
gene are frequent in primary, resected non-small cell lung cancer. Oncogene,
S: 1603-1610, 1990.
19. Mitsudomi, T., Steinberg, S. M., Nau, M. M.. Carbone, D., D'Amico, D.,
We would like to thank Dr. L. J. Old for providing various cell lines.
The technical assistance of Ms. H. Suzuki is also greatly appreciated.
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3509
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Molecular Analysis of the Protein Tyrosine Phosphatase γ Gene
in Human Lung Cancer Cell Lines
Tetsuy Tsukamoto, Takashi Takahashi, Ryuzo Ueda, et al.
Cancer Res 1992;52:3506-3509.
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