Heparin and Hormonal Regulation of mRNA Synthesis and Abundance of Autocrine Growth Factors: Relevance to Clonal Growth of Tumors
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1991, p. 108-116 Vol. 11, No. 1
0270-7306/91/010108-09$02.00/0
Copyright © 1991, American Society for Microbiology
Heparin and Hormonal Regulation of mRNA Synthesis and
Abundance of Autocrine Growth Factors: Relevance
to Clonal Growth of Tumors
ISABEL ZVIBEL, ELAINE HALAY, AND LOLA M. REID*
Departments of Molecular Pharmacology and Microbiology and Immunology,
Albert Einstein College of Medicine, Bronx, New York 10461
Received 8 November 1989/Accepted 12 October 1990
Highly sulfated, heparinlike species of heparan sulfate proteoglycans, with heparinlike glycosaminoglycan
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
chains, are extracellular matrix components that are plasma membrane bound in growth-arrested liver cells.
Heparins were found to inhibit the growth and lower the clonal growth efficiency of HepG2, a minimally
deviant, human hepatoma cell line. Heparan sulfates, closely related glycosaminoglycans present in the
extracellular matrix around growing liver cells, had no effect on the growth rate or clonal growth efficiency of
HepG2 cells. Neither heparins nor heparan sulfates had any effect on the growth rate or clonal growth efficiency
of two poorly differentiated, highly metastatic hepatoma cell lines, SK-Hep-l and PLC/PRF/5. Heparin's
inhibition of growth of HepG2 cells correlated with changes in the mRNA synthesis and abundance of
insulinlike growth factor II (IGF II) and transforming growth factor beta (TGF,I). HepG2 cells expressed high
basal levels of mRNAs encoding IGF II and TGFP that were inducible, through transcriptional and
posttranscriptional mechanisms, to higher levels by specific heparin-hormone combinations. For both IGF II
and TGF,, the regulation was multifactorial. Transcriptionally, IGF II was regulated by the additive effects
of insulin, glucagon, and growth hormone in combination with heparin; TGF(i was regulated primarily by the
synergistic effects of insulin and growth hormone in combination with heparin. Posttranscriptionally, the
mRNA abundance of the IGF II 4.5- and 3.7-kb transcripts was affected by insulin. Heparin induction of all
IGF II transcripts was also dependent on triiodotyronine and prolactin, but it is unknown whether their
induction by heparin was via transcriptional or posttranscriptional mechanisms. Heparin-insulin combinations
regulated TGFPi posttranscriptionally. The poorly differentiated hepatoma cell lines PLC/PRF/5 and SK-Hep-l
either did not express or constitutively expressed low basal levels of IGF I, IGF II, and TGFIA, whose mRNA
synthesis and abundance showed no response to any heparin-hormone combination. We discuss the data as
evidence that matrix chemistry is a variable determining the expression of autocrine growth factor genes and
the biological responses to them.
Previous studies from our laboratory (10) have indicated passage HepG2 cells, which are highly differentiated and
that the extracellular matrix contains a set of factors af- nonmetastatic (29), were obtained from Barbara Knowles
fecting the clonal growth efficiency of tumor cells, a charac- (Wistar Institute, Philadelphia, Pa.).
teristic important for the ability of tumor cells to metastasize Culture conditions. (i) Substrates. Cells were plated di-
and colonize specific tissues. The matrix components re- rectly onto 100- or 150-mm tissue culture plastic dishes
sponsible for this phenomenon include species of highly (Falcon).
sulfated, heparinlike heparan sulfate proteoglycans or their (ii) Media. The hepatomas were cultured in RPMI 1640
glycosaminoglycan chains, heparinlike heparan sulfates (10). (GIBCO, Grand Island, N.Y.) supplemented with penicillin
We are now trying to elucidate the mechanism by which (100 ,ug/ml) and streptomycin (100 ,ug/ml). This medium was
heparins and heparinlike heparan sulfates or their proteogly- further supplemented with 10% fetal bovine serum (GIBCO
can forms can differentially affect the clonal growth effi- Hyclone) to produce serum-supplemented medium (SSM) or
ciency of metastatic versus nonmetastatic carcinomas. Our with a defined mixture of trace elements, hormones, and
working hypothesis, tested in these studies, has been that growth factors (see below) to produce a serum-free, hormon-
these heparins or heparinlike molecules regulate the synthe- ally defined medium (HDM). The HDM used for the hepa-
sis of autocrine growth factors thought to be involved in tomas was that designed for growth of hepatoma cells on
low-density growth. tissue culture plastic and described in detail elsewhere (11,
19). It contains insulin (100 ,ug/ml; Sigma), transferrin (10
MATERIALS AND METHODS ,ug/ml; Sigma), glucagon (10 ,ug/ml; Sigma), hydrocortisone
Human hepatoma cell lines. PLC/PRF/5, established by (108 M; Sigma), triiodotyronine (10-' M; Sigma), prolactin
Alexander et al. (1), expresses some liver-specific functions (2 mU/ml; Sigma), growth hormone (GH) (10 ,uU/ml; Sigma),
(29) and was obtained from I. Millman (Fox Chase Cancer linoleic acid bound to bovine serum albumin (10 ,ug/ml;
Center, Philadelphia, Pa.). SK-Hep-1, a relatively anaplastic Pentax), zinc (10-10 M), selenium (3 x 10-1o M), and copper
cell line (15), was a gift from Jorgen Fogh (Sloan Kettering (10-' M). The trace elements were obtained from Johnson
Institute, Walker Laboratories, New York, N.Y.). Early- Matthey Chemicals (London, England).
Sources for heparins and heparan sulfates. Reference stan-
dards for bovine lung-derived heparins and for bovine lung-
*
Corresponding author. derived heparan sulfates were provided by Larry Rosenberg,
108VOL . 1 l, 1991 HEPARIN AND HORMONAL REGULATION OF mRNA SYNTHESIS 109
who obtained them from Martin Matthews and J. A. Ci- (New England Nuclear), and the RNA-containing filters
fonelli, University of Chicago. Commercially available bo- were prehybridized and then hybridized with the appropriate
vine lung-derived heparins were obtained from Sigma. The probes. The cDNA clones complementary to specific
biological activity of reference standards was quite repro- mRNAs (listed below) were radioactively labeled by primer
ducible, but that of commercially available heparins varied extension as described by Feinberg and Vogelstein (14).
widely from batch to batch. Nevertheless, the scarcity of [32P]dCTP (specific activity, 3,200 Ci/mmol) was included to
glycosaminoglycan reference standards forces investigators obtain a specific activity of 8 x 108 to 12 x 108 cpm/,Lg of
to use the commercial heparins of weaker and variable DNA. DNAs used in hybridization included those comple-
specific activity. These findings are analogous to those mentary to mRNAs encoding insulinlike growth factor I
reported by Castellot et al. (5) in their structure-function (IGF I) and insulinlike growth factor II (IGF II), transform-
analysis of heparin effects on inhibition of growth of smooth ing growth factor alpha (TGFa), transforming growth factor
muscle cells. The data shown are from Sigma's bovine beta (TGF,B), and 18S RNA.
lung-derived heparin, lot 53F-0532. The cultures were The cDNA probe for human IGF I was plasmid phigf 1,
treated with 20 to 50 p.g of this heparin per ml, a concentra- containing a human liver-derived cDNA with a PstI insert of
tion previously shown to regulate tissue-specific gene 662 bp of prepro-IGF I (2). The cDNA encoding human
expression in normal and neoplastic liver cells (18, 47). prepro-IGF II was plasmid phigf 2, containing an internal
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
Clonal growth assays. Cells were plated in triplicate for PstI insert of 1,090 bp (2, 3). The plasmid for mouse TGFa
each condition at 100, 103, 104, and 105 on 60-mm tissue contained an EcoRI insert of 925 bp. These three plasmids
culture plates and in SSM. After 18 h, the plates were gently were the kind gifts of G. I. Bell (Howard Hughes Institute,
rinsed and refed. The test media consisted of SSM with and University of Chicago). The murine TGFPi was an EcoRI
without either bovine lung-derived heparin (50 ,ug/ml) or cDNA insert of 1,600 bp (a kind gift of R. Derynck, Genen-
bovine lung-derived heparan sulfate (50 p.g/ml). The cultures tech, South San Francisco, Calif.). The cDNA encoding
were incubated for 2 to 3 weeks with weekly medium DHFR derived from hamster cells contained an insert of
changes and then stained with 1% acridine orange. The 1,900 bp and was a gift of P. Melera (University of Maryland,
number of colonies per plate was counted. Clonal growth Baltimore).
efficiency was calculated as (number of colonies/number of Nuclear transcript run-on assays. The method is a modifi-
cells seeded) x 100. The experiment was replicated five cation of that described by Clayton and Darnell (7). After 96
times. h in culture, one or two 150-mm plates were scraped with a
Growth curves. Cells were plated in triplicate for each rubber policeman into PBS; the cells were washed twice
condition at 105 cells per 60-mm dish in SSM. After 24 h, the with PBS and centrifuged at 800 x g for 5 min. Then 5 ml of
plates were rinsed with phosphate-buffered saline (PBS) and isotonic buffer (140 mM NaCl, 10 mM Tris [pH 7.4], 1 mM
fed SSM with or without bovine lung-derived heparin (50 MgCl2) and 0.5% (final concentration) Nonidet P-40 were
jig/ml) or bovine lung-derived heparan sulfate (50 ,ug/ml). added for 5 min on ice. The nuclei were pelleted by centrif-
Medium changes were done twice weekly. Triplicate plates ugation at 1,000 x g for 5 min, and the supernatant was used
per condition were used for cell counts on days 1, 3, 7, 10, for cytoplasmic RNA extraction. The nuclei were washed
and 14. The average cell numbers per day were plotted on with 5 ml of 10 mM Tris (pH 8.1)-20% glycerol-140 mM
semilog paper. The doubling time was determined from the KCI-5 mM MgCl2-1 mM MnCl2-14 mM P-mercaptoethanol,
slope of the curve during log-phase growth of the cells. The resuspended in 1 ml of the same buffer, and frozen in liquid
growth curves were repeated three times. nitrogen.
Molecular hybridization assays. Nuclear transcript run-on On the day of the experiment, the nuclei were thawed at
assays and Northern (RNA) blots (see below) were used to 37°C, centrifuged at 1,000 x g for 5 min, and suspended in
determine the synthesis and abundance of mRNAs encoding 200 IlI of complete reaction buffer (10 mM Tris [pH 8.0], 140
various autocrine growth factors. The hepatomas were mM KCI, 5 mM MgCl2, 14 mM dithiothreitol, 1 mM each
plated onto 100- or 150-mm culture dishes under the condi- ATP, GTP, and CTP, 20% glycerol, 100 ,ug of creatine
tions specified, and the medium was changed 6 h after plating phosphokinase per ml, 10mM creatine phosphate, and 1 mCi
and then again after 48 h. The cells were assayed after % h of [32P]UTP [specific activity, 3,000 Ci/mmol; Amersham]
in culture. In each experiment, cells were pooled from two to per ml).
three dishes per culture condition. The experiments were The pellets were incubated in the complete reaction buffer
run at least three times. The data from all autoradiograms of at 30°C for 15 min with gentle shaking. Then 1.5 ml of 500
either run-on assays or Northern blots were scanned with an mM NaCl-10 mM Tris (pH 7.4-S50 mM MgCl2 was added
Quantimet densitometer (model 920; Manufacturer's Cam- along with DNase so that the final concentration was 100
bridge Instrument). The data for each of the autocrine U/ml. Then EDTA was added to a final concentration of 10
growth factor genes were normalized to that of a common mM (pH 8.0), sodium dodecyl sulfate (SDS) was added to a
gene used as an internal control. Dihydrofolate reductase final concentration of 0.5%, and proteinase K was added to
(DHFR) was the internal control for the nuclear transcript a final concentration of 0.4 mg/ml. The samples were incu-
run-on assays; 18S RNA was the internal control for the bated for 30 min at 37°C, after which 4.5 ml of ETS buffer (10
Northern blots. The expression of these two genes was mM Tris [pH 7.5], 0.5% SDS, 10 mM EDTA) was added.
found not to alter under the experimental conditions used. The RNA was purified from the nuclei by standard hot
Northern blots. The cells were washed twice with 10 ml of phenol-chloroform extractions, followed by ethanol precip-
cold PBS, removed from culture dishes with a rubber itation. To eliminate unincorporated label, the samples were
policeman, and pelleted; cytoplasmic RNA was isolated by trichloroacetic acid precipitated, followed by at least two
the isotonic buffer-Nonidet P-40 lysis method, followed by rounds of ethanol precipitation. The same number of counts
phenol-chloroform and chloroform extractions (31). RNA per minute from each RNA sample was added to hybridiza-
samples were resolved by electrophoresis through 1% agar- tion bags containing nitrocellulose filters to which 5-,ug
ose, submerged-slab, denaturing formaldehyde gels in amounts of different cDNA plasmids were bound. The filters
MOPS buffer (31). RNA was transferred to GeneScreen had been prehybridized overnight and were then hybridized110 ZVIBEL ET AL. MOL. CELL. BIOL.
TABLE 1. Influence of heparin and heparan sulfate on growth of hepatoma cellsa
Doubling time (h)
Cell line Plating
density Control colonies
No. ofHP HS Control HP HS
HepG2b 102 2.8 ± 1.7 0 5.3 ± 2.3
(7 3) (5 2) (10 ± 3)
103 14 5 0 12 ± 3
104 Too many to count 64 ± 11 Too many to count
105 Too many to count Too many to count Too many to count 29±2 58±6 31±4
SK-Hep-1 102 28 ± 5 21 ± 3.8 22 ± 5
10-3 Too many to count Too many to count Too many to count
105 Too many to count Too many to count Too many to count 34±4 31±4 28±3
PLC/PRF/5 102 7.3 ± 2.7 15 ± 3.9 15.3 + 4
103 46 6.8 53 7.2 55 ± 7.5
105 Too many to count Too many to count Too many to count 28+3 26±2 24±4
a Data are from three to five experiments for the clonal growth efficiency assays and three experiments for growth rates (doubling times). HP, Bovine
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
lung-derived heparin (NIH reference standard obtained from M. Matthews and J. A. Cifonelli), used at 50 ,ug/ml. HS, Bovine lung-derived heparan sulfate
(reference standard obtained from M. Matthews and J. A. Cifonelli), used at 50 to 100 Rg/ml.
b Data are from experiments with the original heparin-sensitive subline of HepG2. Data in parentheses (102 seeding density) are for the same subline after
responsiveness to heparins had waned.
for 48 h at 37°C in a buffer containing 5 x SSC (SSC is 0.15 levels in PLC/PRF/5 and SK-Hep-1 cells; only faint bands
M NaCl plus 0.015 M sodium citrate), 0.1% SDS, lx for IGF II were observed.
Denhardt solution, 50% formamide, 50 mM sodium phos- Heparin modulation of autocrine growth factor expression
phate, and 500 ,ug of yeast tRNA per ml. The nitrocellulose in the human hepatoma cell lines. Heparin treatment of the
filters were washed four times at 60°C for 20 min each time cells altered the expression of the autocrine growth factors in
in 1 x SSC-0.1% SDS and then twice at 60°C for 45 min each HepG2 but not in PLC/PRF/5 and SK-Hep-1 cells. Repre-
time in 0.2x SSC-0.1% SDS. They were incubated 30 min at sentative findings from one of three experiments are shown
37°C with 10 gig of RNase A per ml in 2x SSC and then in Fig. 2. HepG2, PLC/PRF/5, and SK-Hep-1 cells were
washed for 1 h at 37°C with 2x SSC. The filters were cultured in SSM or in HDM in the presence or absence of
exposed to Kodak X-Omat film with two intensifying screens bovine lung heparin (20 jig/ml). Steady-state mRNA levels of
at -700C. IGF II and TGF, were increased in HepG2 cells cultured
with heparin (Fig. 2). By contrast, heparin had no effect on
RESULTS any of the autocrine growth factors' mRNA synthesis or
abundance in either SK-Hep-1 or PLC/PRF/5 cells (Fig. 2).
Heparin effects on clonal growth efficiency of metastatic and Thus, only the HepG2 cell line proved regulatable by hep-
nonmetastatic hepatoma cells. Heparins, derived from bovine arin.
lung (Table 1) or porcine intestine (data not shown), were After 1.5 years of studies on the HepG2 subline in the
found to inhibit the growth of HepG2 cells plated at cell laboratory (passage number unknown), the heparin regulat-
densities above 104/60-mm dish and to eliminate HepG2 ability waned. All sublines of HepG2 tested from the Amer-
survival at all cell densities at or below 103/60-mm plate. ican Type Culture Collection and from a number of labora-
Heparins had no effect on the growth rate or clonal growth tories also proved insensitive or relatively insensitive to
efficiency of PLC/PRF/5, SK-Hep-1, or the American Type heparins. However, an early-passage (passage 82) HepG2
Culture Collection-derived subline of HepG2. Bovine lung-
derived heparan sulfates had no effect on growth at any
density of any of the cell lines. U- Ln Lr)
Autocrine growth factors produced by metastatic and non- LL- I-
LL
metastatic hepatoma cell lines. To test our hypothesis that c\J L a.
cr - c
C'%JcL
-
0.
heparins affect production of autocrine growth factors, we C-D -, 0
cL.j IX C:D D
screened for various autocrine growth factors in three hu- 0:L. _)
a-
0
I CL c Co U
0 W )
r
I
CL c)
man hepatoma cell lines: HepG2, a minimally deviant cell
line, and two poorly differentiated hepatoma cell lines,
PLC/PRF/5 and SK-Hep-1. Northern blots of the cytoplas-
mic RNA from the cell lines cultured for 96 h in SSM were
hybridized with cDNA probes for a battery of autocrine 28S- '- W'1-"I
growth factors (Fig. 1). HepG2 expressed significant basal
levels of IGF II and TGF, and lower levels of IGF I. The
IGF I probe bound to a transcript of 5.3 kb in all three cell 18S-
lines, an mRNA size identical to that reported for human
fetal liver (20). In the adult human liver, the mRNA sizes GFI IGFIE TGFa TGF
observed for IGF I are 7.7, 5.3, and 0.9 kb. Similarly, the FIG. 1. Northern blots with cytoplasmic RNA (20 ,ug per lane)
IGF II mRNA species present in HepG2 cells were the fetal extracted from hepatoma cells grown for 4 days in SSM. Cytoplas-
ones: 6.0-, 4.5-, 3.7-, and 2.2-kb species. However, the mic RNA was extracted and run in 1% agarose-formaldehyde gels,
2.2-kb transcript was not expressed consistently. In the blotted to GeneScreen, and hybridized with [32P]dCTP primer
human adult liver, the size of IGF II mRNA has been shown extension-labeled cDNA coding for the specified autocrine growth
to be 5.3 kb (2). Only TGF, was expressed at significant factor. Shown are representative blots from one of five experiments.VOL . 1 l, 1991 HEPARIN AND HORMONAL REGULATION OF mRNA SYNTHESIS ill
A HepG2 PLC/PRF/5 SK/Hep-1
I II I-
I A ^
CD 0 -a -C -a
-a
kb 0 VI
LI
0 0
m~ c1r) :: I
6.0- m .I .I I I
E'E'c ,ii
4.5 - I I I
3.7 -
6.0 -
4.5 -
3.7 -
Heparin: - + - + - + - + + +
(209g/rnI) SSM HOM SSm HDM SSM HDM
B
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
B
"*""ww"_wwwu
18S
Bss CL
0. 0 0.
C N.(L
L
I
__
4
-
0)
U
() l_
CD
c1c. I + +
= =:
kb
2.5 - -- _ _- _0 _ 6
HepG2 SKHepI
FIG. 2. Northern blots of cytoplasmic RNA from human hepa-
toma cell lines cultured for 4 days in SSM or HDM in the presence FIG. 3. Northern blots of cytoplasmic RNA from the human
or absence of bovine lung heparin (hep) (20 ,ug/ml). Cytoplasmic hepatoma cell line HepG2 cultured in HDM from which different
RNA was extracted after 4 days, and 20 ,ug of cytoplasmic RNA per hormones were omitted. Bovine lung heparin (20 ,ug/ml) was added
lane was run in a 1% agarose-formaldehyde gel and hybridized with to half of the cultures incubated with each of these defined media.
primer extension-labeled cDNA probes. Shown are representative The Northern blots were prepared by using 5 ,ug of cytoplasmic
blots from one of three experiments. (A) IGF II probe; (B) the same RNA per lane and hybridized with 32P-labeled cDNAs. The blots are
blot after stripping and rehybridizing with an 18S RNA probe; (C), representative of those from three experiments. (A) IGF II probe;
TGF,3 probe. (B) the same blot stripped and rehybridized with an 18S RNA probe;
(C) TGFI probe. hep, Bovine lung heparin; ins, insulin; gluc,
glucagon; T3, triiodotyronine; pro, prolactin.
subline obtained from Barbara Knowles (Wistar Institute)
showed heparin sensitivity even greater than that of the
subline with which we had originally been working. Except each regulated by distinct hormone-heparin combinations
for the medium controls (see Fig. SC and 6), the data shown (Fig. 3A and 4A). The addition of heparin to complete HDM
are either from our original subline or from this early- resulted in an increase in the abundance of all three mRNA
passage subline. transcripts of IGF II. The omission of insulin from HDM
Effects of hormones and heparin on steady-state mRNA eliminated the heparin induction of the 6.0-kb transcript. The
levels for IGF II and TGFji in HepG2 cells. Other studies in lack of insulin also resulted in an increase in the basal
our laboratory (18, 37, 47) have shown that heparin can expression of the 4.5-kb and especially of the 3.7-kb tran-
affect mRNA synthesis and abundance of liver-specific scripts; the addition of heparin did not induce the mRNA
genes by acting in concert with hormones, each gene being abundance further. The omission of glucagon from HDM
regulated by two to three specific hormones. We investi- had no significant effect on either the basal or heparin-
gated whether autocrine growth factor genes are similarly inducible levels of the three mRNA species encoding IGF II.
affected by heparin. Replicate plates of HepG2 cells were Deletion of triiodotyronine, GH, or prolactin from HDM did
grown for 4 days in HDM or in HDM in which individual not affect the basal levels of expression but eliminated the
hormones were omitted and in the presence or absence of heparin-induced mRNA accumulation of all three mRNA
bovine lung-derived heparin (20 to 50 ,ug/ml). In parallel, we transcripts of IGF II.
tested the effects of each of the hormones added alone and TGF, mRNA levels (Fig. 3C and 4B) were also increased
with or without heparin in serum-free RPMI 1640. The by heparin supplementation of HDM. In cultures in HDM
RNAs from these cultures were hybridized with radiolabeled from which GH was omitted, the TGF, basal level was
probes for IGF II and TGFP. We detected three IGF II elevated, and heparin addition did not induce it further. In
mRNA transcripts of 6.0, 4.5, and 3.7 kb and only sometimes cultures in HDM from which insulin was omitted, the basal
a 2.2-kb species. The 6.0-, 4.5-, and 3.7-kb transcripts were level of TGF, was reduced and heparin induction was112 ZVIBEL ET AL. MOL. CELL. BIOL.
A IGF I A C
(L CL)
0.4 H 3.7kb Transcript ~~~~~~~~~~~~~~~~~~~~C_ . L
0.3 DHFR
0.2 [triri
H H IGF II
I ..
..
t 0.1 TGFP S. - _ -
B
I H H 4.5kb Transcript
to 0.3
) CDL
= 0.2
-c 4- 4 + +
.- 0.1 (/) U) u) en U) c.
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
a c c c C c c
a: H ep
+ +, + +: 4 +
H kb
6.0TrHnscript
0.4 | H~~H H~~~~~O Conttrol DHFR
0.3 arin IG FII _r - -
ig/mi) TGF,
0.2 _s - - _w _ _
I;~~~~~~~~~~ Hepc
0.1 c RPM1i R PM i. heF9
oGEM
HDM -Insulin -Glucogon -T3 -Growth -Prolactin
Hormone
B TGF-0 0 Control ifj.F II
X Heparin TGF :. _ _-
0.4 (SOg/ml)
FIG. 5. Nuclear transcription run-on assays of HepG2 cells
U~~~~~~~~~~~ cultured for 4 days. The blots are representative of those from one
03 H of three experiments. (A) Expression in cells in HDM and in
derivative HDM from which specific hormones were deleted. Half
of the cultures maintained in each medium were also treated with
bovine lung heparin (50 ,ug/ml). Note the expression of DHFR used
0.2
0.1
-
as an internal control. (B) Expression in cells in serum-free RPMI
1640 supplemented with one or two hormones at a time in the
presence or absence of bovine lung heparin (50 ,ug/ml). (C) Expres-
sion in HepG2 cells (the American Type Culture Collection subline)
HDM -Insulin -Glucagon -Growth cultured in serum-free RPMI 1640 (no hormones or growth factors)
Hormone alone or supplemented with bovine lung heparin (50 ,ug/ml). These
FIG. 4. Histograms showing the relative mRNA abundances of control experiments could not be repeated with the early-passage
the TGF3 and the three IGF II transcripts in HepG2 cells cultured (passage 82) HepG2 subline obtained from Barbara Knowles since
in HDM with or without specific hormone deletions and with or that subline did not survive in RPMI 1640 without hormones. a-tub,
without heparin. The histograms derive from densitometry readings a-Tubulin. Other abbreviations are as for Fig. 3.
of autoradiograms from one of three replicate experiments. The data
were normalized to 18S RNA expression under the same conditions.
glucagon and especially with GH (Fig. 5A and B; Fig. 6A). In
eliminated. HepG2 cells cultured in the presence of insulin experiments in which hormones were deleted one by one
alone in a serum-free medium had high mRNA abundance from HDM (Fig. 5A), the omission of either insulin, gluca-
for TGF,, and addition of heparin increased TGFO and gon, or GH caused a decrease in the inductive effect of
mRNA abundance even more. heparin on the transcription rate of IGF II compared with
Effects of heparin and hormones on the transcription rates that in cells in complete HDM (Fig. 5A).
of IGF II and TGFI mRNAs in HepG2 cells. Both TGF,3 and Multiple hormones also affected the transcription rate of
IGF II were regulated by the additive or synergistic effects of TGFP. The transcription rate of TGFI mRNA (Fig. 5A and
multiple hormones in combination with heparin. By con- B; Fig. 6B) was induced weakly (twofold) by heparin in
trast, RPMI 1640 alone or RPMI 1640 with heparin showed combination with insulin plus glucagon. However, the peak
no significant effect on transcription rates of IGF II or TGFP transcription rates (13-fold induction) were observed in
(Fig. 5C). Heparin treatment of cells cultured in complete heparin-treated cells in complete HDM or in a serum-free
HDM resulted in a three- to fourfold increase in the mRNA medium supplemented only with insulin and GH (Fig. 6B).
synthesis rate. Heparin-insulin combinations produced a Deletion of insulin from the complete HDM resulted in a loss
small increase in the transcription rates of IGF II mRNA of heparin induction of the transcription rate. Deletion of
(Fig. 5A and B; Fig. 6A). Additive effects were observed in glucagon resulted in a small reduction in the transcription
cells treated with heparin and with insulin combined with rate.VOL . 1 l, 1991 HEPARIN AND HORMONAL REGULATION OF mRNA SYNTHESIS 113
IGF 11 proliferation of others such as smooth muscle cells and
epithelia (5, 6, 26, 38, 39, 54). The mitogenic effect on
Relative Transcription Rate endothelia was due to fibroblast growth factor, which re-
7 1A mained fully active after binding with high affinity to a
EI Control _ Heparin (lung) pentasaccharide sequence in the heparin species made by
endothelia (52). The heparin inhibition of growth of smooth
5 muscle cells has been found to be due to a saccharide
sequence in certain heparins that binds to and results in
4-
down-regulation of the epidermal growth factor receptor
3 without effects on the insulin receptor or platelet-derived
growth factor (5, 38, 39, 54). In recent studies, Conrad and
associates have suggested that heparin, by itself, can also
regulate cell growth through unique changes in its chemical
structure that occur in a density-dependent fashion (13) and
through its translocation to the nucleus of the cell (26).
*Ins +Ins +Ins HDM HDM-InsHDM-GlucHDM-GH Heparin effects on the expression of autocrine growth fac-
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
RPMI
+Gluc +GH
tors in metastatic versus nonmetastatic carcinoma cell lines.
TGF B Since autocrine growth factors are thought to be critical for
low-density growth of tumors (46), we tested the hypothesis
that heparins inhibit growth of minimally deviant tumors by
regulating the synthesis of autocrine growth factors in the
cells. As predicted by our hypothesis, autocrine growth
factor synthesis was regulatable by heparins in the HepG2
cell line but not in two metastatic cell lines, SK-Hep-1 and
PLC/PRF/5. Contradicting the hypothesis were the data
showing that the highest basal levels of the autocrine growth
factors were evident in the highly differentiated, nonmeta-
static HepG2 cells, and heparins induced the autocrine
growth factors in HepG2 cells to even higher levels. Thus,
heparins, which we previously (10) showed caused cell
differentiation and inhibition of growth in normal hepato-
*lns *lns *lns HDM HDM-lnsHDM-GlucHDM-GH RPMI cytes and in minimally deviant tumor cells, also result in
*Gluc +GH greatly elevated levels of specific autocrine growth factors.
FIG. 6. Histograms showing the effects of heparin-hormone Heparin effects on autocrine growth factors are through
combinations on the transcription rates of mRNAs for the genes potentiation of hormonal regulation. In past reports, there
indicated on top. The data were normalized to DHFR expression has been a paucity of data to identify circulating hormones
under the same conditions. The run-on assays were performed three
times with similar results. Representative experiments were chosen
that might regulate IGF II or TGFPi (36, 53). In adult liver,
and used for calculating the histograms. The data for RPMI 1640 IGF I but not IGF II is under transcriptional control by GH
with or without heparin were from an independent experiment using (32). In HepG2, heparin regulation of synthesis of autocrine
the original subline of HepG2; these controls could not be repeated growth factors was via potentiation of hormone effects.
with the early-passage (passage 82) subline since that subline did not TGF, and each of the transcripts of IGF II required the
survive in RPMI 1640 without hormones. The calculations were presence of specific hormones for heparin induction of
made as follows: (optical density for each autocrine growth factor mRNA synthesis or abundance. Moreover, the heparin
gene optical density for pGEM)/(optical density for DHFR under effects were neutral, stimulatory, or inhibitory on expression
the same condition optical density for pGEM). (A) IGF II; (B)
-
of TGFi or of specific transcripts of IGF II, depending on
TGF,B. Ins, Insulin; Gluc, glucagon. which heparin-hormone combination was used. Heparin in
the presence of GH and insulin was stimulatory for TGF,B
expression and involved predominantly a transcriptional
DISCUSSION control mechanism. By contrast, the heparin effect on IGF II
Highly sulfated heparan sulfate proteoglycans, with hep- mRNA levels involved both transcriptional and posttran-
arinlike glycosaminoglycan chains, are plasma membrane- scriptional mechanisms. Therefore, the specificity of the
associated extracellular matrix components of growth-ar- influence of heparin is dictated by the specific hormones.
rested liver cells (13, 26-28). Heparins were found to inhibit The differential regulation of the abundance of the three
growth and to lower the clonal growth efficiency of a IGF II transcripts by distinct groups of hormones and
minimally deviant hepatoma cell line, HepG2. Heparan growth factors (Fig. 3 and 4) could offer an explanation for
sulfates, similar to the poorly sulfated glycosaminoglycan why the different transcripts are found in specific tissues and
chains on heparan sulfate proteoglycans produced by grow- in tissues of different developmental stages. The human IGF
ing liver cells (13, 41), had no effect on HepG2 growth or II gene is transcribed from three promoters, which are both
clonal growth efficiency. Neither heparins nor heparan sul- developmentally regulated (8) and tissue specific (25, 44).
fates had any effect on the growth or clonal growth efficiency The factors affecting IGF II mRNA synthesis from these
of two highly metastatic hepatoma cell lines, PLC/PRF/5 and three promoters are not known. Our data are the first to
SK-Hep-1. The insensitivity of the poorly differentiated suggest possible regulatory signals. We found that insulin,
hepatomas to heparin regulation probably results from their glucagon, and GH were required for heparin induction of the
degradation by tumor cell-derived glycosidases (33). transcription rate of IGF II. The promoters identified for
Heparins have been shown to promote the growth of some IGF II are P1, found active only in adult liver and giving rise
cell types such as endothelia (16, 52) and to inhibit the to a 5.3-kb mRNA; P2, which yields a 6.0-kb as well as a114 ZVIBEL ET AL. MOL. CELL. BIOL.
2.2-kb transcript in fetal tissues; and P3, which yields a hypothesize that the plasma membrane glycosaminoglycan
4.8-kb mRNA species that is also present in many fetal chemistry may influence the signal transduction mechanisms
tissues. Our studies show that the major transcripts of IGF II by growth factors such as IGF II and TGF,. At low growth
in HepG2 cells are 6.0, 4.5, and 3.7 kb. A 2.2-kb transcript factor concentrations or in the presence of heparan sulfates
was observed in some experiments. Thus, insulin, GH, and (produced when cells are in a state of growth), these factors
glucagon likely affect the P2 and P3 promoters; it is unclear would be mitogens. At high concentrations or in the pres-
whether the P1 promoter is affected. In addition, elimination ence of heparinlike glycosaminoglycans (produced when
of insulin resulted in a relative increase in the mRNA cells are at high density or in the quiescent state), the factors
abundance of the 4.5-kb transcript and especially of the would be differentiation signals. Highly metastatic tumor
3.7-kb transcript through posttranscriptional mechanisms, cells either do not produce high enough concentrations of the
suggesting that there may be a negative response element in growth factors or never generate the highly sulfated, hep-
the coding transcript sensitive to insulin and resulting in arinlike heparan sulfate proteoglycans that are hypothesized
lowered mRNA stability. Although seemingly contradictory to drive the signal transduction process towards a differen-
findings, perhaps the data implicate a feedback loop that tiation pathway. This possibility is supported by the fact that
maintains a stable level of IGF II. Loss of insulin results in tumor progression and the loss of differentiation in hepato-
increased stability; presence of insulin results in lowered mas is associated with loss in the activities of an epimerase
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
stability but increased synthesis. Triiodotyronine and pro- and sulfatransferases responsible for the conversion of the
lactin were also found necessary for heparin induction of all poorly sulfated to the highly sulfated forms of heparan
three transcripts. However, we have not tested whether sulfate proteoglycans (41). Other growth factors have been
their effects are via transcriptional or posttranscriptional shown to have biphasic functions: epidermal growth factor
mechanisms. and insulin are both mitogens for adult hepatocytes at low
Although heparin showed a direct effect on the TGF, density (11) or in the presence of heparan sulfates. However,
mRNA stability, its primary effects were on TGFP mRNA they do not affect growth but rather affect the synthesis of
synthesis, again via specific hormone-heparin combinations. liver-specific mRNAs in hepatocytes cultured at high density
The most potent signal for TGF,B transcription was a syner- or in the presence of heparins (19). If our hypothesis proves
gistic effect of insulin, GH, and heparin. true, synergies between hormones or growth factor and
Role of IGF H in neoplasia and differentiation. Although membrane-associated glycosaminoglycan chemistry could
the presence of both IGF I and IGF II in tumors and be part of the mechanism of normal liver cell differentiation
embryonal tissues is well documented (4, 34, 49, 55), the in vivo as well as a differential regulator of minimally deviant
actual data are unclear as to whether IGF II is critically versus metastatic tumors.
involved in neoplastic transformation, in tumor progression,
or in differentiation (4, 9, 20, 30, 32, 34, 42, 45, 48). IGF II ACKNOWLEDGMENTS
mRNA levels are very high in fetal tissues such as embry- We thank Dinish Williams for excellent technical assistance.
onic liver (4) but are low in human adult liver (20) and Excellent secretarial assistance was given by Rosina Passela.
undetectable in rat liver after birth (4). During rat hepato- This research was supported primarily by grant 1897 from the
carcinogenesis, IGF II gene transcription was reactivated Council for Tobacco Research. Funding for some supplies and for
from three different promoters, whose activities differed in technicians who helped with the liver perfusions, with animals, and
efficiency in each of the analyzed tumors (51). In normal rat with glassware washing was through grants from the American
tissues, however, the three promoters were coordinately Cancer Society (BC-439) and National Institutes of Health (NIH/
regulated (50). An alternate explanation is that the IGF II NCI P30-CA13330 and AM17702-12). Lola Reid received salary
levels in tumors result from expansion of liver progenitor support through a career development award NIH CA00783. Isabel
cells expressing IGF II (12, 17, 37) rather than induction due Zvibel received partial salary support through the Molin Founda-
to oncogenic transformation (12). tion.
TGFj expression in normal and neoplastic tissues. TGFP is REFERENCES
a strong inhibitor of the proliferation of epithelial cells, 1. Alexander, J. J., G. MacNab, and R. Saunders. 1978. Studies on
including hepatocytes (22), but it can have different effects, in vitro production of hepatitis B surface antigen by a human
depending on what other factors are present (40). TGF, is hepatoma cell line. Perspect. Virol. 10:103-117.
known to antagonize the mitogenic effects of stimulating 2. Bell, G. I., D. S. Gerhard, N. M. Fong, R. Sanchez-Pescador, and
growth factors, such as the effects of fibroblast growth factor L. B. Rall. 1985. Isolation of the human insulin-like growth
on vascular endothelial cells (21) or the effects of epidermal factor genes: insulin-like growth factor II and insulin genes are
growth factor on hepatocytes (22) or on myc-transfected contiguous. Proc. Natl. Acad. Sci. USA 82:6450-6454.
fibroblasts (40). 3. Bell, G. I., J. P. Merryweather, R. Sanchez-Pescador, M. M.
One of the TGFi effects is the transcriptional activation of Stempien, L. Priestley, J. Scott, and L. B. Rail. 1983. Sequence
of a cDNA clone encoding human preproinsulin-like growth
genes for extracellular matrix components and their recep- factor II. Nature (London) 310:775-777.
tors (23, 24). TGFP inhibition of the differentiation of myo- 4. Brown, A. L., D. E. Graham, S. P. Nissley, D. J. Hill, A. J.
blasts to myotubes is mediated by an increase in collagen I, Strain, and M. M. Rechler. 1986. Developmental regulation of
fibronectin, and integrin receptor expression (23, 24, 35). insulin-like growth factor II mRNA in different rat tissues. J.
Recent studies showed that TGF,B can have not only inhib- Biol. Chem. 261:13144-13150.
itory but also growth stimulatory effects on highly metastatic 5. Castellot, J. J., Jr., J. Choay, J.-C. Lormeau, M. Petitou, E.
cells (43). Perhaps the variability in responses is dictated by Sache, and M. J. Karnovsky. 1986. Structural determinants of
which matrix chemistry is induced by TGFP, a fact that the capacity of heparin to inhibit the proliferation of vascular
should be cell type specific and dependent on synergies with smooth muscle cells. II. Evidence for a pentasaccharide se-
quence that contains a 3-0 sulfate group. J. Cell Biol. 102:1979-
other signals. 1984.
Do synergies between plasma membrane-associated gly- 6. Castellot, J. J., Jr., D. L. Cochran, and M. J. Karnovsky. 1985.
cosaminoglycans and growth factors dictate the physiological Effect of heparin on vascular smooth muscle cells. I. Cell
responses to the growth factors? Our results lead us to metabolism. J. Cell. Physiol. 124:21-28.VOL . 1 l, 1991 HEPARIN AND HORMONAL REGULATION OF mRNA SYNTHESIS 115
7. Clayton, D. F., and J. E. Darnell, Jr. 1983. Changes in liver- Proc. Natl. Acad. Sci. USA 84:6330-6334.
specific compared to common gene transcription during primary 26. Ishilara, M., N. S. Fedarko, and H. E. Conrad. 1986. Transport
culture of mouse hepatocytes. Mol. Cell. Biol. 3:1552-1561. of heparan sulfate into the nuclei of hepatocytes. J. Biol. Chem.
8. DePagter-Holthuizen, P., M. Jansen, F. M. A. van Schaik, R. van 261:13575-13580.
der Kammen, C. OosterwUk, J. L. Van den Brande, and J. S. 27. Kjellen, L., A. Oldberg, and M. Hook. 1981. Cell surface
Sussenback. 1987. The human insulin-like growth factor II gene heparan sulfate: mechanisms of proteoglycan-cell association.
contains two development-specific promoters. FEBS Lett. 214: J. Biol. Chem. 255:10407-10413.
259-264. 28. Kjellen, L., I. Pettersson, and M. Hook. 1981. Cell-surface
9. D'Ercole, A. J., A. D. Stiles, and L. E. Underwood. 1984. Tissue heparan sulfate: an intercalated membrane proteoglycan. Proc.
concentrations of somatomedin C: further evidence for multiple Natl. Acad. Sci. USA 78:5371-5375.
sites of synthesis and paracrine or autocrine mechanisms of 29. Knowles, B. B., C. C. Howe, and D. P. Aden. 1980. Human
action. Proc. Natl. Acad. Sci. USA 81:935-939. hepatocellular carcinoma cell lines secrete the major plasma
10. Doerr, R., I. Zvibel, D. Chiuten, J. D'Olimpio, and L. M. Reid. proteins and hepatitis B surface antigen. Science 209:497-499.
1989. Clonal growth of tumors on tissue-specific biomatrices 30. Little, M. H., G. Ablett, and P. J. Smith. 1987. Enhanced
and correlation with organ site specificity of metastases. Cancer expression of insulin-like growth factor II is not a necessary
Res. 49:384-392. event in Wilms' tumour progression. Carcinogenesis 8:865-868.
11. Enat, R., D. M. Jefferson, N. Ruiz-Opazo, Z. Gatmaitan, L. 31. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
Leinwand, and L. M. Reid. 1984. Hepatocyte proliferation in cloning: a laboratory manual, p. 191-193. Cold Spring Harbor
vitro: its dependence on the use of serum-free, hormonally Laboratory, Cold Spring Harbor, N.Y.
defined medium and substrata of extracellular matrix. Proc. 32. Mathews, L. S., G. Norstedt, and R. D. Palmiter. 1986. Regula-
Natl. Acad. Sci. USA 81:1411-1415. tion of insulin-like growth factor I gene expression by growth
12. Fausto, N., J. E. Mead, L. Braun, N. L. Thompson, M. Panzica, hormone. Proc. Natl. Acad. Sci. USA 83:9343-9347.
M. Goyette, G. I. Bell, and P. R. Shank. 1987. Proto-oncogene 33. Nakajima, M., T. Irimura, D. DiFerrante, N. DiFerrante, and
expression and growth factors during liver regeneration. Symp. G. L. Nicolson. 1983. Heparan sulfate degradation: relation to
Fundamental Cancer Res. 39:69-86. tumor invasive and metastatic properties of mouse B16 mela-
13. Fedarko, N. S., and H. E. Conrad. 1986. A unique heparan noma sublines. Science 220:611-613.
sulfate in the nuclei of hepatocytes: structural changes with the 34. Norstedt, G., A. Levinovitz, C. Moller, L. C. Eriksson, and G.
growth state of the cells. J. Cell Biol. 102:587-599. Andersson. 1988. Expression of insulin-like growth factor I
14. Feinberg, A. P., and B. Vogelstein. 1984. A technique for (IGF-1) and IGF-II mRNA during hepatic development, prolif-
radio-labeling DNA restriction endonuclease fragments to high eration and carcinogenesis in the rat. Carcinogenesis 9:209-213.
specific activity. Anal. Biochem. 137:266-267. 35. Penttinen, R. P., S. Kobayashi, and P. Bornstein. 1988. Trans-
15. Fogh, J., W. C. Wright, and J. D. Loveless. 1977. Absence of forming growth factor 13 increases mRNA for matrix proteins
HeLa cell contamination in 169 cell lines derived from human both in the presence and in the absence of changes in mRNA
tumors. J. Natl. Cancer Inst. 58:209-213. stability. Proc. Natl. Acad. Sci. USA 85:1105-1108.
16. Folkman, J., and M. Klagsbrun. 1987. Angiogenic factors. 36. Ramasharma, K., and C. Hao Li. 1987. Human pituitary and
Science 235:442-447. placental hormones control human insulin-like growth factor II
17. Fu, X. X., C. Y. Su, Y. Lee, R. Hintz, L. Biempica, R. Snyder, secretion in human granulosa cells. Proc. Natl. Acad. Sci. USA
and C. E. Rogler. 1988. Insulin-like growth factor II expression 84:2643-2647.
and oval cell proliferation associated with hepatocarcinogenesis 37. Reid, L. 1990. Stem cell biology, extracellular matrix, and liver
in woodchuck hepatitis virus carriers. J. Virol. 62:3422-3430. differentiation. Curr. Opinions Cell Biol. 2:121-130.
18. Fujita, M., D. C. Spray, H. Choi, J. C. Saez, T. Watanabe, L. C. 38. Reilly, C. F., L. M. S. Fritze, and R. D. Rosenberg. 1986.
Rosenberg, E. L. Hertzberg, and L. M. Reid. 1987. Glycosami- Heparin inhibition of smooth muscle cell proliferation: a cellular
noglycans and proteoglycans induce gap junction expression site of action. J. Cell. Physiol. 129:11-19.
and restore transcription of tissue-specific mRNAs in primary 39. Reilly, C. F., L. M. S. Fritze, and R. D. Rosenberg. 1987.
liver cultures. Hepatology 7:1-9. Antiproliferative effects of heparin on vascular smooth muscle
19. Gatmaitan, Z., D. M. Jefferson, N. Ruiz-Opazo, L. Biempica, I. cells are reversed by epidermal growth factor. J. Cell. Physiol.
Arias, G. Dudas, L. Leinwand, and L. M. Reid. 1983. Regulation 131:149-157.
of growth and differentiation of a rat hepatoma cell line by the 40. Roberts, A. B., M. A. Anzano, L. M. Wakefield, N. S. Roche,
synergistic interactions of hormones and collagenous substrata. D. F. Stern, and M. B. Sporn. 1985. Type 1 transforming growth
J. Cell Biol. 97:1179-1190. factor: a bifunctional regulator of cellular growth. Proc. Natl.
20. Han, V. K. M., P. K. Lund, D. C. Lee, and A. J. D'Ercole. 1988. Acad. Sci. USA 82:119-123.
Expression of somatomedin/insulin-like growth factor messen- 41. Robinson, J., M. Viti, and M. Hook. 1984. Structure and
ger ribonucleic acids in the human fetus: identification, charac- properties of an under-sulfated heparan sulfate proteoglycan
terization, and tissue distribution. J. Clin. Endocrinol. Metab. synthesized by a rat hepatoma cell line. J. Cell Biol. 98:946-953.
66:422-429. 42. Schmid, C., T. Steiner, and E. R. Froesch. 1983. Preferential
21. Hotta, M., and A. Baird. 1987. Differential effects on low enhancement of myoblast differentiation by insulin-like growth
density lipoprotein metabolism by transforming growth factor ,B factors (IGF I and IGF II) in primary cultures of chicken
mediates its effects on steroidogenesis in bovine adrenocortical embryonic cells. FEBS Lett. 161:117-121.
cells in vitro. Endocrinology 121:150-159. 43. Schwarz, L. C., M. C. Gingras, G. Goldberg, A. H. Greenberg,
22. Houck, K. A., J. L. Cruise, and G. Michalopoulos. 1988. and J. A. Wright. 1988. Loss of growth factor dependence and
Norepinephrine modulates the growth-inhibitory effect of trans- conversion of transforming growth factor-P 1 inhibition to
forming growth factor P in primary rat hepatocyte cultures. J. stimulation in metastatic H-ras-transformed murine fibroblasts.
Cell. Physiol. 135:551-555. Cancer Res. 48:6999-7003.
23. Ignotz, R. A., and J. Massague. 1986. Transforming growth 44. Shen, S.-J., M. Diamon, C.-Y. Wang, M. Jansen, and J. Ilan.
factor-beta stimulates the expression of fibronectin and collagen 1988. Isolation of an insulin-like growth factor II cDNA with a
and their incorporation into the extracellular matrix. J. Biol. unique 5' untranslated region from human placenta. Proc. Natl.
Chem. 261:4337-4345. Acad. Sci. USA 85:1947-1951.
24. Ignotz, R. A., and J. Massague. 1987. Cell adhesion protein 45. Shinizu, M., F. Torti, and R. A. Roth. 1986. Characterization of
receptors as targets for transforming growth factor P action. the insulin and insulin-like growth factor receptors and respon-
Cell 51:189-197. sitivity of a fibroblast/adipocyte cell line before and after differ-
25. Irminger, J.-C., K. M. Rosen, R. E. Humbel, and L. Villa- entiation. Biochem. Biophys. Res. Commun. 137:552-558.
Komaroff. 1987. Tissue-specific expression of insulin-like 46. Sporn, M. B., and A. B. Roberts. 1985. Autocrine growth factors
growth factor II mRNAs with distinct 5' untranslated regions. and cancer. Nature (London) 313:745-747.116 ZVIBEL ET AL. MOL. CELL. BIOL.
47. Spray, D. C., M. Fujita, J. C. Saez, H. Choi, T. Watanabe, E. factor II gene during hepatocarcinogenesis. Carcinogenesis
Hertzberg, L. C. Rosenberg, and L. M. Reid. 1987. Glycosami- 9:1779-1783.
noglycans and proteoglycans induce gap junction synthesis and 52. Uhlrich, S., 0. Lagente, J. Choay, Y. Courtois, and M. Lenfant.
function in primary liver cultures. J. Cell Biol. 105:541-551. 1986. Structure activity relationship in heparin: stimulation of
48. Toilefsen, S. E., J. L. Sadow, and P. Rotwein. 1989. Coordinate non-vascular cells by a synthetic heparin pentasaccharide in
expression of insulin-like growth factor II and its receptor cooperation with human acidic fibroblast growth factors. Bio-
during muscle differentiation. Proc. Natl. Acad. Sci. USA chem. Biophys. Res. Commun. 139:728-732.
86:1543-1547. 53. Underwood, L. E., and A. J. D'Ercole. 1984. Insulin and
49. Tricoli, J. V., L. B. Rail, C. P. Karakousis, L. Herrera, N. J. insulin-like growth factors (somatomedins) in fetal and neonatal
Petrelli, G. I. Bell, and T. B. Shows. 1986. Enhanced levels of
insulin-like growth factor messenger RNA in human colon development. Clin. Endocrinol. Metab. 13:69-89.
carcinomas and liposarcomas. Cancer Res. 46:6169-6173. 54. Wright, T. C., Jr., T. V. Johnstone, J. J. Casteilot, and M. J.
50. Ueno, T., K. Takahashi, T. Matsuguchi, H. Endo, and M. Karnovsky. 1985. Inhibition of rat cervical epithelial cell growth
Yamamoto. 1988. Transcriptional deviation of the rat insulin- by heparin and its reversal by EGF. J. Cell. Physiol. 125:499-
like growth factor II gene initiated at three alternative leader- 506.
exons between neonatal tissue and ascites hepatomas. Biochim. 55. Yee, D., K. J. CuHlen, S. Paik, J. F. Perdue, B. Hampton, A.
Biophys. Acta 950:411-419. Schwartz, M. E. Lippman, and N. Rosen. 1988. Insulin-like
Downloaded from http://mcb.asm.org/ on March 28, 2021 by guest
51. Ueno, T., K. Takahashi, T. Matsuguchi, K. Ikejiri, H. Endo, and growth factor II mRNA expression in human breast cancer.
M. Yamamoto. 1988. Reactivation of rat insulin-like growth Cancer Res. 48:6691-6696.You can also read
