Ce document numérisé est le fruit d'un long travail approuvé par le ...

18
C. Huin et al. / Biology ofthe Ce1l94 (2002) 15-27
Table 1
Primers se1ected for SI nuclease protection assay.
Probe Primer up (5'-3') Primer down (5'-3') Length (bp) Reference
hPPARa GCCCAGGCTGAAGCTCAGGG GACACGGAAAGCCACTCTTGC 244 Sher et al. (1993)
hPPARYl TCGGACCCCGAGCCCGAG TCAAACGAGAGTCAGCCTTTAAAC 133 Fajas et al. (1997)
hPPARY2 CCCATCTCTCCCAAATATTT CACAGAGATCGCATTCGGCCC 282 Fajas et al. (1997)
hPPARY3 GAAAGAAGCCGACACTAAACCAC ACACCTCATCTTTACGACCTCTTC 222 Fajas et al. (1998)
G3PDH CCCATCACCATCTTCCGA GGAAACTGTGGCCTGTAG 380 Tso et al. (1985)
and down primers are indicated in table 1. Amplification
was calTied out by 30 cycles (95 oC, 30 s; 60 oC, 30 s;
72 oC, 30 s) foUowed by an extension step at 72 oC for
5 min. After purification, the PCR fragments were cloned
into the pBluescript II KS+ vector to obtain the recombinant
plasmids pKS+/PPARa, pKS+/PPARYJ, pKS+/PPARY2 and
pKS+/G3PDH then sequenced. The PPARYJ probe (133 nt)
contained exon Al (94 nt) and a part of exon El (39 nt)
according to the definition of the genomic structure of the
human PPARy gene by Fajas et al. (1998). The PPARY2
probe (282 nt) spanned exon B (243 nt) and the 5' end of
exon 1 (39 nt) whereas the PPARY3 probe (222 nt) contained
the fuUlength exon A2 (76 nt) and exon 1 (146 nt).
The plasmids were linearized as foUows: pKS+/PPARYJ
and pKS+/PPARY2 with Bam Hl, pKS+/PPARY3 and
pKS+/G3PDH with Eco RI, then labeled with (a_ 32 p] dCTP
by down extension using Taq polymerase and the specifie
primer (Table 1). Total RNA (5 [log) extracted from Caco-2
ceUs at different durations of culture, was first treated by
RNAse free RQ-1 DNAse (Promega, La JoUa, CA, USA)
then hybridized with the labeled single-stranded DNA
probes (10 5 cpm pel' sample) at 60 oC for 16 h. Non
hybridized DNA was digested by SI nuclease (50 U pel'
sample) for 1 h at 37 oC then extracted by phenol!
chloroform procedure (Sambrook et al., 1989). The
DNAIRNA hybrids were resolved by electrophoresis on a
5% polyacrylamide gel under denaturing conditions. Then,
the gel was exposed to a X-OMAT Kodak film for 24 h. The
resulting bands were quantified by densitometry and the
values were standardized versus the cOlTesponding G3PDH
values.
2.8. Statistical analysis
Data concerning the actJVltles of sucrase-isomaltase,
alkaline phosphatase, AOX and catalase were expressed as
means ± standard deviations from three independent experiments.
Densitometric analyses of SI nuclease protection
assay blots were calTied out from four independent experiments.
Evaluation of statistical significances was assessed
using analysis of variance (ANOVA) and the Fisher protected
least significant difference test (multiple comparisons)
(Winer, 1971). Statistical significance is indicated in
each figure.
3. Results
3.1. Assessment of Caco-2 cel! differentiation
The specifie activities of sucrase-isomaltase and alkaline
phosphatase (two brush border membrane enzymes), AOX
and catalase (two peroxisomal enzymes) were determined in
homogenates of Caco-2 ceUs cultured for 5, 10 and 15 days.
As shown in table 2, the specifie activities of sucraseisomaltase
and alkaline phosphatase increased steadily during
ceU culture. They were increased by 2.3 and 5.7 fold,
respectively at day 15 of culture when compared to control
values (5 days of culture). The pattern of peroxisomal
enzymes was somewhat different as AOX and catalase
specifie activities increased by 1.7 and 3 fold, respectively,
between day 5 and day 10 of ceU culture then remained
constant.
Using specifie polyclonal antibodies, the protein levels of
villin, a molecular marker of brush border development, and
of AOX, PBE, catalase and PMP70 (both peroxisomal
proteins) were analyzed by Western blotting. An unique
band with molecular weight of 90 kDa was detected for
villin in Caco-2 ceU homogenates. Its intensity increased
about 2.3 fold foUowing 15 days of culture (Fig. 1). Whatever
the duration of culture, three bands were immunodetected
for AOX at 65.6, 56 and 42 kDa, respectively. This is
consistent with previous western blot data for this enzyme
(Duclos et al., 1997). Scanning densitometry of the intensity
of the 56 kDa subunit revealed a graduaUy increase giving
a maximum of about 3.3 fold at 15 days of culture (Fig. 2).
PBE and catalase exhibited only one band estimated at
Table 2
Specifie activities of enzymes from cu1tured Caco-2 cells.
Sucrase-isoma1tase 65.1 ± 3.9
Alka1ine phosphatase 0.95 ± 0.1
AOX 0.74 ± 0.04
Cata1ase 12.3 ± 0.2
Duration of Caco-2 cell culture
Enzymes 5 days 10 days
80.7 ± 5.5
35.2±4.1
1.10 ± 0.1
34.6±0.5
15 days
155.2 ± 10.1
55 ± 4.5
l.11 ± 0.05
35.6±0.6
They were determined as described in Materials and methods and
expressed as mU mg- 1 protein. Values represent means ± standard deviations
from three independent experiments. ANOVA and multiple comparison
analysis show a significant difference (p < 0.01) for aIl enzymes
activities measured at day 15 versus day 5.