Cell Growth. & Differentiation. 799. Human. Lung Cancer Cell Lines Exhibit Resistance to Retinoic Acid Treatment. Joseph Geradts,'. Jeou-Yuan. Chen, Edward.
Vol.
4, 799-809,
October
Cell Growth
1993
Human Lung Cancer Cell Lines Exhibit to Retinoic Acid Treatment
Joseph Geradts,’ Jeou-Yuan Chen, Edward K. Russell, James R. Yankaskas, Luis Nieves, and John D. Minna2 National Medical
Cancer Center,
Institute-Navy Medical Oncology Bethesda, Maryland 20889 ti G.,
Branch, National E. K. RI; Division
Pulmonary Diseases, University of North Carolina, Chapel Hill, Carolina 27599 U. R. Y.J; and Simmons Comprehensive Cancer University
Dallas,
of Texas
Southwestern
Texas 75235-8590
Medical
Naval of
North Center,
Retinoic acid (RA) and nuclear retinoic acid receptors in a variety
of human
and
RA has been
proposed as a chemopreventive agent for bronchogenic carcinoma. Normal human tracheobronchial epithelial cells show dramatic induction of RARmRNA and significant growth inhibition after RA treatment. In contrast, 1 7 of 22 small cell lung cancer (SCLC) and 9 of 1 5 non-SCLC lines treated with 1 M RA showed no significant
growth
inhibition.
Of
interest,
5 SCIC
lines
with high levels of myc gene family expression related to c-, N-, or L-myc gene amplification exhibited growth inhibition (28-87%), whereas 2 non-SCLC lines adually showed growth stimulation after treatment with 1 M RA. The lines varied greatly in their constitutive expression of RAR-i mRNA, and 1 5 of 20 SCLC and 8 of 1 5 non-SCLC lines failed to show RAR-f mRNA indudion after RA treatment. Six cell lines showed possible alterations in the coding region of RAR-3 by complementary
(PCR) analysis isoforms,
since
DNA
(cDNA)/polymerase
using primers other
common
regions
would
chain
readion
to the RAR-31,2,3 undergo
cDNA/PCR
amplification whereas the DNA binding domain would not. Nonetheless, no abnormal band shift patterns in cDNA amplified by PCR were found by single strand conformation polymorphism analysis covering all 1344 base pairs of the RAR-3 open reading frame. Finally, no abnormalities
in RAR-a
were identified including
gene
structure
or expression
by Southern and Northern
lines with
cytogenetic
cases,
RA can
affect
growth
and
morpho-
In addition, retinoic acid can produce dramatic remissions in APL, an effect correlated with rearrangement ofthe RAR-a (1 5, 1 6). A commonly used retinoid is all-trans-retinoic acid, which seems to be the active compound derived from vitamin A and n-carotene metabolism (1 7).
Abstrad
cancer,
on normal bronchial epithelium by inducing mucous and blocking squamous differentiation (3, 4). Vitamin A deficiency is linked to increased susceptibility to lung cancer (6), and administration of vitamin A protects against the development of bronchial metaplasia, dysplasia, and carcinoma logical properties oflung cancer cells in vitro(8-l 0). Growth of other tumor cells is also modulated by retinoids (11-14).
FL. N., J. Y. C., J. D. M.J
(RARs) have been implicated malignancies including lung
799
Resistance
(4, 7). In some
Center,
& Differentiation
blot analysis,
abnormalities
We conclude that abnormalities of the RAR-3 common in human lung cancer cell lines.
of 1 7q21. system
are
Introdudion
Retinoids affect growth and differentiation of normal bronchial epithelial cells in vivo (1) and in vitro (2-5). RA3 acts
Received 6/3/93; accepted 7/20/93. 1 Present address: Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, NC 27599. 2 To whom requests for reprints should be addressed, at Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8590. 3 The abbreviations used are: RA, aIl-trans-retinoic acid; RAR, RA receptor; RXR, nuclear RA-binding orphan receptor; SCLC, small cell lung cancer;
The physiological effects of RA are mediated by nuclear RARs, which appear to act as transcription factors. Three major forms of RAR have been characterized so far: RAR-a (1 8, 1 9), RAR-3(20, 21), and RAR-y(22). The major receptor types are differentially expressed in various tissues (1 9, 2123). For each type of RAR, multiple isoforms have been described which differ in their NH2-terminal domains (24-27). The isoforms also show differential spatiotemporal expression, creating a complex, tissue-specific assortment of nuclear RAR transcription factors (24, 25, 27, 28). Abnormalities in RAR structure or expression have been implicated in a variety of human malignancies: RAR-a in APL and embryonal carcinoma (1 6, 29); RAR-f3 in hepatomas and squamous cell carcinoma of the cervix (14, 30, 31); and RAR-’y in oral squamous cell carcinoma and embryonal carcinoma (32, 33). Previous reports also noted abnormalities in RAR-3 gene structure and expression in human lung cancer (3436). The latter observation is intriguing because the RAR-f3 gene is located in chromosome region 3p24 (37), a site commonly showing allele loss in lung cancer (38-40). Thus, one hypothesis is that RA mediates the normal differentiation of bronchial epithelial cells, and in this pathway RARfunctions as a recessive oncogene. In this scenario, normal RAR-f3 would serve under RA regulation to send a signal for differentiation, whereas a mutant form of RAR-f.3 would be
altered
in this function.
this pathway
could
Alternatively,
be abnormal,
response to RA differentiation been reports of RA inducing
other
leading
components
of
to an inappropriate
signals. Although there have differentiation in lung cancer
cells, for many years we have noted in unpublished observations that RA usually has no effect on human lung cancer cell growth in vitro. Thus, we decided to systematically in-
vestigate a large number of lung cancer cell lines for their growth and morphological response to RA and to determine whether or not they had structural alterations in their RAR-ct
NSCLC,
non-SCLC;
cheobronchial
APL, acute
epithelial
cells;
promyelocytic cDNA,
leukemia;
complementary
HTBE, DNA;
human PCR,
tra-
polym-
erase chain reaction; kb, kilobase(s); bp, base pair(s); ORF, open reading frame; SSCP, single strand conformation polymorphism analysis; 505, sodium dodecyl sulfate;SSC, standard saline citrate;FBS, fetalbovine serum; UTR, untranslated region; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PS, sense primer; Pas, antisense primer.
800
RA Effects
on
Human
Lung
Cancer
Cell
Lines
and RAR-f3 genes. In addition, in nonneoplastic cells and many tumor model systems, RAR-J3 mRNA expression is markedly induced by RA whereas RARunder the control of RA up-regulates its own transcription (1 4, 23, 34, 35, 41). Thus, we also studied the regulation of RARexpression in lu ng cancer cells after treatment with RA. From these studies, we found that the vast majority of lung cancer cell lines growing in vitro exhibit a profound resistance to the effects of RA. We also tested for structural abnormalities in RAR-3 cDNA to account for this resistance.
Results Effed of RA on Growth
Cell Type Normal
HTBE
Neuroblastoma
H82 H21 1
U
H446 of Lung Cancer
Cells in Vitro.
In is
studies with human lung cancer cell line NCI-H82, which reported to respond to RA, we found that morphological effects were reproducibly seen with RA concentrations of 1 pM. These results are similar to those of Doyle eta!. (9), who found that RA caused NCI-H82 to change from “variant” to “classic” morphology (42, 43). In the absence of RA, this SCLC Variant cell line grew in suspension as loose, irregular clusters of cells. RA caused formation of tightly clustered, spheroid colonies, which became evident after about 1 week in culture (data not shown). NCI-H82 is also the lung cancer cell line displaying the greatest growth-inhibitory effect of RA (see below). We tested the effect of RA on the growth of NCI-H82 cells and a human neuroblastoma cell line (CCL-l 27), since neuroblastoma lines have been reported to show RA-induced growth inhibition (9-13), and reproducibly found that RA induced growth inhibition (80% for H82 and 30% for CCL1 27) with these tumor cell lines (Fig. 1). For comparison, we also investigated the effect of RA on the growth of primary HTBE cells in culture and found that 1 M RA had a marked growth-inhibitory effect (>75%) (Fig. 1 ). SCLC line NCI-H82 is amplified for, and overexpresses, c-mycassociated with its variant morphology (42). We therefore tested three other SCLC lines with variant morphology (42, 43), either amplified and overexpressing c-myc (H211 and H446) or amplified and overexpressing N-myc (H526). All of these exhibited modest but statistically significant growth inhibition (28-53%) in response to 1 M RA (Fig. 1). One extrapulmonary small cell carcinoma (NCI-H510, exhibiting classic morphology but amplified and overexpressing L-myc) also showed growth inhibition with RA (Fig. 2A). In contrast, 14 classic SCLC lines, 1 unclassified SCLC line, and 2 lines of small cell carcinomas arising in extrapulmonary sites (NCIHl 341 and Hl 870) failed to show significant growth inhibition when treated with RA (Fig. 2A). Nine of 15 NSCLC lines showed no growth changes with 1 M RA(Fig. 2B). Four lines showed small but significant growth inhibition (2841%), including adenocarcinomas, squamous, and large cell carcinomas, whereas 2 other NSCLC lines (Hl 666 and Hl 651) actually showed growth stimulation with RA treatment (Fig. 28). Thus, the majority (70%) oflung cancer lines demonstrated nogrowth-inhibitoryeffectofRA, and, in most cases, when any growth inhibition was seen, it was quantitatively modest. Search for Gross Abnormalities in the R,4R-j3 and RAR-a Genes by Southern Blotting. Other authors using Southern blotting reported structural abnormalities ofthe RAR-j3 gene in human lung and liver cancer (30, 34) and of the RAR-ct gene in APL and embryonal carcinoma cells (1 6, 29). Thus, we probed DNAdigests of46 cell lines with the whole ORF of RAR-2 (44). None of these exhibited any abnormalities (data not shown). Many tumor DNA samples were digested
-RA
H526
H-I
10
0 Fold Fig.
1.
Growth
After
8 Days
±
20 SD
Effect of 1
M RA on the growth of HTBE cells, neuroblastoma line and SCLC lines exhibiting variant morphology. Cell numbers were after 8 days of growth. Values are means ± SD (bars). All lines significant inhibition of growth with RA treatment (P < 0.05).
CCL-1 27, determined exhibited
with two or more restriction enzymes, which included Mspl, EcoRl, Hindlll, and Psd. We observed the previously descnibed MspI polymorphism for RAR-f3 (45): 5 lung cancer cell lines expressed the 7.7-kb band, 28 lines expressed the 8.1 -kb band, and 1 cell line expressed both alleles (data not shown). Only a single cell line (NCI-H735) was identified which had an abnormal 6-kb band on Mspl digest (data not shown). PstI, EcoRI, and HindIlI digests of cell line H735 displayed a normal pattern of bands on Southern blot, strongly suggesting that the abnormal band represents a previously undescnibed Mspl polymorphism. DNAs from 30 lung cancer cell lines, 6 of which carry cytogenetic abnormalities involving 1 7q2l , the locus of the APL RAR-a rearrangement, were also tested for structural abnormalities in the RAR-a gene, using the whole ORF of RAR-a as a probe. The previously described RAR-a Ps!I polymorphism (46) was noted (2.9- or 3.3-kb bands); however, no abnormalities were found (data not shown). Expression of RAR-fi mRNA in Lung Cancer Cell Lines and the Effed of RA on mRNA Expression. We tested for constitutive expression of all RAR-3 isoforms in a large number of SCLC, extrapulmonary small cell carcinoma, and NSCLC cell lines by Northern blotting. Because many lung cancer cell lines were low level expressors of RAR-3, we exposed the hybridized blots for 10 days. Fig. 3 shows examples of
RAR-
mRNA
expression.
There was a wide
range of RAR-f3
mRNA expression in the lung cancer lines (Fig. 3; Tables 1 and 2). Two sizes (2.9 and 3.3 kb) of RAR-f3 mRNA transcripts were found in some ofthe lung cancer lines, whereas others
expressed
only
the 3.3-kb
transcript.
However,
in the cell
lines expressing very low levels of 3.3-kb RAR-3 mRNA, we cannot rule out the presence of a 2.9-kb band whose level may have been too low for detection by the Northern blotting method (see below). In 24 NSCLC lines studied, 13 (54%) showed no bands, 4 (1 7%) showed one band, whereas 7 (29%) showed two bands (Table 2). There was an absence of RAR-j3 mRNA bands in 7 of 8 lines with squamous differentiation and extremely low levels in the eighth squamous carcinoma line, findings similar to those reported by Houle eta!. (36). In 35 small cell carcinoma lines, 8(23%)
Cell
A Cell
Cell
Line
& Differentiation
B
Line
Effect of RA on the Growth Non-Small Cell Lung Cancer
Hi65i
H2171 Hi 870 H1314 H510 H2196 Hi 994 Hi 963 Hi876 Hi628 Hi 522 Hi5i4 Hi045 H774 H7i i H220 H209 Hi28 H69
Growth
-
Hi334
of Lines
*
Hi i 55 H460 Hi703 Hi563
Effect
of RA on Growth
Small
Cell
and
Lung
of
Cancer
Extrapulmonary
H226 Hi 944
Lines
Hi869
.
Hi792
.
+RA
-RA
Hi666
Hi466
#{149}-RA
Hi 355 ,,-.
Fold
Growth
After
Days ± SD
8
H838
Fold
-4
Growth
After
8
Days ± SD
H522 0
5
0
i-0
Fig. 2. Effect of 1 M RA on the growth of: (A) a panel of SCLC lines and small cell carcinoma lines. The histologic classifications of the various tumor cell lines are given in Tables 1 and legend to Fig. 1. ‘Cell lines showing significant growth inhibition after culturing in RA.
H1792 33kb5kb,(#{216}
GAPDHUI
HTB11
H838
H1628
H128
1et#{248}
H1618
H1334
-:
“UP
Fig. 3. Northern blot analysis ofthe expression of RAR-f3 in lung cancer cell lines and in the neuroblastoma control cell line HTB11. Total RNA (15 pg) isolated from cells exposed to 1 M RA (+) and vehicle I-) (24 h to 11 days) was fractionated, blotted onto nitrocellulose, and hybridized with a probe containing the whole ORF of RAR-f32. The same filters were stripped and rehybridized with a GAPDH probe.
exhibited no detectable transcript, 24 (68%) exhibited one transcript, and only 3 (9%) showed the two transcripts (Table 1 ). In nonneoplastic tissues and many tumor cells, expression of the RAR-f3 gene is up-regulated by RA (23, 35, 41 , 47). Previous studies as well as our preliminary experiments have shown that the up-regulatory effect of RA is very rapid and sustained over a prolonged period of time (23, 35, 48). We tested for RA induction of RAR-3 mRNA in 34 lung cancer cell lines (1 9 SCLC and 1 5 NSCLC). As RA has consistent effects on neuroblastoma cells (41 ), the neuroblastoma line HTB11 was included as a positive control to demonstrate RA induction of RARtranscripts. Very similar results were obtamed for neuroblastoma line CCL-127 (data not shown). Tumor cells were divided into separate cultures and grown in medium containingl M RAorvehicle (0.1% ethanol) for various periods of time, and then RNA was harvested for analysis (we tested periods of 24 h to 11 days exposure to RA). The resulting change in RARtranscript levels was graded as: - (no recognizable or very minor change); weak (2-3-fold increase); and + (at least 4-fold increase) (Fig. 3; Tables 1 and 2). Only five (26%) of the SCLCs and 7 (47%) of the NSCLCs showed any increase in RARmRNA level
iO
lines derived from 2. Values are means
20 extrapulmonary ± SD (bar,)
30
y
40
tumors; (B) a panel of NSCLC the same assays described in
(Tables 1 and 2), whereas 65% of the lines showed no change in RARmRNA with RA treatment. Adenocarcinoma line Hl 792 responded to RA by preferential induction of the 2.9-kb band. In most cases, any increase in RARmRNA levels in lung cancer cells was modest. Reduction in RARmRNA signals was not observed following RA treatment. Poor Correlation of the Effeds of RA on Growth and RARMessage Level in Lung Cancer Cell Lines. In neuroblastoma cells and primary HTBE cultures, growth inhibition was associated with the presence of up-regulation of RAR-3 transcripts by RA. Whereas 5 of 9 lung cancer lines showing growth modulation also showed RARupregulation, several of the lung cancer lines not showing growth-regulatory effects expressed su bstantial constitutive levels of RARmRNA and, in several cases, showed RAinduced RARmRNA up-regulation (Table 3). In addition, RA modulated the growth of some cell lines which did not show RAR-J3 induction and which did not express any detectable RARmessage on Northern blot. This suggests that factors other than RARare involved in mediating the effect of RA on proliferation in some lung cancer cells.
RARmRNA Expression Is Deteded cation from Lung Cancer cDNAs. Since
by PCR Amplifi-
many lung cancer cell lines, with or without RA induction, did not exhibit RARtranscripts by Northern blot analysis, we used PCR amplification of random primed cDNA prepared from total cellular RNAs to search for the presence of very low levels of RAR-f3 transcripts. Normal human tracheobronchial epithelial cells with undetectable levels of RAR-3 mRNA by Northern blot were found to express RAR-j3 mRNA by this procedure after RA induction (Fig. 4). PCR amplifications were performed on random primed cDNAs from 34 lung cancer cell lines using primers common to all RAR-p isoforms, as well as primers specific for RAR-2 and RAR-13 (see “Materials and Methods” for primer details). In all cases, the f3-actin gene could be amplified from cDNAs from all
801
802
RA Effects on Human
Table 1 treatment
Expression with 1 M
Lung Cancer
Cell Lines
of RAR-f3 mRNA retinoic acid
Cell
level s in SCLC
No.of
.
line
lines
be fore
Relative signal strength
.
.
transcripts’
and
after
Table 2 Expression of RAR-f3 with 1 M retinoic acid’
Induction . by retinoic
(uninduced)
.
line
acidc
H60
0
-
H69
2
3+
NT Weak
H128
1
2+
-
H838
H182
1
2#{247}
-
H1355
H209
0
-
-
H1466
H711
1
2+
-
H719
1
+
H735
1
H738
1
H1045
(PCR +)
+
+ -
0
-(PCR+)
-
H1648
0
-
NT
NT
H1666
0
-(PCR+)
-
+
-
H1792
2
2#{247}
b
+
NT
H1869
0
-
NT
0
-(PCR+)
-
H1944
2
3+
+
H1105
0-+2’
-
+
H2122
1
2+
NT
H1185
0
-
NT
H1238
0
-
-
H1284
1
2+
-
H125
0
-
NT
H1339
1
±
NT
H596
0
-
NT
H1450
1
+
NT
H647
0
-
NT
H1514
1
+
NT
H1522
1
+
NT
H1618
1
2#{247}
-
H157
0
-(PCR-i-)
H1628
p
-(PCR-s-)
-
H226
0
-
H1788
1
+
NT
H1264
1
±
NT
H1876
1
+
-
H1563
0
-(PCR+)
-
H1881
1
+
H1703
0
-
H1994
0
-
H2028
1
#{247}
NT
H2059
1
+
NT
H460
2
2+
+
H2141
1
2+
NT
H1155
0
-(PCR-i)
-
H1334
2
+
+
H1385
2
2+
-
+
-
(PCR +)
(PCR
Adenosquamous
-)
NT (PCR
0__,1*
H196
1
±
NT
H446
1
+
Weak
-(PCR+)
H1048
1
+
NT
H1341
1
2+
-
H1870
1
+
-
NSCLC,
2
Northern
Weak
blots.
,
number
transcript;
2, 2.9- and 3.3-kb
of transcript
sizes after
RA
intensity on Northern blots: -, no band seen after 1 0 days exposure; faint or clear bands seen after 1 0 days exposure; 2+, bands seen after 72 h exposure; 3+, bands seen after 24 h exposure. PCR +, RAR-3 cDNA detected by PCR analysis; PCR -, no amplifiable cDNA in RA-untreated or +,
cells.
1
M RA treatment, changes in RAR-f3 significant increase in RAR-f3 mRNA; weak, mRNA; +, >4-fold increase in RARmRNA.
cell lines, as a positive
control.
lines
(H1450
and
NT, not tested; increase in
Most of the lung cancer
lines without detectable RARanalysis showed RAR-j3 mRNA cDNAeven without RA induction
cell
mRNA: 2-3-fold
mRNA on Northern by PCR amplification (Figs. 4 and 6, Tables
Hl28)
footnotes 2.9-kb band.
1 to Table
1 for
explanation
and
definitions.
-
+
1, 3.3-kb
not subclassified
H1651
not subclassified
by
NT
Large cell
b
0
found
(PCR +)
-
+
H510
detected;
NT
#{247})
(PCR
(PCR +)
-
+)
2
Squamous
H82
transcripts induction. b Signal
Two
+
-(PCR+)
no RAR-)3 transcript
After
.
1
H2171
C
Induction by retinoic . acid
0
SCLC,
-treated
signal
strength . (uninduced)
.
Weak
Extrapulmonary
or
Relative
afte r treatment
4+
aSee
±
and
0*2*
H522
Variant
a0,
transcripts
Adenocarcinoma H23
Classic
es before
No. of
Cell
b
in NSCLC-lin
had evidence
-, no RAR-f3
cell blot from 1-3).
of gross
structural changes at the message level. Amplification of cDNA from cell line Hl450 consistently yielded a band which was shorter (-700 bp) than the expected size (777 bp) (Fig. 48). Other workers have reported on genomic DNA abnormalities in SCLC line Hl 28 (34). Although we did not detect genomic DNA changes in Hi 28, we did find changes
in Hl 28 cDNAwhich yielded clear signals when the central portion ofRAR-2 was amplified but notwhen other primers were used, suggesting structural abnormalities both in the 5’ and in the 3’ portion of the ORF (data not shown). PCR/SSCP Analysis of RAR-f3 cDNA. To search for subtle abnormalities of the RAR-j3 mRNA isoforms, PCR amplifications of the random primed cDNAs derived from the
RAR-3
mRNAs
were
performed
followed
by SSCP analysis
(Fig. 5). Five sets of primer pairs were utilized to map the regions common to all RARisoforms in PCR reactions (see “Materials and Methods” for primer details). In addition, another set of primers was also included for mapping to the variable 5’-coding region of the isoforms in which the 5’primer was designed to amplify specifically the RAR-2 or RAR-31,3 isoforms, respectively. As shown in Fig. 6, A-C, among 28 lung cancer cell lines examined, no abnormality of band shift was observed by SSCP analysis. However, several lung cancer lines (Hl 563, Hl 61 8, Hi 792, and Hi 869) failed to amplify the C region where the DNA binding domain resides using primers Ps5601/Pas5602 (Fig. 6C), whereas other regions produced normal SSCP patterns. Amp!ifying parts of the D + E regions using primers Ps5603/ Pas5604, no abnormal band shift was observed (Fig. 6D).
However,
when we extended
the 5’ end ofthe
further upstream by using primers set of PCR products was observed
PCR products
Ps5611/Pas5604, an extra (labeled ‘), suggesting the
Cell
Table 3 Correlation mRNA levels in lung Lung cancer cell line
of effect of retinoic cancer cell lines”
acid
on growth
Relative signal th sreng (uninduced)
No. of transcripts
H82
0-*1’
-
H510
0
-
H1944
2
H460
2
H226
0
-
H446
1
H1334
and
.
Induction transcripts
of
RAR-)3
Effect growth
on (%(
-
-58
3+
+
-41
2+
+
-37
-
-33
+
Weak
-28
2
+
+
-28
H1651
1
+
-
+26
H1666
0
-(PCR+(
-
#{247}102
H1792
2
2#{247}
h
NSDC
H838
1
#{247}
#{247}
NSD
H522
2
4#{247}
Weak
NSD
H69
2
3#{247}
Weak
NSD
(PCR
+(
H2171
2
#{247}
Weak
NSD
H711
1
2#{247}
-
NSD
H1341
1
2#{247}
-
NSD
H1870
1
-
NSD
H1876
1
#{247} #{247}
-
NSD
H209
0
-
(PCR
-
NSD
H1045
0
-
(PCR
-
NSD
H1155
0
-
(PCR
-
NSD
H1355
0
-(PCR+)
-
NSD
H1466
0
-
(PCR
-
NSD
H1563
0
-
(PCR
-
NSD
H1628
0
-
(PCR
-
NSD
H1994
0
-
(PCR
#{247}( #{247}( #{247}( #{247}(
-
NSD
H1238
0
-
(PCR
-)
-
NSD
2
#{247}
#{247}
-30
#{247}( #{247}( #{247}(
1
.a700bP.EI__
B
Neuroblastoma CCL-127
HTBE
cells
a See footnotes b
Significant NSD,
2 to Table
induction
no significant
#{247} (PCR
±
1 for explanation of 2.9-kb
growth
and
-i-)
& Differentiation
A
-87
i-(
(PCR
Growth
549
bp-
-78
definitions.
band.
effect
compared
to controls.
B-actin possible presence of potentially different RAR-/3 isoforms such as 134 (Fig. 6G). Cell lines H82, H209, H838, and Hi 651 expressed only the lower two bands with these primers, which are associated with f31,2, and f33 mRNA. Cell lines H128, H526, H1045, H1563, H1792, Hi869, and H1870 expressed only the upper two bands, whereas other lines (H522 and Hi 944) expressed both sets, and one line (H522), in addition to expressing both sets, had an additional band as well. Expression of RAR-a in Lung Cancer Cell Lines. In contrast to RAR-f3, RAR-a was expressed in 1 5 of 1 9 SCLC cell lines and in all NSCLC lines tested (Table 4). The lines expressing RAR-ct always displayed both 3.5-kb and 2.6-kb
RAR-ct transcripts
(23, 24). None
of the 22 cell lines tested
displayed induction of RAR-a expression by RA (Table 4). RAR-a also was not inducible in primary HTBE cells in vitro (Table 4). This is in agreement with previous studies which reported relatively high steady-state levels of RAR-a message which were not modulated by RA (23, 41).
Discussion We tested a large panel of cell lines representing all major histological types of lung cancer and found that the majority do not exhibit significant growth inhibition after treatment with RA. The majority of the cell lines also do not show
Fig. 4. (Hemi-)nested PCR amplification of cDNA, probed with RAR-13. A cDNAs from lung cancer cell lines H1450 and H510 were amplified using the RAR-f32-specific 5’ primer P1 739 and the antisense primer P1 740. The PCR products were diluted 1 :1 00 and subjected to a hemi-nested second round amplification using primers P1739 and P1749, which amplify the 5’ half of the ORF of RAR-f32. The second round PCR products were electrophoresed, blotted onto a nylon membrane, and hybridized with a probe contaming the whole ORF of RAR-f32. The expected size is 777 bp (H510( with Hi 450 showing a truncated cDNA. 8, cDNAs from lung cancer cell lines and primary HTBE cells were amplified using primers Pt 739 and P2657 (sense) and P2421 (antisense), which amplify the ORF of RAR-f3 isoforms 1 ,3 and 2, respectively. The PCR products were diluted 1 :1 00 and subjected to a second round of PCR amplification using primers Pt 743 and P1 749, which specifically amplify a 549-bp segment common to all isoforms near the center of the coding region of RAR-f3. The second round PCR products were processed as above (A). As a control, the f3-actin gene was amplified from the same cDNAs; the corresponding ethidium bromide-stained gel bands are shown.
increased seen in However, RARhuman mainder analysis. siveness reading
RAR-3 mRNA levels after treatment with RA, as is cultures of normal tracheobronchial epithelial cells. most ofthe lung cancer cell lines actually express mRNA at levels higher than those found in normal tracheobronchial epithelial cells, whereas the reexpress comparable levels detected by cDNNPCR We thus tested to see whether this lack of responto RA resulted from mutations in the RAR-/3 open frame. By SSCP analysis, we found no alterations in
803
804
RA
Effects
RAR-3
on
Human
Lung
Cancer
Cell
Lines
mRNA:
I 5’-UTR
A
IBI C 1 D
I
IFI
E d
(225 bp)
b) Ps 5610/Pea
5609
I
e
(222 bp)
(306 bp) (306 bp)
g
(327 bp)
(368 bp)
Fragmenis
unnunu
Sense Primer
Anhi-sense
(152) Ps1739
()32) Pas5609
b
(t1,3)Ps561O
(1,3)Pas56O9
C
Ps5601
Pas5602
d
Ps5603
Pas5604
e
Ps5605
Pas5608
I
Ps5607
Pas5606
9
Ps5611
Pas5604
#{216}P’
*_:T_
*
$:*
.
I) Ps 5607/Pea
the regions ofthe ORF of RAR-f3 common to all isoforms and the regions specific to /32 or 3t,3 isoforms as examined by PCR/SSCP analysis. However, a few cell lines failed to amplify the DNA binding C region ofthe RAR-f3 cDNA, and one gave an abnormal sized PCR product. These findings mdicate a frequent dysfunction of the RA response pathways in lung cancer cell lines which in some cases is correlated with abnormalities of RAR-3 cDNA but in the majority is not associated with alterations in the RAR-f3 open reading frame. Lechner eta!. (2) reported that low levels of RA (3 X 1 0_to M) enhanced growth of normal HTBE cells cultured in serumfree medium, whereas serum supplementation had a growth-inhibitory effect. Wu eta!. (3) also reported a growthpromoting effect of RA in concentrations from 1 o to 1 O M. Preliminary experiments in our laboratory showed that human lung cancer cell lines exhibited no growth or morphological effects at a wide range of RA concentrations. We thus tested 1 M RA, which had been shown to induce biological effects in a number of model systems (1 3, 34, 35). At this concentration, primary cultures of normal HTBE cells displayed marked inhibition of growth in serum-free medium. In contrast, proliferation ofthe majorityofhuman lung cancer cell lines was not affected. However, among SCLC cell lines, growth inhibition in responseto RAtreatment was found in variant but not classic cell lines. The finding that the
morphological
characteristics after exposure to RA is in agreement with the observations of others, who reported conversion of SCLC variant lines including H82 to a classic morphology associated with growth inhibition and decreased c-myc expression (8-10). In NSCLC cell lines, no correlation was noted between histological subtype (large cell, squamous, or ad-
:
5606
Jtt4
--
---.-
*
it) Ps 561 I/Pea 5604
r
-
.-
-
,*,,,,w .
different
s
Primers
Fig. 5. Schematic diagram of RAR-f3 mRNA and the amplified PCR products used in the SSCP analyses. Top open bar, the mRNA derived from the RAR-)3 gene, which consists of the 5-’-UTR and A-F regions. The lines underneath the mRNAindicatethe relative positionsandthelengthsofthe DNAfragments generated by PCR reactions for SSCP analyses. The sense and antisense primers used for generating the DNA fragments are listed. Due to the divergence of the mRNA sequences around the 5’-UTR and A region among the RAR-f3 isoforms, specific sense primers were used to generate the f32- and f3, ,3-related DNA fragments spanning the A region as indicated.
cell line H82 assumes
:
:i:
;;*ae
a
SCLC variant
nn=nu 5609
(246 bp)
b(
PCR
unn: Ps l739lPas
ii)
*‘
-.-.
.
,.
.--
*, *
..lw-.-
Fig. 6. SSCP analysis of PCR-amplified RAR-j3 cDNA derived from 28 lung cancer lines. Random-primed cDNA from human lung cancer cell lines was subjected to the PCR amplification by the primer sets listed in Fig. 5, and the PCR products were analyzed on a 6% polyacrylamide gel (1 2 by 1 6 in) under nondenaturing conditions. The SSCP patterns in (a) to (g) correspond to the PCR fragments (a) to (g) Illustrated in Fig. 5. The single asterisks (‘) indicate the positions of the separated single-stranded DNA fragments, whereas the double asterisks (“) indicate the positions ofthe re-annealed double-stranded PCR products. ‘ ‘ indicates presence of different RAR-)3 isoforms other than 13i,
132,
or f33.
enocarcinoma) and growth response to RA. Whereas some cell lines showed growth inhibition by RA, growth of two lines was enhanced by RA. Sever and Locker (14) reported similar data on hepatocellular carcinoma cell lines. The biological effects of RA are thought to be mediated by nuclear RARs (23, 28). Several lines of evidence suggested that structural alterations in the RAR-f3 gene may be involved in neoplasia. In fact, RAR-f3 was originally discovered by virtue of hepatitis B virus insertion into this gene in a hepatocellular carcinoma (20, 30). The gene for RAR-f3 is located at 3p24, a region of the human genome which frequently shows allele loss in lung cancer (37-40). Because of this, we and others entertained the possibility that RAR-f3 may function as a recessive oncogene. Previous reports suggested that structural alterations of the RAR-j3 gene may occur in cancer but are infrequent events (30, 34). Although known (45) and unsuspected Mspl polymorphisms were found, we were unable to detect evidence of rearrangements affecting the ORF of the RARgene in 45 lung cancer cell lines by Southern blotting. We did not find the DNA structural abnormality in cell line Hi 28 which was reported by
Cell Growth
Table
4
Expression
of RAR-a
in lung
cancer
cell
lines
on
Northern
mRNA.
blots”
However,
exists between Cell
No. of
line
Relative
transcripts
SCLC H60 H69
signal
2
by retinoic
+
NT
2
±
-
Hi 28
0
-
-
H 182
0
-
-
H209
2
+#{247}
NT
H446
0
-
-
H5i
0
2
++
-
H7i
1
2
+
-
H7i
9
2
++
NT
H735
0
-
-
Hi284
2
+
-
Hi 339
2
+
NT
Hi6i8
2
+
-
Hi628
2
+
-
Hi 788
2
+
NT
Hi876
2
±
-
Hi994
2
+
-
H2i4i
2
+
NT
H2i7i
2
+#{247}
-
acid
RAR-3 message by Northern with squamous differentiation,
is expressed at high levels in squamous cells (22, 28). There is also evidence that RAR-3 and RAR-’y1 mRNA levels are inversely correlated (49). Thus, in squamous carcinoma cells, RAR-f3 expression may be suppressed by high levels of RAR-’y1 . In normal cells and some lung cancer cells, RAR-f3 is expressed as two transcripts (2.9 and 3.3 kb), which may
different
isoforms
(25, 26). The absence
ofthe
or differential 2.9-kb
RAR-f3
polyadenylation transcript
in many
H226 H460
2
+
NT
2
+
-
H522
2
++
-
H1355
2
+
-
Hi 385
2
+
-
Hi466
2
++
-
Hi 563
2
++#{247}
-
mal bronchial
H 1666
2
+
-
H 1703
2
+
NT
Hi 792
2
++
-
H 1944
2
+
-
trast, in studies of 35 cell lines, 75% of SCLC and 53% of NSCLC cell lines showed no change in RARmessage level when grown in the presence of 1 M RA. Of interest, tu-
H2i22
2
+
NT
HTBii
2
+++
CCL-i 27
2
++
HTBE
of our lung cancer
KK
footnotes
2
to Table
lines is currently
unexplained, et a!. (34).
and
epithelial
cells
(14, 23, 35, 41, 50). In con-
morigenic HeLa cells also fail to increase RAR-3 mRNA in response to RA, whereas up-regulation occurs in nontumorigenic hybrid cells (31). In neuroblastoma and normal HTBE cells, an increase in RAR-3 mRNA levels is associated with growth modulation by RA. However, we found no correlation between RAR-j3
#{247}
i for explanation
cell
similar findings were reported by Gebert An RA response element is present in the promoter(s) of the RAR-f3 gene and under the control of RA, RAR-3 will up-regulate its own expression including expression in nor-
cells
070591
expression
-
human
and definitions.
or induction
lung
cancer
cell
on hepatoma
cell
lines
and growth lines.
Similar
probes
but
not with
for this discrepancy is not an abnormality in Hl28 of cDNA occurred with
primer
pairs
near
the 5’ and
3’
ends ofthe ORF. In addition, the PCR amplification product obtained from cell line H1450 cDNA was consistently than
of a deletion
that derived
within
from
the other
the 5’ portion
cell
lines,
of the RAR-3
indicative
coding
re-
gion. Gebert eta!. (34) reported relatively high levels of RAR-j3 mRNA expression in whole human lung obtained at pulmonary resection and in rabbit tracheal epithelial cells. In contrast, Nervi et a!. (35) and the current study found that
RAR-3 expression in human bronchial epithelial cells was extremely low or undetectable by Northern blot analysis. RARlevels may be high in bronchial epithelium during embryogenesis and may change over time (28). Our results indicate that basal levels of RAR-13 mRNA in normal HTBE cells are very low, requiring detection by PCR amplification of cDNA. Relative to normal HTBE cells, many lung cancer constitutively
express
many
fold
higher
levels
of RAR-f3
and HeLa
modulation
by RA in
results
obtained
were
(14, 31). Multiple isoforms existfor all three classes of RAR, and two additional RA receptors, RXR-a and RXR-f3, have been described (24,
Gebert et a!. (34), and the reason obvious. However, we did find cDNA such that PCR amplification
lines
levels
to this
We did not comprehensively study RAR-’y expression in our lung cancer cell lines, but it is known that this receptor
Neuroblastomas
shorter
mRNA
cell lines. An exception
transcripts have so far been described (25, 26). In the lung, isoforms j33 and 13i predominate, whereas isoform 32 is present at low levels (25). This ratio seems to be preserved in lung cancer cells. The j34 isoform is generated in the mouse by alternative splicing and the use ofa CUG initiator codon. Thisfourth isoform could accountfor the SSCPfindings using primer set g spanning the D and E regions of the ORF.
represent
central
in RAR-3
805
and only trace amounts in the remaining line. These data confirm and extend previous reports of RAR-f3 expression in bronchogenic carcinomas (34-36). Four isoforms of RAR-g3
NSCLC
aSee
variation
is the absence of detectable analysis in 7 of 8 cell lines
Induction
strength (uninduced)
marked
lung cancer
& Differentiation
cells
25, 27, 28, 51 , 52). Whether the effect of RA on cell growth is mediated by one given isoform or a complex combination
of different receptors remains to be determined. It has already been shown that there is interaction between different RARs and that expression of RAR-f3 can be influenced by binding of RAR-a or RAR-’y to the RA response 51). In contrast to RAR-f3, RAR-a is ubiquitously
element expressed.
(50, In
other systems, replacement of the A domain of RAR-a by a large portion of the PML gene occurs in most, if not all, APL patients carrying the t(l 5; 1 7) translocation (1 6), whereas structural aberrations of RAR-a were also found in embryonal carcinoma cells (29). We found most lung cancer cell lines to express both the 2.9- and 3.5-kb RAR-a mRNA. Structural changes in RAR-a were not found in lung cancer,
and the majority of the cell lines failing to show growth inhibition by RA expressed substantial levels of RAR-a mRNA. In conclusion, lung cancer cell lines of all histological types frequently fail to respond morphology, growth modulation, is in contrast to normal human
to RA, either by change in or RAR-j3 induction. This tracheobronchial epithelial
806
RA Effects on Human
cells, great
Lung Cancer
Cell Lines
which are dramatically growth inhibited and exhibit increases in RAR-fJ mRNA after treatment with RA.
Thus, lung cancer cells have developed some abnormalities in the RA signaling pathways. Genomic alterations of RAR-3 are uncommon, but abnormal expression is frequently observed, and abnormalities in RAR-j3 cDNA were also found.
Due
to the multitude
of RA-binding
difficult to dissect the molecular in cancer cells. For example,
shown
to cooperate
52); the role
receptors,
with and antagonize
of cytosolic
it may
be
mechanisms of RA action RARs and RXRs have been
RA-binding
each other (49, 51, proteins
is not yet de-
harvested and processed for RNA extraction. At least 1 O tumor cells were obtained for each RNA preparation. Statistical Analysis. For each data point, mean and SD from four to six cell counts were calculated. Student’s ttest was performed in the evaluation of the proliferative effect of RA. For data pairs with P 0.05, the difference in growth rate was considered not statistically different. Where differences in growth rates are reported in the tables,the P-value ranged between 0.01 and
