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Molecular Biology and Genetics |
Department of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IGF-II is a paternally expressed, maternally imprinted, embryonic growth factor that is a potent modifier of growth in vivo (5) . In mice, after disruption of the paternal Igf2 allele, total body weight is reduced by 40% at birth (6) . Systemic IGF-II levels fall after birth, and postnatal growth is then regulated by the related ligand, IGF-I (5) . Increased systemic availability of IGF-II, either due to biallelic expression, increased delivery from a transgene, or disruption of the IGF-II/M6P receptor, results in overgrowth phenotypes in mice (7, 8, 9) . Similar effects occur in human overgrowth syndromes such as Beckwith-Wiedemann, where the biallelic expression of IGF2 results from either LOI or unipaternal disomy (10) . Increased expression of IGF-II in transgenic mice not only increases tumor frequency in organs that express the transgene, but also at distant sites, suggesting that both local and systemic supply can promote tumor progression (11 , 12) .
IGF-II is well known for promoting a cell number increase in vitro. The mechanism may predominantly relate to cell survival rather than cell division (13 , 14) . IGF-II and the related ligand, IGF-I, exert cell survival and growth effects via heterotetrameric IGF-I and insulin receptors, which mediate signal transduction through the PI3 kinase/Akt pathway also modified by the recently identified phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumor suppressor (15) . Serial analysis of gene expression has identified IGF-II as the most abundant mRNA overexpressed in human colorectal cell lines and tumors compared to normal tissue (16) . IGF-II is also overexpressed in Wilms tumor, rhabdomyosarcoma, neuroblastoma, germ cell tumors, adrenocortical carcinoma, breast, and hepatocellular carcinoma (reviewed in Ref. 17 ). Furthermore, increased maternal allele expression (allele ratio of <3:1 taken as LOI) has also been detected at surprisingly high frequency in normal human leukocytes and colonic mucosa (12%), particularly in cases with microsatellite instability in associated tumors (91%; Ref. 18 ). This significant finding suggests that increased local IGF-II supply may predispose to the development of early onset colorectal cancer before the appearance of either an adenoma or tumor, especially in individuals with defects in DNA mismatch repair (19) .
We examined the effect of IGF-II supply in crosses between mice with genetically altered IGF-II expression and the ApcMin/+ model of colorectal cancer. Increased IGF-II delivery to the alimentary tract was achieved using a bovine keratin 10 promoter-driven transgene (K10Igf2), which results in the phenotype of colon, skin, and uterine overgrowth (9) . Although the predominant growth effect is in tissues that express the transgene, there are also subtle metabolic effects due to increased circulating IGF-II (20) . Decreased IGF-II supply in the alimentary tract was achieved using mice with a disruption of the paternal allele of Igf2 (Igf2+m/-p) (6) . Except for the leptomeninges of the brain, Igf2 mRNA expression from the maternal allele is normally undetectable in these animals. Therefore, the source of any locally delivered IGF-II to the alimentary tract should be derived from increased maternal allele expression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice with increased IGF-II supply (K10Igf2) were at >10th generation inbred onto 129/SvJ (9) . Mice with disruption of the paternal allele of Igf2 (-p) but an intact maternal allele (+m, denoted Igf2+m/-p) were a gift from A. Efstratiadis and were also at >10th generation inbred onto the same 129/SvJ (6) . DNA was extracted from tails (day 7) and liver (at the time of dissection). After incubation (12 h; 55°C) with 0.5 µg/ml proteinase K in lysis buffer [50 mM Tris (pH 8.0), 100 mM EDTA, 100 mM NaCl, 1% SDS] and RNase A (1 h; 37°C), DNA was extracted with phenol/chloroform, precipitated with ethanol, and resuspended in TE buffer [10 mM Tris (pH 7.4), 1 mM EDTA]. Animals were genotyped for the presence of ApcMin/+, K10Igf2/+, and Igf2+m/-p by established PCR protocols (2 , 23) . All breeding used male 129/JSv IGF-II mutant mice and female C57Bl/6J ApcMin/+. Female K10Igf2/+ are poor mothers because they develop an imperforate uterus, and the disrupted allele in Igf2+m/-p is paternally inherited. All litters were cross-fostered to F1 mothers before postnatal day 7 (C57Bl/6J,CBA/Ha or C57Bl/6J,129/SvJ). All animal procedures were approved by the Home Office of the United Kingdom government, departmental ethics committee and were carried out in accordance with the United Kingdom Coordinating Committee on Cancer Research guidance for the welfare of animals in experimental neoplasia (second edition; Ref. 23 ).
Adenoma Scoring and Collection.
Depending on age, ApcMin/+ become moribund
as a result of chronic anemia and intestinal obstruction. After daily
monitoring of initial litters for signs of anemia and distress, the
dates of dissection of experimental crosses were refined so that
animals did not suffer unduly. Small intestinal adenoma and colonic
adenoma were therefore scored for number and diameter either at
postnatal day 80 or 150 for the cross between
ApcMin/+ x K10Igf2/+
and ApcMin/+ x Igf2+m/-p, respectively. The stomach,
small intestine, and colon were dissected free of mesentery and opened
along the longitudinal axis using a jig and blade designed by us to aid
rapid processing. Intestinal contents were cleared with PBS, and the
small intestine was divided into three equal-length segments and laid
open with the colon on the absorptive side of Benchkote (Whatman).
Intestines were fixed in 4% (4 g/100 ml) paraformaldehyde in PBS (24
h) followed by 70% ethanol (v/v). Using a dissecting microscope
(x10–30) and calipers, adenoma number and diameter were obtained for
the entire length of the small intestine and colon. Adenoma analysis
was performed without knowledge of genotype by one person (A. B.
H) and confirmed independently by another (J. A. H.).
Material for cryosections was either placed face down relative to the
cutting surface for the small intestine or rolled (for the colon),
immediately embedded in TissueTek (Sakura Fintek, Zoeterwoude,
the Netherlands), and stored at -40°C. Small intestine surface area
was calculated by summation of the multiples of length of each fixed
segment by width, which was measured at the midpoint of each segment.
Colon surface area was calculated by multiplying the length of the
fixed material from the anorectal junction to the point of insertion of
the small intestine, omitting the appendix, by the width at the
midpoint. Statistical analysis is described in figure and table
legends. Calculations were performed using the Minitab 10Xtra (Minitab
Inc.).
Histopathology.
Distal colon samples and small intestinal segments were
paraffin-embedded and sectioned (5 µm), and every fifth section was
stained with H&E. Stained sections were viewed without knowledge of
genotypes (A. B. H) and checked by an independent histopathologist
(D. Rowlands, Department of Histopathology, University of Birmingham,
United Kingdom). Sections for immunohistochemistry were cleared with
xylene and rehydrated in an ethanol series to PBS, and endogenous
peroxidases were quenched with 3%
H2O2 in PBS (v/v; 15 min).
Sections were incubated at 4°C with primary antibodies in PAT
(PBS/0.1% BSA/0.1% Tween 20) for 16 h. The following antibodies
were used: antihuman APC (C-20) rabbit polyclonal to the COOH-terminus
of human APC (amino acid 2824–2843), antihuman APC (N-15) rabbit
polyclonal to the amino terminus of human APC (amino acid 2–16), and
IGF-IRß (sc-713) antihuman rabbit polyclonal to a
carboxy-terminal peptide, all from Santa-Cruz Biotechnology Inc.
(Santa Cruz, CA). Proliferation was assessed with the HsMCM2/BM28
rabbit polyclonal antibody to human MCM2, a cell cycle protein marker
specific for G1-S-phase cells, a gift from I.
Todorov (24
, 25)
. Sections were blocked with 2% (v/v)
horse serum in PAT (30 min), incubated with biotinylated antirabbit
antibody from horse (Vector Laboratories Inc., Burlinghame, CA), and
visualized with a peroxidase ABC Vector Elite kit with
3,3'-diaminobenzidine substrate. Sections were dehydrated,
counterstained with methyl-green, washed in acetone plus 1% (v/v)
acetic acid, and mounted.
In Situ Hybridization.
DNA containing the distal coding region and 3' untranslated
region of Igf2 exon 6 was amplified from a pGEM plasmid
containing Igf2 cDNA (derived from a gift from P. Rotwein).
Primers BH1, 5'-NNG AGC TCA GCC TCT TCG GAG ATG TC-3'
(position 541–558, M14951) and BH2, 5'-NNG GTA CCA ACA GCC
TGA TGT GGG GA-3' (position 738–721, M14951) amplified a 198-bp
fragment. Sequence analysis confirmed the fragment was mouse
Igf2. Engineered SacI and KpnI
restriction sites in primers BH1 and BH2, respectively (italicized)
were used to clone digested gel-purified product into pBluescript
KS-. Linearized plasmid was used to transcribe sense and
antisense probe with digoxigenin-UTP (Roche Diagnostics Ltd, Essex,
United Kingdom). Ten-µm cryosections were mounted on adhesive-treated
slides (3-aminopropyltriethoxy-silane), were dried with a gentle heat
from a hair dryer for 2–3 min, and were immediately placed in fresh
4% (w/v) paraformaldehyde/PBS (diethylpyrocarbonate-treated) at 4°C
for 20 min. To quench the endogenous alkaline phosphatase, incubation
in 2 N HCl for 20 min at 4°C significantly
reduced background (26)
. Slides were washed once in PBS,
passed through a PBS/ethanol dilution series to 100% ethanol, and air
dried. Slides were hybridized with 10 ng of denatured probe/section at
25°C for 16 h in filtered hybridization buffer (10%; w/v)
dextran sulfate, 2x SSC, 50% (w/v) formamide, 10% (w/v) SDS, 1
mM DTT, 10 µg/ml salmon sperm, 10 µg/ml tRNA,
and 20 units of RNase inhibitor (Sigma, United Kingdom). Sections were
washed with 1x SSC [150 mM NaCl, 15
mM sodium citrate (pH 7.0)], O.1x SSC/50%
(v/v) formamide (2 x 5 min), and incubated with 20
µg/ml RNase A for 30 min at 37°C. Digoxigenin RNA was visualized
with an antidigoxigenin-alkaline phosphate Fab fragment (Roche
Diagnostics Ltd, Essex, United Kingdom) using nitro-blue tetrazolium
chloride and 5-bromo-4-chloro-3-indoyl-phosphate staining with 0.5
µg/ml levamisole in buffer [0.1 M Tris-HCl (pH
9.5), 0.1 M NaCl, 50 mM
MgCl2] containing 10% (w/v) polyvinyl alcohol (30–70 kDa) for
16 h at 25°C. Sections were counterstained with 0.02% fast
green and viewed with a JVC color charge coupled device camera
attached to a Leica upright microscope. Control experiments using a
sense probe, excess unlabeled antisense probe, predigestion with RNase
A, tissue from Igf2+m/-p, and omission of
alkaline phosphatase Fab fragment all resulted in a background signal.
Sections from an Igf2 transgene-derived mammary tumor acted
as a positive control in every experiment.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Increased IGF-II Supply Increases Number, Diameter, and Malignant
Progression of Colon Adenoma.
The bovine keratin 10 promoter used to deliver Igf2 mRNA
(K10Igf2/+) was previously found to target transgene
expression to the suprabasal layers of the skin, alimentary canal, and
uterus (9
, 28)
. To our knowledge, this is the only
transgene available that increases IGF-II supply in the colon, although
a similar phenotype has recently been described for an actin-IGF-I
transgene (29)
. Overgrowth of the colon can result in
rectal prolapse in K10Igf2/+ mice that are >6 months old,
yet no intestinal epithelial tumors have been observed, even in the
highest expressing line used ("Blast" line). However, small raised
pale polyps in the distal colon can be seen in these animals (average
of 3.5 polyps, >2 mm in diameter/mouse colon at postnatal day 80;
n = 18). Histological analysis revealed
mucosal collections of lymphocytes (not shown). Therefore, all colon
polyps were checked by H&E-stained paraffin-embedded sections and
cryosections, and only nonlymphoid adenoma counts were reported.
Although K10Igf2 transgene expression has not been resolved
with respect to the four separate cell types of the crypt (see in
situ hybridization), there is evidence for both smooth muscle
thickening and increased crypt depth in the colon, indicating
overgrowth in both compartments (30)
. Apart from systemic
delivery from the blood stream, a further source of IGF-II may be
release from the stomach into the lumen and distal delivery to the
small intestine and colon. Both IGF-I and IGF-II may increase mucosal
cell growth after intraluminal supply (31)
.
Mice that develop intestinal adenoma and increased IGF-II supply
in the colon (ApcMin/+,K10Igf2/+)
lose weight (Fig. 1A)
, rapidly become anemic, and commonly develop rectal
prolapse by 80 days (41%, 7/17 compared to K10Igf2/+ alone
6%, 1/18). To counter the possibility that altered adenoma growth was
simply a reflection of an alteration of small intestine and colon
growth, we corrected for surface area measured in fixed tissue. Adenoma
number and diameter were expressed either without correction (Fig. 2)
or with correction for surface area (Table 1)
. The number of adenoma increased in the colon (P < 0.0001; Fig. 2
), even when correcting for increased colon
growth (P < 0.001; Table 1
). The diameter of
the adenoma also increased disproportionately relative to the increased
colon growth (Table 2)
. Examination of dissected colons revealed large distal adenomas (Fig. 3A)
. Histopathological features within each adenoma showed a
spectrum of changes, with increased progression to carcinoma in
situ and invasion in a significant proportion (Fig. 3B
;
Table 3
). Only one polyp-like lesion was seen in the uterus of female
ApcMin/+,K10Igf2/+ (n = 7 animals); no mammary, skin, or stomach tumors were observed.
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. NH2-terminal APC antibody
stained all adenoma. There were no differences in Apc staining related
to IGF-II supply. Smaller adenoma tended to retain Apc staining in some
cells located within the adenoma rather than on its surface (Fig. 4A)
. Proliferation visualized with an anti-MCM2 antibody
(BM-28) was confined to adenoma and basal crypts (Fig. 4, B and E)
. Again, no differences in ratio of labeled:unlabeled
nuclei (proliferation index) in each high power field were detected
between adenoma of different genotypes (not shown). The distribution of
IGF1Rß was concentrated in the smooth muscle layer, the upper luminal
surface of villi, and the upper zone of small intestine and colonic
crypts (Fig. 4C)
. Large >2-mm colonic adenoma generally
appeared to have a ramifying network of staining confined to the
stromal cell compartment, rather than staining exclusively confined to
adenoma cells (Fig. 4F)
. However, although a proportion of
adenoma had increased staining (18/31 adenoma from 13 animals), some
showed low level staining with anti-IGF1Rß. No differences between
genotypes were detected.
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). There was significant reduction
in adenoma number and diameter in
ApcMin/+, Igf2+m/-p
in the small intestine at 150 days, even allowing for the reduction in
small intestinal growth (P < 0.001; Fig. 2
;
Tables 1
and 2
). The reduction in adenoma size was most pronounced when
comparing adenoma with diameter >2 mm, suggesting that reduced IGF-II
supply limits early adenoma progression as well as total number (Table 2)
. There were too few adenoma in the colon to detect a similar trend.
Histological comparison of small intestinal adenoma from
ApcMin/+,Igf2+m/-p
with
ApcMin/+,Igf2+m/+p
revealed similar histological features, with no differences independent
of adenoma size (not shown).
Igf2 Is Expressed in ApcMin/+ Adenoma, and
Maternal Igf2 Allele Is Expressed in
ApcMin/+,Igf2+m/-p.
Igf2 in situ hybridization of adult wild-type C57Bl/6J
villi, crypts, and smooth muscle layers of small intestine and colon
showed only background signal. In situ hybridization in
K10Igf2/+ (Blast) revealed Igf2 expression in the
upper two thirds of the crypts of the stomach and colon and associated
low level signal in smooth muscle layers (Fig. 4I)
.4
In situ hybridization in adenoma revealed an increased
signal in
ApcMin/+,Igf2+m/+p
(17/21 small intestinal adenoma from six animals),
ApcMin/+, K10Igf2/+ (10/12 colon
adenoma from six animals), and ApcMin/+,
Igf2+m/-p (9/13 small intestinal adenoma from
four animals; Fig. 4
, G-I). Signal intensity appeared
similar irrespective of genotype in parallel processed slides. We
presume that mRNA degradation accounts for the failure to detect signal
in all adenoma because frozen section samples kept for longer than 3
months (at -20°C) frequently showed background signal.
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DISCUSSION |
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Decreased IGF-II supply limited the number of adenoma in the
small intestine, again suggesting either a reduced rate of adenoma
initiation or a decrease in early adenoma progression. The disruption
of the Igf2 paternal allele had a clear effect on the number
of large adenoma, suggesting that IGF-II influences the growth of
established adenoma. The fact that adenoma appeared in the absence of
paternal Igf2 expression may be explained by either the
selection for the autocrine expression of Igf2 from the
maternal allele or by the expression of an alternative growth factor.
It is improbable that adenoma growth will depend on a single growth
factor such as IGF-II. However, results from Igf2 in situ
hybridization show adenoma-specific Igf2 expression and support
selection for increased maternal allele expression. Similar findings
have been described by Christofori et al. (35
, 36)
in a pancreatic tumor model using SV40 T-antigen expression
from a rat insulin promoter (RIP-Tag) and in both TGF
and SV40
T-antigen-induced hepatocellular carcinoma models (37
, 38)
. However, although SV40 T-antigen-induced hepatocellular
carcinoma showed reduced tumor size in combination with
Igf2+m/-p, increased maternal allele
expression was rarely found. Increased IGF-II supply in tumors with
intact Igf2 alleles appeared to be due to selection for
paternal allele disomy and maternal-specific LOH (38)
.
Studies of intestinal adenoma growth in mice with homozygous disruption
of Igf2 are in progress
(ApcMin/+,Igf2-m/-p).
The effects of increased and decreased IGF-II supply support our view that IGF-II supply is a modifier of adenoma number and progression. How IGF-II alters adenoma number is not known, but the mechanisms could include either enhanced survival of cells that have lost the normal Apc allele (ApcMin/-, LOH) or via modification of an increased crypt fission rate detected in the developing intestine of ApcMin/+ (39) . The mitogenic and apoptotic functions of C-MYC, transcriptionally up-regulated as a result of APC dysfunction (40) , often require addition of survival factors, such as IGF-II, particularly in c-myc-induced, p53-dependent, cell death transduced by p19ARF (14 , 41) . Mechanisms of how IGF-II acts as a survival factor include phosphorylation and inactivation of Bad, which normally antagonize Bcl-2 blockage of cytochrome c release (42) . IGF-IR-mediated cell survival functions may also be influenced by systemic levels of the growth hormone-controlled ligand, IGF-I, because an increased frequency of colonic carcinoma can occur in acromegalic patients with excess IGF-I (43) .
Patients with microsatellite instability in colorectal tumors develop LOI of IGF2, which may promote growth of colon tumors. However, it is not known whether LOI in this circumstance has significant functional consequences in terms of increasing the probability of developing early onset colorectal cancer. However, our experiments highlight the potential importance of this observation and of increased local IGF-II expression due to LOI in normal human colonic tissue (18) . It is not known whether increased IGF-II supply in normal colon mucosa predisposes to colonic adenoma without mutation of APC or whether IGF-II supply contributes to the development of polyclonal adenoma via a paracrine/community effect (44) . We found no obvious increase in the proportion of adenoma with normal Apc staining in mice with increased IGF-II supply.
Excess IGF-II expression is not the only perturbation of growth factor pathways in colorectal cancer. Frequent mutations can occur in growth factor receptor genes in human tumor-associated mismatch repair defects, e.g., the TGFß type II receptor (45) and the IGF-II/M6P receptor (46) . In addition to mutation of the type II TGFß receptor, Smad3 and Smad4 transducers of the TGFß pathway are also mutated in human tumors and result in increased malignant progression of intestinal tumors after disruption of murine genes (33 , 47) .
Igf2 expression and subsequent autocrine/paracrine growth effects must offer adenoma cells a selective advantage. Our data provide experimental support for mathematical models concerning natural selection of expanding tumor cell clones expressing autocrine cell survival factors (48) . It is clear that IGF-II supply is tightly regulated in normal tissue, with expression predominantly from one allele during embryonic growth in both the human and mouse (6 , 49) . The addition of a single-expressed Igf2 allele results in overgrowth, and reduced IGF-II supply results in reduced embryonic growth (-40%). We and others have shown that IGF-II is an important regulator of murine tumor growth, both in early adenoma and in the progression to carcinoma (35, 36, 37, 38) . However, this is the first demonstration of the influence of IGF-II supply on intestinal tumor growth in a murine model that closely mimics a human colorectal cancer syndrome and is independent of the SV40 T antigen. We conclude that IGF-II supply is a potent modifier of intestinal tumor growth and that IGF-II may subsequently prove to be an important target for human colorectal cancer therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from The Cancer
Research Campaign United Kingdom 2390/0101 (to A. B. H.).
A. B. H. is a Cancer Research Campaign Senior Clinical
Research Fellow. 
2 To whom requests for reprints should be
addressed, at the Department of Zoology, University of Oxford, South
Parks Road, Oxford OX1 3PS, United Kingdom. Phone: 44-1865-271227; Fax:
44-1865-271228; E-mail: bass.hassan{at}zoo.ox.ac.uk
3 The abbreviations used are: IGF, insulin-like
growth factor; IGF-IR, insulin-like growth factor-I receptor; LOI, loss
of imprinting; SPF, specified pathogen-free; APC, adenomatous polyposis
coli; TGF, transforming growth factor.
Received 10/21/99. Accepted 12/14/99.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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