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16 C. Huin et al.! Biology ofthe Ce1l94 (2002) 15-27
et al., 1994; Tontonoz et al., 1994, 1995; Schoonjans et al.,
1996; Spiege1man et al., 1997) and monocyte/macrophage
(Marx et al., 1998; Ricote et al., 1998; Tontonoz et al., 1998)
differentiation. Until now, the precise role of PPAR~ has not
yet been elucidated.
In a recent study (Huin et al., 2000), we have reported
that the PPAR subtypes exhibit different patterns of expression
in relation to morphogenesis and cell differentiation in
the developing human feta1 digestive tract. Recent reports
indicate that the growth of several different colon carcinoma
cell lines is inhibited by the ligand activation of PPARy
(Brockman et al., 1998) and that transplantable tumors in
mice derived from human colon cancer cells show significant
reduction of growth when mice are treated with
troglitazone, a PPARy ligand (Sarraf et al., 1998). On the
other hand, Lefebvre et al. (1998) have reported that ligand
activation of PPARy in C57BLl6J-APc Mi n/+ mice promotes
the development of colon tumors, and Saez et al. (1998)
found that it accelerates the formation of colonic polyps in
the same mice. Those divergent observations 1ead us to
investigate the expression of PPARs in the human colon
cancer Caco-2 cells since it has been known that they
undergo spontaneous differentiation in culture when they
reach confluence (Pinto et al., 1983). The PPAR expression
was analyzed by immunocytochemistry, Western- and
Northern-blotting and SI nuclease protection assay. The
results indicate that as Caco-2 cell differentiation goes on
characterized by the appearance of bmsh border and peroxisomal
molecular markers, the protein 1evels of PPARa,
PPARYl and PPARY2 increase gradually. The amount of
PPARY3 mRNA increases concomitantly to the resulting
PPARYl protein. On the other hand, the amounts of PPARa
and PPARY2 mRNA are not significantly changed during
Caco-2 cell differentiation suggesting that the increase in
their respective protein is due to an elevation of the
translational rate.
2. Materials and methods
2.1. Cel! culture conditions
The human adenocarcinoma cellline Caco-2 was originally
established by Fogh and Trempe (1975). Cells were
cultured in Dulbecco's modified minimum medium
(DMEM) containing 4.5 g 1- 1 glucose, without glutamine
and supplemented with 20% (v/v) heat-inactivated fetal calf
semm (56 oc for 30 min) and 1% (v/v) non-essential amino
acids (all obtained from Eurobio, Les Ulis, France) using an
incubator at 37 oC with a humidified 10% CO2 atmosphere.
The cells (from passages between 85 and 100) were seeded
at an initial concentration of 6 x 10 4 m1- 1 and subcultured
every 5 days. After 5, 10 and 15 days of culture, the cells
were processed for ultrastructural cytochemistry and immunocytochemistry
or frozen in liquid nitrogen unti1 used.
2.2. Ultrastructural cytochemistry ofcatalase
The presence of peroxisomes in Caco-2 cells was inves·
tigated using the procedure of Novikoff et al. (1972), whid
allows the visualization of the peroxisomal catalase activit)
after reduction of alkaline 3,3'-diaminobenzidine (DAB
Sigma Chemical Co, St-Louis, MO, USA). In brief, Caco-:~
cell monolayers were fixed at 4 oC for 40 min in 0.1 M
sodium cacodylate buffer, pH 7.4, containing 2.8% (v/v:
glutaraldehyde, then washed 3 times for 20 min in presencE
of the same buffer containing 7.5% (w/v) sucrose. Cell~
were treated for 3 h at 37 oC in DAB-medium. The 1attel
was replaced after 90 min. Samp1es were postfixed in lo/c
(v/v) osmium tetroxide buffered with 0.1 M potassium
phosphate, pH 7.4, for 30 min, dehydrated in ethanol and
embedded in araldite/epon (v/v) mixture. Thin sections were
stained with uranyle acetate and lead citrate according to
Reynolds (1963) and observed with a Philips CM12 electron
microscope at 80 kV.
2.3. Enzyme assays
Cell monolayers were rinsed three times with ice-cold
0.1 M potassium phosphate buffer, pH 7.4, scraped with a
mbber policeman and homogenized in 2 mM Tris/HCl,
pH 7.1, containing 30 mM mannitol. Homogenates were
kept for 1 h on ice, then centrifuged for 15 min at
15000 x g. The supernatants were assayed for protein
concentration according to Lowry et al. (1951) using bovine
semm albumin as a standard. Sucrase-isomaltase
(EC.3.2.1.26) activity was determined according to Messer
and Dahlqvist (1966). Alkaline phosphatase (EC.3.1.3.1)
activity was measured according to Garen and Levinthal
(1960). For both enzyme activity, 1 unit is defined as the
activity that hydrolyses 1 [!mo1e of substrate pel' minute at
37 oC under the experimenta1 conditions. Fatty acylcoenzyme
A oxidase (AOX; EC.1.3.99.3) was determined
using the method reported by Hryb and Hogg (1979) with
homogenates prepared in 50 mM Tris/HCl, pH 8.0, containing
0.1% (v/v) Triton X-lOO. Lauroyl-CoA (Sigma Chemical
Co) was used as a substrate and the mo1ecular extinction
coefficient was 6930 M- 1 cm- 1 . One unit is defined as the
activity that converts 1 [!mo1e NAD+ into NADH, H+ per
minute at 37 oC under the experimental conditions. Catalase
(EC. 1.l1. 1.6) activity was assayed according to Baudhuin et
al. (1964). One unit 'Baudhuin' (UB) is defined as the
activity that hydrolyses 1 [!mole of substrate per minute at
4 oC.
2.4. Immunoblot analysis
Cell homogenates were prepared according to two protocols.
Firstly, Caco-2 cells were homogenized in 20 mM
Tris/HCl, pH 8.0, containing 5 mM EDTA and 1% (v/v)
Triton X-lOO, then centrifuged at 15000 x g for 30 min at
4 oc. The protein concentration of the supernatant was