(Received for publication, November 8, 1995)
From the
In this study we have examined the role of insulin-like growth factor-II (IGF-II) in the differentiation of the CaCo-2 human colon carcinoma cell line. We have shown previously that IGF-II is an autocrine growth factor for CaCo-2 cells. IGF-II expression is high in proliferating, undifferentiated CaCo-2 cells and markedly decreases when cells become confluent and start to differentiate. To evaluate whether differentiation of CaCo-2 cells depends on an IGF-II related pathway, we treated cells with a blocking antibody to the IGF-I receptor that mediates most IGF-II biological effects. Treatment of preconfluent CaCo-2 cells with this antibody decreased by 40% autonomous cell proliferation and induced differentiation as shown by an increase in sucrase isomaltase activity and apolipoprotein A-I (apoA-I) mRNA levels. To examine the significance of autocrine IGF-II production in CaCo-2 cell differentiation, we generated stable CaCo-2 cell lines that constitutively express rat IGF-II under the control of a Rous sarcoma virus promoter. Sustained expression of IGF-II resulted in: (a) increased proliferative rate; (b) high IGF-I receptor number, even after reaching confluence; (c) increased capability of anchorage-independent growth; (d) inhibition of the expression of apoA-I and SI mRNAs. Analysis of several independent IGF-II-transfected clones showed an inverse correlation between IGF-II mRNA levels and expression of the differentiation markers, the cells expressing the higher levels of the transfected IGF-II being the less differentiated ones. Our data suggest that perturbation of IGF-II-mediated cell proliferation interferes with the enterocyte-like differentiation pathway of CaCo-2 cells.
The molecular mechanisms that govern cell growth and differentiation of normal and neoplastic human intestinal epithelium are poorly understood. In the small intestine and colon, epithelial cells arise from stem cells located in crypts at the base of villi (small intestine) or near the base of the colonic glands. As intestinal epithelial cells migrate along the crypt-villus axis, they exit from the cell cycle and enter into an alternative pathway in which a specialized phenotype is assumed(1, 2) . Several in vitro models of colon cancer cells have been used to study these processes. Cultured colon cancer cells are reversibly growth inhibited and induced to differentiate by treatment with sodium butyrate(3, 4) , phorbol esters(4) , and polar planar solvents (5) as well as by changes in cell density (6, 7) and glucose concentration(7) . Also, the mechanisms of the balance between proliferation and differentiation of the enterocytes has been studied in an in vivo model of transgenic mice in which the expression of SV40 T Ag is restricted to differentiated enterocytes(8) . Expression of the transgene in villus-associated enterocytes causes them to reenter the cell cycle without producing detectable changes in the state of differentiation(8) .
Investigation of molecules that
regulate cell location along the crypt-villus axis and the time span
necessary for the cells to move from the proliferative to the
differentiative compartment has also focused on the role of polypeptide
growth factors(2) . In particular, insulin-like growth factors
(IGF-I and IGF-II) ()regulate proliferation of intestinal
mucosa(9, 10) . IGFs are secreted as small peptides
that are structurally related to insulin and display multiform effects
on cell growth and metabolism(9, 10) . IGF-I and
IGF-II both exert their mitogenic activity through type I receptor
(IGF-I receptor)(9) . In rodents, IGF-II is expressed at high
levels in the fetus and at lower levels in the adult and functions
primarily as a prenatal growth
regulator(11, 12, 13) . In man, IGF-II is
highly expressed during the fetal life, and its synthesis has been
demonstrated in adult liver and adult extrahepatic
tissues(13) .
Little is known about the physiological role of IGFs during intestinal differentiation. A large percentage of primary human intestinal tumors express IGF-II(10, 13) , and almost all primary human colon cancers and colon cancer cell lines are positive for IGF-I receptors(10) . Recently, expression of IGF-II and of type I and II receptors has been shown in CaCo-2 cells, a human colon carcinoma cell line that spontaneously differentiates in culture with features similar to mature small bowel enterocytes(6, 14, 15, 16, 17) . We have shown previously that IGF-II is an autocrine growth factor for CaCo-2 colon carcinoma cells (14) . Also, IGF-II expression is high in proliferating, undifferentiated CaCo-2 cells and decreases by more than 10-fold when cells become confluent and start to differentiate(14) .
In the present study, we investigated whether IGF-II might inhibit the differentiation of CaCo-2 cells. We showed that the blockade of IGF-I receptor, through which most IGF-II related events are mediated, stimulates CaCo-2 cell differentiation. We also altered the normal pattern of IGF-II expression associated with CaCo-2 cell differentiation by introducing an exogenous IGF-II cDNA under a constitutive promoter. The effect of constitutive IGF-II overexpression in the CaCo-2 cells resulted in unregulated growth and inhibition of the enterocyte-like differentiation of the cells. These data suggest that IGF-II interferes with the differentiation of CaCo-2 cells.
Figure 1:
Effect of the IGF-I
receptor blocking antibody -IR3 on proliferation and
differentiation of preconfluent CaCo-2 cells. CaCo-2 cells at day 3 of
culture were washed and grown in serum-free medium. Two days later,
cells were incubated in serum-free medium alone or with the addition of
10 µg/ml
-IR3 IGF-I receptor-blocking monoclonal antibody or
with the same concentration of the isotypic control antibody MOPC-21
for 48 h. A, inhibition of CaCo-2 basal cell growth by
-IR3 blocking antibody. Mean ± S.D. of three independent
experiments. B, effect of
-IR3 antibody treatment on
sucrase activity of CaCo-2 cells. Values are expressed as
nM/mg of protein. Mean ± S.D. of three independent
experiments. C, effect of
-IR3 antibody treatment on
apoA-I mRNA expression. Northern blot analysis of apoA-1 and 28 S RNA
levels.
Figure 2: Analysis of RSV-IGF-II mRNA expression in parental and IGF-II-transfected CaCo-2 cells. Total RNA (20 µg) obtained from 8-day-old culture of nontransfected CaCo-2 cells (lane 1), pool of neomycin-transfected CaCo-2 cells (lane 2), neomycin-transfected CaCo-2 cell clone 1 (lane 3), pool of IGF-II-transfected CaCo-2 cells (lane 4), IGF-II transfected CaCo-2 cell clones 1 (lane 5), 2 (lane 6), 4 (lane 7), and 9 (lane 8) was analyzed by RNase protection assay. The RNAs were hybridized to a 218-nucleotide (nt) antisense transcript complementary to the transcription start site of the RSV promoter and to the 5` 126 nucleotides of rat IGF-II transcript. Transfected RSV-rat IGF-II- and endogenous human IGF-II- specific protected RNA bands of 165 and 126 nucleotides, respectively, are shown. Thirty µg of total yeast RNA were hybridized to the same probe as a control (lane 9).
Figure 3: IGF-II peptide levels in conditioned media of parental and IGF-II transfected CaCo-2 cells. Western blot analysis of media collected at 4, 14, and 21 days of culture from parental CaCo-2 cells (lanes 1-3, respectively), pool of neomycin-transfected CaCo-2 cells (lanes 4-6, respectively), pool of IGF-II-transfected CaCo-2 cells (lanes 7-9, respectively), and at 21 days of culture from neomycin-transfected CaCo-2 cell clone 1 (lane 10) and IGF-II transfected CaCo-2 cell clone 1 (lane 11), and of a purified human IGF-II (lane 12) using a mouse monoclonal antibody to rat IGF-II. The amount of medium loaded was calculated by normalizing to protein content of the cells at different days of culture. Migration of molecular weight markers is indicated on the right.
Figure 4:
Cell growth of parental, neomycin- and
IGF-II-transfected cells. A, growth cuve of parental, neomycin (NEO)- and IGF-II-transfected cells. Cells were seeded at a
density of 40,000 cells/35-mm dish in DMEM supplemented with 10% FCS.
Hemocytometric cell counts presented are from representative
experiments in which each point is the mean of triplicate
determinations; bars, S.D., which are not shown if too small
for visual display. , CaCo-2;
, neomycin clone 1;
,
IGF clone 1. B, thymidine incorporation in parental, neomycin (NEO)- and IGF-II-transfected cells. Cells were grown in DMEM,
10% FCS. At different days of culture, cells were pulse-labeled for 18
h by addition of [methyl-
H]thymidine,
and incorporation was measured as described under ``Experimental
Procedures.'' Thymidine incorporation is expressed as
counts/min/10
cells. Data presented are from a
representative experiment in which each point is the mean ± S.D.
of triplicate determinations. Open columns, CaCo-2; shaded
columns, neomycin clone 1; striped columns, IGF clone
1.
Figure 5: Analysis of anchorage-independent growth. A, number of colonies of CaCo-2, neomycin (NEO) pool, IGF-II pool, neomycin clone 1, and IGF clone 1 grown in agar after 3 weeks. B, number of colonies larger than 0.12 mm of CaCo-2, neomycin (NEO) pool, IGF-II pool, neomycin clone 1 and IGF clone 1 grown in agar after 3 weeks. Values are means ± S.D. of three wells. Data are representative of four similar experiment.
Figure 6: SI and apoA-I mRNA levels in control and IGF-II-transfected cells. A, Northern blot analysis of SI and apoA-I transcripts in pools of IGF-II- and neomycin (NEO)-transfected control CaCo-2 cells at day 8, 14, and 21 of culture. B, SI and apo-A1 mRNA levels in control and IGF-II-transfected clones at day 8, 14, and 21 of culture. Twenty µg of total RNA were loaded. The same filters were sequentially hybridized to human SI, human apoA-I, and human 28 S rRNA probes. The size of the transcripts in kilobases is indicated on the right. NEO, neomycin.
In this paper we establish a functional basis for the correlation between IGF-II expression and the enterocyte-like differentiation of CaCo-2 cells. The results described here demonstrate that IGF-I receptor blockade induces differentiation in preconfluent CaCo-2 cells; conversely, disregulated IGF-II expression in post-confluent CaCo-2 cells results in inhibition of differentiation.
Several experimental evidences suggest that IGF-II plays an important role during intestinal epithelial cell growth. Normal adult human colonic epithelium express IGF-I and IGF-II receptors(31) . IGFs and their receptors are overexpressed in 30% of colorectal tumors and in several human colon carcinoma cell lines compared with normal colon mucosa(10, 13) . We and others have recently shown that IGF-II, IGF-I receptor, but not IGF-I, are expressed in CaCo-2 cells (14, 15, 16) and that IGF-II is able to stimulate cell proliferation through the IGF-I receptor(14) .
The relationship between IGF-II and cell differentiation has not been well clarified yet. IGF-II seems to play an important role in promoting differentiation of skeletal muscle myoblasts, since an increased expression of IGF-II occurs during terminal stages of muscle development (32) and in vitro myotube formation is dependent on autocrine IGF-II secretion(33) . On the contrary, overexpression of IGF-II or IGF-I receptor genes inhibits myogenic differentiation of myoblasts in culture(34, 35, 36) . IGF-II overexpression is considered a marker of low or undifferentiated hepatocellular carcinomas of woodchucks, where an inverse correlation between IGF-II and albumin expression has been observed in tumor cells compared with normal liver cells(37) . Down-regulation of IGF-II expression has been reported during cAMP analog-mediated differentiation of SaOS-2 human osteosarcoma cells(38) .
In this study, we showed that blockade of IGF-I receptor inhibited cell growth and stimulated differentiation of preconfluent CaCo-2 cells. Thus, induction of CaCo-2 cell differentiation is paralleled by the inhibition of cell growth, obtained by blocking an IGF-II-mediated pathway. It has been shown that CaCo-2 cells differentiate spontaneously, depending on cell density(6) . Our data indicate that differentiation is dependent on the growth status of the cells rather than on the acquisition of cell confluence. Conversely, constitutive IGF-II overexpression in transfected cells resulted in the inhibition of CaCo-2 enterocyte-like differentiation, and this effect was directly related to the levels of exogenous IGF-II transcripts. IGF-II ectopic expression resulted in an unregulated stimulus of cell growth as indicated by delayed inhibition of growth at confluence and by higher thymidine incorporation of IGF-II-transfected cells compared with parental and neomycin-transfected control cells. Thus, the inhibition of the enterocyte-like differentiation of CaCo-2 cells in IGF-II transfected cells might be mediated through a continuous stimulus of cell growth.
Also, IGF-II-transfected CaCo-2 cells showed changes in the malignancy of cell phenotype as assessed by increased clonogenicity in soft agar. The inhibition of differentiation in IGF-II-transfected cells could also be explained by the acquisition of a more malignant phenotype, since it has been shown that transformation of CaCo-2 cells by polyoma middle t or by activated ras oncogenes inhibited enterocyte-like differentiation of the cells(39) .
We have also shown that constitutive IGF-II expression altered the physiological down-regulation of IGF-I receptor associated with CaCo-2 cell differentiation, since receptor number remained elevated in IGF-II-transfected post-confluent cells. IGF-I receptor plays an important role in neoplastic transformation of mammalian cells (40) and high IGF-I receptor expression inhibits muscle differentiation of cultured myoblasts(35, 36) . Therefore, the continuous mitogenic stimulus, the increased capability of anchorage-independent growth, and the inhibition of differentiation of IGF-II transfected cells might all be mediated by the high IGF-I receptor number.
All these data suggest that, at least in vitro, IGF-II-regulated growth and differentiation programs of intestinal epithelial cells are inversely related events. The scenario is more complex in vivo, since transgenic animals expressing SV40 T Ag under the control of an enterocyte-specific promoter showed increased proliferative activity but no changes in the differentiation pattern of the enterocytes(8) . However, this mouse model is not comparable with our and other in vitro experimental systems, since the transgene begins to be expressed when the enterocytes are already committed to differentiate.
Regulation of the balance between growth and differentiation of normal and transformed intestinal epithelial cells appears to be a complex phenomenon, depending on several mechanisms. Based on our data, down-regulation of IGF-II and IGF-I receptor expression seems to play a role for the differentiation process to take place. The mechanisms by which IGF-II interferes with the differentiation program are still unclear. It has been shown that activation of the signal transduction pathway of IGF-I receptor by a mitogenic stimulus leads to the nuclear translocation of protein kinase C(41, 42) . Since we have shown previously that protein kinase A-mediated differentiation of CaCo-2 cells is negatively regulated by the activation of protein kinase C (17) , one can speculate that constitutive IGF-II expression interferes with differentiation through sustained activation of protein kinase C. Further studies are needed to address this issue.