(CANCER
RESEARCH 46,1530-1534,
March 1986]
Differential Expression of p21rasGene Products among Histológica! Subtypes
of Fresh Primary Human Lung Tumors1
Razelle Kurzrock,2 Gary E. Gallick,3 and Jordan U. Gutterman4
Department of Clinical Immunology and Biological Therapy [R. K., J. U. G.J and Department of Tumor Biology, Section of Virology [G. E. G.], M. D. Anderson Hospital
and Tumor Institute, Houston, Texas 77030
ABSTRACT
Activation of the ras oncogene has been associated with a
number of human tumors. In this study, expression of p2Vas in
different histological types of fresh primary bronchogenic carci
nomas was examined. p21fas products were detected in all lung
tissues that were analyzed. Only 1 of 23 tumors demonstrated
aberrant migration of p21'as. In contrast, 10 tumors had substan
tially elevated levels of p2Vas products with respect to the
adjacent normal lung tissues and with respect to the other lung
tumors. There was no correlation between increased ras protein
expression and tobacco exposure of the patient or extent of
disease at the time of diagnosis. However, 9 of 11 tumors with
a squamous component as opposed to only 1 of 12 tumors
belonging to other histological classifications demonstrated in
creased p21ras.These data suggest that, in bronchogenic carci
nomas, mutations associated with structural abnormalities and
aberrant migration of p21ras occur infrequently as compared to
quantitative changes in p21ras. Furthermore, differential expres
sion of c-ras products in primary human lung tumors correlates
with pathological cell type, and may be a common event in
squamous cell carcinomas, but not adenocarcinomas or small
cell carcinomas of the lung.
ras RNA in colon, lung, and renal tumors (7, 8), and increased
ras protein in approximately 50% of colorectal tumors (9, 10)
suggests that in vivo quantitative changes in c-ras expression
may be important in certain human cancers.
Although bronchogenic carcinoma is the most frequently oc
curring fatal cancer in the United States (11, 12), studies of
oncogene expression in these tumors have been limited. To
date, most reports have focused on RNA transcripts as a param
eter of gene expression and have usually been restricted to cell
lines because of the rapid degradation of RNA in fresh solid
tumors (13-15). However, genetic aberrations observed in es
tablished cell lines may not always reflect events occurring in
unmanipulated tumors (4). Furthermore, studies of the protein
products of these genes, which are the molecules to which
cellular changes are ultimately attributable, have not been re
ported for lung tumors. Therefore, we used a monoclonal anti
body that detects the p21 product of the c-Ha-ras, c-Ki-ras, and
N-ras genes to investigate ras expression in 23 paired samples
of fresh primary human lung tumors and adjacent normal lung
tissues. We report differential expression of c-ras which corre
lates with histological classification, with increased p21fas de
tected in squamous cell carcinomas but not in adenocarcinomas
or small cell carcinomas of the lung.
INTRODUCTION
MATERIALS AND METHODS
Aberrant expression of cellular oncogenes belonging to the
ras gene family has been observed in several naturally occurring
cancers. Activation of these genes by specific point mutations
confers the ability to transform NIH-3T3 cells after transfection
and is estimated to occur in 10 to 20% of human tumors (1-3).
Cell Cultures. Uninfected normal rat kidney cells and normal rat kidney
cells infected with Kirsten Sarcoma virus were provided by Dr. E.
Scolnick. Cell lines were grown in Dulbecco's modified Eagle's medium,
However, such somatic alterations are not seen in, and therefore
are unlikely to account for the development of the majority of
tumors of any given type (4). Malignant transformation may also
result from a change in the level of expression of normal cellular
proto-oncogenes. Such tumorigenic potential has been demon
strated in vitro for the c-Ha-ras gene by experiments in which
transfected molecular clones of this gene ligated to a retroviral
long terminal repeat induced high levels of expression of the
normal gene product associated with cellular transformation (5).
Furthermore, recent work demonstrating hypomethylation of the
c-Ha-ras gene in human colon and lung tumors (6), increased
Received 7/8/85; revised 11/8/85; accepted 11/11/85.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Research conducted, in part, by the Clayton Foundation for Research and the
James E. Lyon Foundation for Research.
2To whom requests for reprints should be addressed, at Department of Clinical
Immunology and Biological Therapy, Box 41, M. D. Anderson Hospital and Tumor
Institute, 6723 Bertner Avenue, Houston, TX 77030.
3 Aided by NIH Grant RR5511 -21.
4 Senior Clayton Foundation Investigator.
CANCER RESEARCH
supplemented with 15% fetal calf serum. Upon reaching confluency, cell
pellets were collected by centrifugation, frozen in liquid nitrogen, and
then lyophilized for 12 h.
Procurement of Human Tissue. Tissue was removed from patients
undergoing pneumonectomy or lobectomy for carcinoma of the lung, in
accordance with institutional guidelines. All tissues were obtained im
mediately after surgical resection. Lung tumor tissues which showed
necrotic areas were carefully trimmed. Normal lung tissues were taken
from the far margins of resection of the lung in those patients whose
margins of resection were free of tumor. Biopsies of normal skin were
also obtained and trimmed to separate the epidermis from the dermis.
Tissue was frozen in liquid nitrogen and then lyophilized for 12 h. Tissues
stored in this manner have consistently yielded reproducible .results in
protein analysis.
Patient Data. Data pertaining to patient characteristics were obtained
by review of hospital charts. Tumors from patients who had received
preoperative radiation therapy were not analyzed in this study. Lung
tumors were classified as squamous cell carcinomas, adenocarcinomas,
or small cell carcinoma based on histopathological examination. Tumors
demonstrating both squamous and glandular elements were classified
as adenosquamous carcinoma. In addition, cellularity and proportion of
stromal tissue in each tumor was determined, by reviewing pathology
slides.
Stage of diseasewas designatedaccordingto the criteriaof the
VOL. 46 MARCH
1986
1530
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
GENE PRODUCTS
IN LUNG TUMOR TISSUES
American Joint Committee for Cancer Staging (16). The stages are as
follows: Stage I refers to a primary tumor <3 cm in diameter without
RESULTS
evidence of nodal métastasesoutside the peribronchial or ipsilateral hilar
region, or a tumor >3 cm without any nodal métastases. Stage II refers
to a primary tumor >3 cm with nodal métastases confined to the
peribronchial or ipsilateral hilar region. Stage HI refers to tumors with
direct extension into adjacent structures, i.e., parietal pleura or chest
wall and/or any tumor with distant nodal or visceral métastases.
Immunoprecipitation of Tissue Extracts. A rat monoclonal antibody,
Y13-259 (donated by Dr. E. Scolnick), which reacts with the p21 product
of c-Ha-ras, c-Ki-ras, and N-ras genes (17) was used in all studies.
Lyophilized tissues (2.5 mg) were lysed in 1 ml of detergent-containing
The procedure used in these experiments, whereby immunoblotting was performed on immunoprecipitates of p21'8S from
relatively large amounts of tissue (2.5 mg) rather than on whole
cell extracts (where only about 100 /*g of tissue can be applied
to .the gel) has been shown previously by Gallick ef a/. (9) to
detect even the small amounts of p21rasfound in NIH-3T3 cells
and normal human tissue, with a minimum of spurious bands. In
addition, this technique detects migration differences based on
structural changes caused by alteration of the 12th amino acid
in p21 (9, 24) and accurately reflects quantitative changes in p21
in a normal rat kidney cell line before and after infection with
Kirsten sarcoma virus (Fig. 1). The band in the M, 30,000-35,000
buffer as described by Naso e? al. (18) and then homogenized. The
resulting suspension was cleared by centrifuging at 10,000 rpm for 10
min. The lysates were then incubated for 3 h with 10 /¿\
of the monoclonal
antibody (1.7 mg/ml) at 4°C.Lysates were next reacted for 2 h with 10
6
ni of rabbit antibodies against rat IgG (Cappel Laboratories, Cochranville,
PA), which had been reconstituted according to the manufacturer's
7
instructions. Antigen-antibody complexes were then reacted with 60 M!
of formalin-inactivated Staphylococcus aureus (Cowan strain) for 30 min
at 4°C(19). The immune precipitates were collected by centrifugation,
and the cell pellets were then washed 3 times. Immunoprecipitated
proteins were released by resuspending the samples in SDS5-polyacryl-
LC-
amide gel electrophoresis buffer (1% SDS, 1% 2-mercaptoethanol, 0.001
M EDTA, 0.005 M bromophenol blue, 5% glycerol, and 0.01 M Tris-HCI,
pH 8.0) and boiling for 5 min. Samples were then cooled to room
temperature and centrifugea at 6,000 rpm for 10 min, and the Cowan
pellets were discarded. Immunoprecipitation of tubulin was performed
with a sheep antiserum to human tubulin (20) (donated by Dr. B. Brinkley)
as described above, except that S. aureus protein A was added imme
diately after 4 h of incubation with the anti-tubulin serum.
Fig. 1. Comparison of p21 and tubulin levels in primary human squamous cell
carcinomas of the lung and adjacent normal lung tissue. Immunoblotting of immunoprecipitates was performed as described in "Materials and Methods." A, reactivity
of p21 was determined by reaction of immunoprecipitates with 125Isheep anti-rat
Western Blotting. Western blotting of proteins was done by a modi
fication of the method of Anderson ef al. (21). Immunoprecipitated
proteins were resolved by SDS polyacrylamide gel electrophoresis by
using a 10.5% acrylamide gel. The gels were calibrated according to the
following molecular weight standards: Phosphorylase B (M, 92,500);
bovine serum albumin (M, 66,200); ovalbumin (M, 45,000); carbonic
anhydrase (M, 31,000); soybean trypsin inhibitor (M, 21,500); and lysozyme (M, 14,400). Electrophoretic transfer of proteins from the gel onto
nitrocellulose filter paper (22) was then accomplished at 100 V for 45
min. Blotted protein molecular weight standards were separated from
the rest of the nitrocellulose and stained in a 0.1% amido black solution
for direct visualization. The remaining nitrocellulose filter was rinsed in
TNE/NP40 buffer (0.15 M NaCI, 0.002 M EDTA, 0.1% NP40, 0.05 M Tris,
pH 7.5) and then preblocked for 3 h at 37°C in a plastic bag containing
IgG. Lane 1, NRK cells; Lane 2, NRK cells infected with Kirsten sarcoma virus;
Lanes 3 and 4, lung tumor and normal tissues, respectively, (patient M,) (Table 1);
Lanes 5 and 6, lung tumor and normal tissues (patient Ci); Lanes 7 and 8, lung
tumor and normal tissues (patient P,). LC denotes light chain of IgG. B, reactivity
of tubulin was determined by reaction of immunoprecipitates with 125IStaphylococ
cus aureus protein A. Lanes 1 and 2, lung tumor and normal tissues, respectively
(patient M,); Lanes 3 and 4, lung tumor and normal tissues, respectively (patient
Ci); Lanes 5 and 6, lung tumor and normal tissues, respectively (patient P,); Lane
7, no cell extract.
8
456
9
45K-
Mini.
2 ml per gel lane TNE/NP40 with 3% bovine serum albumin on a rocking
platform. After blocking, a 1:100 ratio of anti-p21 sera to fresh buffer
was added and then incubated at 4°Cfor 12 h on a rocking platform.
Filters were then washed five times with TNE/NP40 buffer, placed in a
plastic bag, and incubated with rocking for 1 h with 1 ^Ci per gel lane
125l-sheep anti-rat IgG (specific activity, 5-20 ¿iCi/mg;Amersham, Chi
22K-
h M
.
»'
» •-P21
cago, IL), diluted in 2 ml per gel lane TNE/NP40 buffer containing 3%
bovine serum albumin. Filters were washed 4 times with TNE/NP40 and
then once in TNE/NP40 containing 1.2% glycerol. After drying, filters
were autoradiographed at -70°C with Kodak Lightning-Plus intensifying
screens (23). Quantitation of differences in the level of p21ras was
performed by densitometry analysis. Immunoblotting of tubulin was done
by the same procedure, except that immunoprecipitated proteins were
resolved by SDS polyacrylamide gel electrophoresis using an 8% acryl
amide gel. Reactivity of tubulin in Western analyses was determined by
reactivity with 0.1 ftC\ per gel lane 125l-labeledS. aureus protein A (specific
Fig. 2. Comparison of p21 levels in primary human lung carcinoma of different
histological subtypes with adjacent normal lung tissue and normal skin tissue.
Immunoblotting of immunoprecipitates was performed as described in "Materials
and Methods." Reactivity of p21 was determined by reaction of immunoprecipitates
with 125Isheep anti-rat IgG. Lane 7, no cell extract; Lanes 2 and 3, small cell
carcinoma of the lung and adjacent normal lung tissue, respectively (patient Z,)
(Table 1). Lanes 4 and 5, squamous cell carcinoma of the lung and adjacent normal
lung tissue, respectively (patient Wi). Lanes 6 and 7, adenocarcinoma of the lung
and adjacent normal lung tissue, respectively (patient X,); Lanes 8 and 9. normal
skin tissues; LC denotes light chain of IgG.
activity, >30 mCi/mg; Amersham).
5 The abbreviations used are: SDS, sodium dodecyl sulfate; EGF, epidermal
growth factor; TNE, Tris-NaCI-EDTA; NP40, Nonidet P-40.
CANCER
RESEARCH
VOL.
46 MARCH
1986
1531
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
p21"s GENE PRODUCTS
IN LUNG TUMOR TISSUES
Table 1
Selected properties of primary lung tumors
of
history
ratio: tumor/
disease8IIIIII11IIillII1illinIIilllillill1IIII11II1IISmoking
(pack/yr)6301203003060453030501335450905007050120503050p21
normal
lungc>10.0>10.04.1>10.05.15.04.25.04.3
diagnosisAdenosquamous
(yr)/sex57/F68/M56/M76/M58/F65/M63/M46/M68/M64/M49/M49/F56/F65/F49/F58/M58/F65/M55/M62/M58/F56/M54/FHistological
PatientM,C,JiP,s,S2w,0,Y,T,E,P*P.E,T,S3W2H,N,X,K,Z,D,Age
carcinomaAdenosquamous
carcinomaAdenosquamous
carcinomaSquamous
carcinomaSquamous
carcinomaSquamous
carcinomaSquamous
carcinomaSquamous
carcinomaSquamous
carcinomaSquamous
carcinomaSquamous
carcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcino
carcinomaSmall
cell
carcinomaSmall
cell
cell carcinomaStage
" Criteria of the American Joint Committee for Cancer Staging (16).
6 Pack/yr = number of 20-cigarette packages smoked per day x number of years of smoking.
c Quantitation was done by densitometry.
molecular weight region of the gel, seen in all lanes, including
the lane in which no cell extract was immunoprecipitated (Fig. 2,
lane 1), represents reactivity of 125l-sheep anti-rat IgG with light
chain of the rat monoclonal antisera.
Examples of immunoblotting of immunoprecipitates of human
lung tumors and adjacent normal lung tissue are shown in Figs.
1 and 2. To date, ras gene expression has been analyzed in
samples of both fresh primary lung cancers and normal lung
tissue from each of 23 patients (Table 1). The tumor tissues
were histologically classified as squamous cell carcinomas (8
samples), adenosquamous carcinomas (3 samples), adenocar-
Fig. 2 shows examples of p21'as levels in a small cell carcinoma
(lanes 2 and 3), squamous cell carcinoma (lanes 4 and 5), and
adenocarcinoma (lanes 6 and 7). In contrast to the results
observed in squamous carcinomas, only 1 of 9 bronchogenic
adenocarcinomas and none of 3 small cell carcinomas demon
strated enhanced ras expression in tumor relative to normal
tissue. As determined by densitometry, the median ratio of p21ras
in the 11 squamous tumors analyzed, relative to adjacent normal
tissue, was 5.1 (range, 0.5 to >10). The median ratio of p21'as
in the 12 nonsquamous lung tumors analyzed as compared to
adjacent normal tissue was 0.95 (range, 0.5 to 4.0). When equal
amounts of protein were immunoprecipitated (in contrast to equal
dry weights) similar differences between tumors were observed
(data not shown). In addition, when densitometry comparisons
of p2Vas levels between different histological types of tumors
were performed, the 9 squamous tumors with increased p2Tas
tumor/normal ratio also showed elevation of p21rasranging from
3 to >10-fold as compared to adenocarcinomas or small cell
carcinomas analyzed on the same gel. Therefore, p21ras levels
cinomas (9 samples), and small cell carcinomas (3 samples). As
expected, p21'as was present in all lung tissues examined. A
p21'as which migrated more slowly than the normal was detected
in only one of the lung tumors examined. This tumor was
classified as an adenocarcinoma, and the aberrant migration
pattern was not seen in the normal lung tissue adjacent to this
tumor. Ten tumors demonstrated a greater than four-fold eleva
tion in p21'as with respect to adjacent normal lung tissue (Table
1). Interestingly, 9 of the tumors with increased p21'as had a
were increased in squamous tumors as compared to other lung
tumors or as compared to adjacent normal lung tissue.
Review of pathology slides revealed that the stromal compo
not have a squamous component was the adenocarcinoma
nent of both the adenocarcinomas and squamous cell carcino
(mentioned above), which demonstrated an aberrant migration
mas constituted approximately 45 to 55% of the tumor tissue.
pattern. Three examples of the increased p2Vas levels in squa
The small cell carcinomas appeared somewhat more cellular
mous or adenosquamous lung carcinomas are shown in Fig. ÃŒA than the other tumor types, with the stromal component consti
(lanes 3 to 8). In contrast, Fig. 1ß(lanes 1 to 6) illustrates that
tuting 30 to 40% of the tumor tissue. Therefore, variations in the
although there is some patient to patient variability when tissue
amount of stromal tissue between tumor types cannot account
for the increased levels of p21ras in squamous carcinomas as
extracts identical to those in A were blotted and reacted with
antiserum to human tubulin, the levels of this protein are similar
compared to adenocarcinomas or small cell carcinomas.
To determine the expression of p2Vas in normal squamous
between tumor and normal tissue in each of these patients.
Thus, variations in the extent of nonspecific proteolysis occurring
epithelium, the epidermal layer of normal skin derived from 2
additional patients was obtained. The level of p21ras in normal
in tumor versus normal tissue are unlikely to account for the
dramatically increased levels of p21ras seen in these squamous
skin epidermal tissue was equivalent to that in normal lung tissue
squamous cell component on histological examination (Table 1).
Furthermore, the single tumor with increased p21ras which did
(Fig. 2, lanes 8 and 9). This is consistent with our previously
lung tumors relative to adjacent normal lung tissue.
CANCER
RESEARCH
VOL. 46 MARCH
1986
1532
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
p21'as GENE PRODUCTS
IN LUNG TUMOR TISSUES
reported results (9) showing about a two-fold variation in p21ras
adenocarcinoma.
expression among various normal tissues, including lung, liver,
and colon. These data suggest that increased p21rasis a property
adenocarcinoma may have occurred because of previous acti
vation of the c-ras gene by a point mutation, since this tumor
was the one in which slowed migration of p21 was detected.
Elevation of p21ras was not observed in normal squamous epi
of malignant but not of normal squamous epithelium.
Alternatively,
elevation of p21'as in this one
DISCUSSION
thelium derived from skin biopsies, suggesting that increased
p21'as is a property of squamous cell carcinoma but not of normal
Activation of cellular oncogenes has been implicated in induc
tion of In vitro malignant transformation of NIH-3T3 cells and
may be related in vivo to several human cancers (1-3, 5-10). In
particular, transfection experiments have revealed that altered
p21 products of the c-ras gene seem to be responsible for the
mous cell carcinoma but not of normal keratinocytes.
Recent work suggests that the autonomous growth of malig
nant cells may be due to aberrant expression of controlling
growth factors, growth factor receptors, or their intracellular
messenger system, any of which may be encoded directly by
oncogenes or may interact with oncogene protein products (28).
In particular, experiments demonstrating that EGF stimulates the
guanine nucleotide binding of the c-Ha-ras and v-Ha-ras protein
(29) suggest a functional link between p21ras and the receptor
transforming potential of individual lung cancer cell lines (25) and
fresh lung tumors (26). Quantitative changes in oncogene
expression may also be associated with bronchogenic cancers.
In small cell lung cancer cell lines, elevated expression of the cmyc (14) and c-myb (15) genes have been observed, with the
former correlating with progression. Hypomethylation of the cHa-ras gene and enhanced expression of c-Ki-ras and c-Ha-ras
mRNA have also been described in individual lung tumors (6, 8).
However, to date, systematic comparison of oncogene expres
sion in fresh lung tumors of different histological types has not
been performed. Therefore, in this study, fresh tissues from
bronchogenic carcinomas classified as squamous cell, adenosquamous, adenocarcinomas, and small cell carcinomas were
analyzed for expression of the p21 product of the ras gene by
immunoblotting with a broadly reactive monoclonal antisera to
p21'as. Although this technique has been shown previously to
detect migration differences caused by a point mutation at the
12th amino acid of p21 (9), only 1 of 23 (4.3%) lung tumors
demonstrated such a migration difference. These results in fresh
lung tumor tissue are in accord with previous work in lung cancer
cell lines suggesting that this point mutation occurs infrequently
and therefore probably does not play a role in the development
of most lung cancers (4). In contrast, quantitative changes in
p2Vas were commonly observed in this study, with 10 of 23
(44%) lung tumor tissues analyzed showing a greater than four
fold elevation of p21 in the tumor with respect to the adjacent
normal tissue. Increased p2Vas was not associated with age or
sex of patient, history of tobacco use, or stage of disease (Table
1). We have reported previously that p21'as expression in colorectal cancer corresponds with stage of disease, with higher
levels of expression detected in intermediate stage primary tu
mors as compared with metastatic lesions, or with late stage
primary disease in which métastasesare present (9). Therefore,
it is possible that differential expression of p21'as may be asso
ciated with progression of colorectal carcinoma but not of lung
carcinoma. However, it is important to bear in mind that the total
of 3 different gene products are being assayed, i.e., c-Ha-ras, cKi-ras, and N-ras, and the same one may not be increased in
colon and lung tumors.
Interestingly, differential expression of c-ras did correlate with
the histological classification of the lung tumors (Table 1). Nine
of 11 (82%) tumors with a squamous component as opposed to
only 1 of 12 (8.3%) nonsquamous cell carcinomas of the lung
demonstrated increased p21ras.There is a large body of evidence
suggesting that the major histological subtypes of lung carci
noma may be developmentally related with some tumors exhib
iting characteristics of more than one subtype (27), possibly
accounting for the elevated p21'as in a single apparently "pure"
system stimulated by EGF. The structure of the ras proteins is
homologous to the a-subunit of the G-regulatory proteins (30)
that transduce signals from certain receptor-ligand systems into
changes in the intracellular levels of adenylate cyclase, in part,
by binding to guanine nucleotides (31, 32). Studies performed
with yeast further support the concept that Aas proteins are
controlling elements of adenylate cyclase (33). Therefore, it is
possible that overexpression of the ras gene may be involved in
the genesis of squamous cell carcinomas by acting as a signal
transducer on cyclic AMP in a similar fashion to G-proteins and/
or by its interaction with the EGF receptor system. Furthermore,
amplification of the EGF receptor gene has been described in
squamous carcinoma cell lines (34-36), and recent work indi
cates that increased EGF receptor is a consistent feature of
squamous cell carcinomas (37). Since p21'as has been shown to
regulate the synthesis of transforming growth factors (38-40)
the actions of which are mediated directly or indirectly by inter
action with EGF or EGF receptor (41), it would be of interest to
see if transforming growth factors are aberrantly expressed in
those squamous cell carcinomas with increased p2Vas. Finally,
while our data suggest that high levels of p21'as are a common
feature in squamous cell carcinomas but not adenocarcinomas
or small cell carcinomas of the lung, further studies of ras
expression in premalignant states are required to determine if
increased p21ras is necessary for the development of the malig
nant phenotype or is a consequence of other initiating events.
REFERENCES
1. Pulciani, S., Santos, E.. Lauver, A. V., Long, L. K., Aaronson, S. A., and
Barbacid. M. Oncogenes in solid human tumours. Nature (Lond.), 300: 539542,1982.
2. Fujita, J., Yoshida, O., Yuasa, Y., Rhim, J. S., Hatanaka, M., and Aaronson,
S. A. Ha-ras oncogenes are activated by somatic alterations in human urinary
tract tumours. Nature (Lond.), 309: 464-466, 1984.
3. Eva, A., Tronick, S. R., Gol, R. A., Pierce, J. H., and Aaronson, S. A.
Transforming genes of human hematopoietic tumors: frequent detection of
ras-related oncogenes whose activation appears to be independent of tumor
phenotype. Proc. Nati. Acad. Sci. USA, 80. 4926-4930,1983.
4. Feinberg, A., Vogelstein, B., Droller, M. J., Baylin, S. B., and Nelkin, B. D.
Mutation affecting the 12th amino acid of the c-Ha-ras oncogene product
occurs infrequently in human cancer. Science (Wash. DC), 220: 1175-1177
1983.
5. DeFeo, D., Gonda, M., Young, H. A., Chang, E. H., Lowy, D. R., Scolnick, E.
M., and Ellis, R. W. Analysis of two divergent rat genomic clones homologous
to the transforming gene of Harvey murine sarcoma virus. Proc. Nati. Acad.
Sci. USA, 78: 3328-3332,1981.
6. Feinberg, A. P., and Vogelstein, B. Hypomethylation of ras oncogenes in
primary human cancers. Biochem. Biophys. Res. Commun., 777:47-54,1983.
7. Spandidos, D. A., and Kerr, I. B. Elevated expression of the human ras
CANCER RESEARCH VOL. 46 MARCH
1533
1986
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
p2VK
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
GENE
PRODUCTS
IN LUNG
RESEARCH
TISSUES
26. Santos, E., Martin-Zanca, D., Reddy, E. P., Pierotti, M. A., Della Porta, G., and
Barbacid, M. Malignant activation of a K-ras oncogene in lung carcinoma but
not in normal tissue of the same patient. Science (Wash. DC), 223: 661-664,
1984.
27. Baylin, S. B. Implications of differentiation relationship between endocrine and
non-endocrine cells in human lung cancer. In: Peptide Hormones as Mediators
in Immunology and Oncology, pp. 1-12. R. D. Hesch (ed.), London: Academic
Press, 1985.
28. Heldin, C. H., and Westermark, B. Growth factors: mechanism of action and
relation to oncogenes. Cell, 37: 9-20,1984.
29. Kamata, T., and Feramisco, J. R. Epidermal growth factor stimulates guanine
nucleotide binding activity and phosphorylation of ras oncogene proteins.
Nature (Lond.), 370: 147-150, 1984.
30. Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Oilman, A. G.
Homologies between signal transducing G proteins and ras gene products.
Science (Wash. DC), 226: 860-862,1984.
31. Gilman, A. G. G-proteins and dual control of adenylate cyclase. Cell, 36: 577579, 1984.
32. McGrath, J. P., Capon, D. J., Goeddel, D. V., and Levinson, A. D. Comparative
biochemical properties of normal and activated human ras p21 protein. Nature
(Lond.), 370: 644-649, 1984.
33. Toda, T., Uno, I., Ishikawa, T., Powers, S., Kataoka, T., Broek, D., Cameron,
S., Broach, J., Matsumoto, K., and Wigler, M. In yeast, ras proteins are
controlling elements of adenylate cyclase. Cell, 40: 27-36, 1985.
34. Merlino, G. T., Xu, Y-H., Ishii, S., Clark, A. J. L., Semba, K., Toyoshima, K.,
Yamamoto, T., and Pastan, I. Amplification and enhanced expression of the
epidermal growth factor receptor gene in A3431 human carcinoma cells.
Science (Wash. DC), 224: 417-419,1984.
35. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tarn, A. W., Lee,
J., Yarden, Y., LJbermann, T. A., Schlessinger, J., Downward, J., Mayes, E. L.
V., Whittle, N., Waterfield, M. D., and Seeburg, P. H. Human epidermal growth
factor receptor cDNA sequence and aberrant expression of the amplified gene
in A431 epidermal carcinoma cells. Nature (Lond.), 309: 418-425,1984.
36. Merlino, G. T., Xu, Y-H., Richert, N., Clark, A. J. L., Ishii, S., Banks-Schlegel,
S., and Pastan, I. Elevated epidermal growth factor receptor gene copy number
and expression in a squamous cell cell line. J. Clin. Invest., 75: 1077-1079,
1985.
37. Cowley, G., Smith, J. A., Gusterson, B., Hendler, F., and Ozanne, B. The
amount of EGF receptor is elevated on squamous cell carcinomas. Cold Spring
Harbor Symp., 7: 5-10, 1984.
38. Kaplan, R. L., Anderson, M., and Ozanne, B. Transforming growth factor(s)
production enables cells to grow in the absence of serum: an autocrine system.
Proc. Nati. Acad. Sci. USA, 79: 485-489, 1982.
39. Weissman, B. E., and Aaronson, S. A. BALB and Kirsten murine sarcoma
viruses alter growth and differentiation of EGF-dependent BALB/c mouse
epidermal keratinocyte lines. Cell, 32: 599-606,1983.
40. Salomon, D. S., Zwiebel, J. A., Noda, M., and Bassin, R. H. Flat revenants
derived from Kirsten murine sarcoma virus-transformed cells produce trans
forming growth factors. J. Cell. Physiol., 727: 22-30, 1984.
41. Derynck, R., Roberts, A. B., Winkler, J. E., Chen, E. Y., and Goeddel, D. V.
Human transforming growth factor-alpha: precursor structure and expression
in E. coli. Cell, 38: 287-297,1984.
oncogene family in premalignant and malignant tumours of the colorectum. Br.
J. Cancer, 49: 681 -688, 1984.
Slamon, D. J., deKernion, J. B., Verma, I. M., and Cline, M. J. Expression of
cellular oncogenes in human malignancies. Science (Wash. DC), 224: 256262, 1984.
Gallick, G. E., Kurzrock, R., Kloetzer, W. S., Arlinghaus, R. B., and Gutterman,
J. U. Expression of p21â„¢*in fresh primary and metastatic human colorectal
tumors. Proc. Nati. Acad. Sci. USA, 82: 1795-1799, 1985.
Thor, A., Hand, P. H., Wunderlich, D., Caruso, A., Muraro, R., and Schlom, J.
Monoclonal antibodies define differential ras gene expression in malignant and
benign colonie diseases. Nature (Lond.), 311: 562-565,1984.
Minna, J. D., Higgins, G. A., and Glatstein, E. J. Cancer of the lung. In: Cancer:
Principles and Practice of Oncology, pp. 396-463. V. T. DeVita, Jr., S. Hellman,
and S. A. Rosenberg (eds.), Philadelphia: J. P. Lippincott Co., 1982.
Silverberg, E. Cancer Statistics, 1980. Cancer (Priila.), 30: 23-38, 1980.
McCoy, M. S., Toóle, J. J., Cunningham, J. M., Chang, E. H., Lowy, D. R.,
and Weinberg, R. A. Characterization of a human colon/lung carcinoma
oncogene. Nature (Lond.), 302: 79-81, 1983.
Little, C. D., Nau, M. M., Carney, D. N., Gazdar, A. F., and Minna, J. D.
Amplification and expression of the c-myc oncogene in human lung cancer cell
lines. Nature (Lond.), 306: 194-196,1983.
Griffin, C. A., and Baylin, S. B. Expression of the c-myb oncogene in human
small cell lung carcinoma. Cancer Res., 45: 272-275, 1985.
Mountain, C. F., Carr, D. T., and Martini, N. Staging of lung cancer. In: American
Joint Committee for Cancer Staging and End Results Reporting. Chicago:
Task Force on Lung Cancer, 1980.
Furth, M. E., Davis, L. J., Fleurdelys, B., and Scolnick, E. M. Monoclonal
antibodies to the p21 products of the transforming gene of Harvey murine
sarcoma virus and of the cellular ras gene family. J. Virol., 43: 294-304,1982.
Naso, R. B., Arcement, L. J., and Ariinghaus, R. B. Biosynthesis of Rauscher
leukemia viral proteins. Cell, 4: 31-36,1975.
Kessler, S. W. Rapid isolation of antigens from cells with a staphylococcal
protein a-antibody adsorbent: parameters of the interaction of antibody-antigen
complexes with protein A. J. Immunol., 775: 1617-1624, 1975.
Brinkley, B. R., Fistel, S. H., Marcum, J. M., and Pardue, R. L. Microtubules in
cultured cells; indirect immunofluorescent staining with tubulin antibody. Int.
Rev. Cytol., 63: 59-95, 1980.
Anderson, S. K., Furth, M., Watt, L., Ruscetti, S. K., and Sherr, C. J.
Monoclonal antibodies to the transformation-specific glycoprotein encoded by
the feline retroviral oncogene v-fms. J. Virol., 44: 696-702, 1982.
Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and some appli
cations. Proc. Nati. Acad. Sci. USA, 76: 4350-4353,1979.
Bonner, W. M., and Laskey, R. A. A film detection method for tritium-labelled
proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem., 46: 8388, 1974.
Tabin, C. J., Bradley, S. M., Bargmann, C. l., Weinberg, R. A„
Papageorge, A.
G., Scolnick, E. M., Dhar, R., Lowy, D. R., and Chang, E. H. Mechanism of
activation of a human oncogene. Nature (Lond.), 300:143-149,
1982.
Der, C. J., and Cooper, G. M. Altered gene products are associated with
activation of cellular ras* genes in human lung and colon carcinomas. Cell, 32:
201-208,1983.
CANCER
TUMOR
VOL.
46 MARCH
1986
1534
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1986 American Association for Cancer Research.
Differential Expression of p21ras Gene Products among
Histological Subtypes of Fresh Primary Human Lung Tumors
Razelle Kurzrock, Gary E. Gallick and Jordan U. Gutterman
Cancer Res 1986;46:1530-1534.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/46/3/1530
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 14, 2017. © 1986 American Association for Cancer Research.