(Received for publication, June 6, 1995; and in revised form, August 16, 1995)
From the
The expression of multidrug resistance/P-glycoprotein genes mdr1b(mdr1) and mdr1a(mdr3) is elevated during hepatocarcinogenesis. To investigate the regulation of mdr1b gene expression, we used transient transfection expression assays of reporter constructs containing various 5`-mdr1b flanking sequences in hepatoma and non-hepatoma cells. We found that nucleotides -233 to -116 preferentially enhanced the expression of reporter gene in mouse hepatoma cell lines in an orientation- and promoter context-independent manner. DNase I footprinting using nuclear extracts prepared from hepatoma and non-hepatoma cells identified four protein binding sites at nucleotides -205 to -186 (site A), -181 to -164 (site B), -153 to -135 (site C), and -128 to -120 (site D). Further analyses revealed that, while site B alone played a major part for the enhancer function, sites A and B combined conferred full enhancer activity. Site-directed mutagenesis results also supported these results. Gel retardation experiments using oligonucleotide competitors revealed that the site B contains a dominant binding protein. This is the first report demonstrating a cell type-specific enhancer in the mdr locus. The role of this enhancer in the activation of mdr1b gene during hepatocarcinogenesis is discussed.
The role of the multidrug resistance transporter P-glycoprotein
(P-gp) ()in the development of resistance to a wide variety
of lipophilic compounds in animal cells has been well documented (for
reviews, see (1, 2, 3) ). P-gp is a membrane
protein containing multiple transmembrane domains and two
intracellularly localized nucleotide binding sites. It is generally
believed that P-gp functions as an efflux pump, through which cytotoxic
lipophilic compounds are expelled. P-gp is encoded by a small gene
family consisting of three members in rodents (4, 5, 6) but only two in
humans(7, 8) . However, only two rodent genes (mdr1a or mdr3 and mdr1b or mdr1) (9, 10) and one human gene (MDR1) (11) are related to the multidrug resistance function in
cultured cells.
Tissue-specific expression of different mdr genes has been observed in the rodents, i.e. mdr1a in intestine and blood-brain barrier, mdr1b in kidney and adrenal gland, and mdr2 in the liver. Homozygous disruption of the murine mdr1a resulted in accumulation of cytotoxic drugs in the brain and increased drug sensitivity to the animals(12) . Likewise, disruption of the murine mdr2 led to an impairment of hepatic phospholipid secretion in homozygous animals(13) , consistent with the idea that mdr2 product is a phospholipid transporter(14) .
Overexpression of MDR1 gene has been detected in human malignant biopsies(15) , and in some cases, correlation between elevated MDR1 expression and poor response to chemotherapeutic agents has been reported(16) . In animals, enhanced expression of mdr1a and mdr1b mRNA has been seen during hepatocarcinogenesis(17, 18, 19, 20, 21, 22, 23, 24) . However, the underlying mechanisms for the activation remain to be determined. To explore the mechanisms that control the expression of mdr gene during hepatocarcinogenesis, we have isolated a genomic DNA containing the 5` portion of the mouse mdr1b. Using a transient expression assay, we report here the identification of an enhancer sequence proximal to the promoter of this gene that functions preferentially in hepatoma-derived cell lines.
A fragment containing nucleotides (nt) -1069 to +97 of mdr1b gene was synthesized by PCR DNA using the subcloned DNA as template, SP6 promoter sequence (in the vector) as left side primer, and oligonucleotide 3 as the right side primer. The PCR product was cloned into the PstI-HincII sites of pGEM3Z, released by XbaI/HindIII digestion, and recloned into the XbaI-SmaI sites of a vector containing the chloramphenicol acetyltransferase (CAT) gene(26) , generating -1069M1CAT. A KpnI-PstI fragment was excised from -1069M1CAT and subcloned into the KpnI-PstI sites of the CAT vector to generate the first 5`-deletion mutant, -586M1CAT. Three additional 5`-deletion recombinant DNA were constructed by PCR using -1069Ml3Z as template and oligonucleotide primers 4-6 (all contain a KpnI site), each paired with SP6 promoter sequence (in the vector) as left side primer. The PCR products were digested with KpnI/PstI and inserted into the CAT vector(26) , generating recombinants -233M1CAT, -158M1CAT, and -116M1CAT, respectively.
Construction of 3`-deletion mutants was performed by the same strategy using oligonucleotides 7-9, each paired with oligonucleotide 4 and -1069M1CAT as template. The PCR products were cut with KpnI and cloned into the KpnI site of -116M1CAT, yielding -233(del-130/-116)M1CAT, -233(del-155/-116)M1CAT, and -233(del-172/-116)M1CAT, respectively.
Pairwised oligonucleotides containing complementary DNA sequences, i.e. oligonucleotides 14 and 15, 16 and 17, 18 and 19, and 20 and 21, were annealed, and each were cloned into the BamHI site of pBLCAT2 vector, generating -210/-185TKCAT, -185/-155TKCAT, -153/-135TKCAT, and -127/-120TKCAT, as well as their reverse version of recombinants, respectively.
The mobility shift assays were performed essentially as described previously(32) . DNase I footprinting was carried out using the protocol described by Ohisson and Ediund(33) .
Figure 1:
Transient transfection assay of
promoter function of the mouse mdr1b gene. A, various
lengths of the 5`-flanking sequences from -1069 to -116
plus 58 nucleotides from transcription start site (arrows)
were inserted into the CAT vector (constructs a-e). Constructs f-h represent the 3`-deletion mutants with
the deleted sequences indicated by broken lines. The putative
transcription factor binding sites (SPl and TATA) and (AT) repeats (35, 36) are shown on
-1069M1CAT. These recombinant DNA were transfected into BprC1
cells and NIH3T3 cells (B), and the levels of expression were
assayed using cotransfected pH2B-
gal plasmid DNA as internal
control for calibration of transfection efficiency. The results are
average of three experiments.
Recombinant -1069M1CAT (Fig. 1, construct a) was transfected into these cells.
2 days after transfection, the CAT activity in the transfected cells
was determined. Levels of CAT expression in BprC1 cells were comparable
to those in Hepalclc and Hepal-6 cells (not shown) but were about 2-
and 20-fold higher than those in NIH3T3 cells and HeLa cells (not
shown), respectively, using the cotransfected pH2B-gal as an
internal control (Fig. 1B). Similar results were
obtained with pCMV-
gal, in which the expression of lacZ gene is controlled by cytomegalovirus enhancer/promoter (not
shown). These results suggest that a sequence within nt -1069 can
direct reporter gene expression preferentially in hepatoma-derived
cells. Cell type-specific activity of mdr1b upstream sequences
have also been demonstrated previously by transient transfection
assay(35, 36) .
The levels of CAT expression from -1069M1CAT, -586M1CAT, and -233M1CAT recombinants (Fig. 1B, constructs a-c) in the transfected BprC1 cells were not significantly different. However, deleting nt -233 to -158 and further downstream (Fig. 1B, constructs d and e) resulted in a reduction of more than 90% of CAT activity. These results suggest that sequences downstream from nt -233 are important for the expression of the mdr1b gene in mouse hepatoma cells. Similar results were seen in Hepal-6 and Hepalclc hepatoma cells (data not shown), in which high levels of mdr1b expression were also seen(25) .
When the same set of recombinant plasmids were transfected into NIH3T3 cells and HeLa cells, different expression patterns emerged. In the transfected NIH3T3 cells (Fig. 1B), the level of CAT activity progressively decreased as the lengths of 5`-mdr1b region decreased. Furthermore, only less than 20% reduction in the levels of CAT expression was found between -233M1CAT (construct c) and -158M1CAT (construct d). In the transfected HeLa cells, there was no significant difference in the CAT activity between -233M1CAT and -158M1CAT recombinants (not shown). These results suggest that positive cis-regulatory element(s) located downstream from -233 nt of the mdr1b gene functions preferentially in hepatoma cell lines.
To determine the 3` boundary of the cis-regulatory element, we carried out similar analysis using -233MlCAT constructs with progressive deletions from -116 to -172 (Fig. 1A, recombinants f, g, and h). Removing sequences between -116 and -130 or between -116 to -155 resulted in reduction of no more than 30% CAT activity in BprC1 cells. Removal of additional sequences, i.e. -155 to -172, resulted in reduction of 45% of activity. These results, together with those from the 5`-deletion assay, suggest that the sequence located between -233 and -172 plays a major role for the expression of the reporter constructs in BprC1 cells, whereas the sequence between -172 and -155 may also be contributory but comparatively less substantial.
Figure 2: Transient transfection CAT expression assay of the mdr1b enhancer in hepatoma BprC1 and NIH3T3 cells in different promoter contexts. mdr1b sequences -233 to -116 were inserted into mdrlCAT (a, b) or pBLCAT2 (d, e) in both the forward (a, d) and reverse (g, e) orientations. The resultant recombinants and the vector (c, f) were transfected into BprC1 and NIH3T3 cells as indicated. The levels of CAT expression of these recombinants were shown in A (average of three experiments). B shows the structure of the recombinant DNA.
To investigate whether nt -233 to -116 can enhance reporter gene expression from a heterologous promoter, we inserted this sequence in both forward and reverse orientations into pBLCAT2 (Fig. 2B, constructs d-f). This vector contains a CAT reporter gene driven by the basal promoter from the thymidine kinase (TK) gene. As shown in Fig. 2A, the forward version of construct (construct d) displayed a 10-fold increase in the CAT activity in BprC1 cells, whereas it displayed about a 2-fold increase for the reverse version (construct e). Again, all of these constructs exhibited a very low enhancement of CAT activity in NIH3T3 cells. These results indicate that nt -233 to -116 can enhance the reporter gene expression in an orientation- and promoter context-independent manner. Furthermore, this sequence showed hepatoma specificity in directing the expression of the reporter gene even in a heterologous promoter. Therefore, this sequence can be considered as an enhancer preferentially active in the hepatoma cells.
Figure 3:
DNase I
footprinting assay of enhancer binding proteins in nuclear extract
prepared from BprC1 cells. The DNA fragment containing the enhancer
sequence (nt -233 to -116) (panel A) was
end-labeled with P on either the coding strand (lanes
1-3) or the noncoding strand (lanes 4-6). The
labeled DNA was mixed with (lanes 2, 3, 5,
and 6) or without (lanes 1 and 4) BprC1
nuclear extract in the presence of poly dI
dC competitors and
digested by DNase I. Lanes G and T contain molecular
size markers generated by dideoxynucleotide sequencing ladders. The two
footprints (sites A and B) in both coding and noncoding strands are
shown by brackets. For detecting footprints downstream from
-160, end-labeled oligonucleotides with sequence -185 to
-50 (coding strand, lanes 1-4)) and -210 to
-80 (noncoding strand, lanes 5-7) were used as
probes. Two footprints, C and D, are shown by brackets (panel B). Another footprint downstream from -120
is shown by bars.
To determine whether additional protein binding sites located downstream from -164 that could not be adequately resolved under the gel electrophoretic conditions were favorable for detection of sites A and B, we carried out footprinting analyses using end-labeled fragments spanning nt -50 to -185 for the coding strand and -210 to -80 for the non-coding strand (Fig. 3B). At least two additional protein binding sites, located approximately at -153 to -135 (site C) and -128 to -119 (site D), were detected. For the reason as mentioned above, site C could be better detected in the non-coding strand (by the characterized flanking hypersensitive sites) than in the coding strand. The reason for this is not clear but could be due to the unfavorable cleavage bias of A and C residues by the nuclease (see lanes 1 and 2 in Fig. 3B). These results demonstrated multiple protein binding sites in the mdr1b enhancer region. Fig. 3B also shows one footprint (indicated by bars) downstream from -120 nt. The functional aspect of this footprint was not further characterized.
Figure 4:
CAT expression analysis of various
portions of the mdr1b enhancer. CAT expression from various
recombinant plasmid DNA (a-g) in BprC1 and NIH3T3 cells
are shown. In all cases, F and R refer to forward and
reverse versions, respectively, of the mdr constructs that were used.
The structures of the plasmid DNA are schematically shown. CAT
expression was normalized to the -galactosidase activity using the
cotransfected pH2B-
gal plasmid DNA. mdr1b sequences in
these constructs are as follows: a, -233 to -116; b, -210 to -155, c, -210 to
-185; d, -185 to -155; e,
-162 to -116; f, -153 to -135; and g, -127 to -120.
To further substantiate this finding, we carried out site-specific mutagenesis of nucleotides downstream from -233 in -233M1CAT. A total of 11 mutants were prepared (Fig. 5A). These constructs were transfected into BprC1 cells. The CAT activities in the transfected cells were determined (Fig. 5B). Mutants b through e, which contain specific mutations between nt -226 and -200, had CAT activities comparable to that of the wild-type construct (construct a). Mutants f through i, which contain mutations spanning from the 3`-half of site A to the 5`-half of site B, however, exhibited a 90% reduction of CAT activity in BprC1 cells but no reduction in NIH3T3 cells. Mutations in sites C and D (mutants j-l) exhibited minor reduction in CAT activities in BprC1 cells in relative to those found in mutant e, and no significant reduction in NIH3T3 cells. These results collaborate the notion that sites A and B are important for the enhancer function as mentioned above.
Figure 5: Site-directed mutagenesis of mdr1b enhancer and CAT assay. A, DNA sequence of the mdr1b enhancer region. The nucleotides chosen for mutagenesis are indicated by boldface, and the corresponding mutated nucleotides are shown below the arrows and underscored. Locations of footprints, sites A through D, are indicated by brackets. B, quantitative analysis of the CAT activities of these mutants (b through l), in reference to the activity of -233M1CAT (a) in BprC1 cells (hatched bars) and in NIH3T3 cells (open bars), are shown. The results are from three independent transfection experiments.
Figure 6:
Gel mobility retardation assay of
protein-DNA complexes. End-labeled probe nt -233 to -116
containing sites A through D (panels A and D),
-210 to -155 (sites A and B, panels B and E), and -162 to -116 (sites C and D, panels C and F) were mixed with nuclear extract prepared from
BprC1 cells (panels A-C) or from NIH3T3 cells (panels D-F) in the presence of poly dIdC and
various amounts of unlabeled, double-stranded enhancer DNA competitors,
whose nucleotide sequences span A (-210 to -185), B (-185 to -155), C (p153 to -135),
and D (-127 to -120) elements as
indicated.
The precise reasons that oligonucleotides containing site A and site D sequences failed to show competitions in gel shift assays are not clear but could be due to the following possibilities: (i) protein binding to these sites, as detected by DNase footprinting assays (Fig. 3), may require neighboring sequences, since the probes used in these two assay systems were not entirely the same; (ii) protein bindings to site A and site D may require protein occupancies to site B and site C for stabilization; (iii) site A and site D may not be genuine protein binding sites; instead, they may be created by protein-protein interactions, using sites B or C as anchoring points, thereby masking nuclease accessibilities of the neighboring sequences. Likewise, the failure of detecting enhancing activities for site C and site D in CAT assay (Fig. 4) suggests that proteins recognizing these sites may serve only a structural role. Further studies are needed to clarify these possibilities.
Recent studies from several laboratories have demonstrated that several putative positive and negative cis-regulatory elements are present in the promoter regions of the rodent and human mdr genes(35, 36, 37, 38, 39, 40, 41) . In addition, several protein binding sites have been located at the promoter region of the murine mdr1b gene(42, 43) . The study presented here identified an enhancer located between nt -233 and -116 in the mouse mdr1b gene in which four DNase I footprints are located. Further analyses showed that site A and site B together possess full enhancer function, but site B plays a predominant role. Strikingly, these sequences can promote preferentially in hepatoma-derived cell lines. This is the first cell type-specific enhancer found in the mdr locus (a cell type-specific enhancer upstream from the human MDR1 gene was reported(44) , but association to the MDR1 could be due to cloning artifact(45) ).
Site A contains the imperfect inverted
repeat sequence 5`-ACTTACCTGAACACGTAAAG (underscored). Mutations in the
first half repeat (Fig. 5, mutant e) failed to abolish
the function of the enhancer, whereas the second half did (Fig. 5, f and g), suggesting that the 5`
boundary of the enhancer may begin at the second half of site A. We
searched DNA sequences recognized by transcription factors in the
GenBank and found that the second half repeat of site A contains a
subsequence resembling the sequence motif CGT(A/C)A that is critical
for binding of a group of cellular transcription factors, i.e. ATP, CREB, E4F1, or E4TF3, and EivF. Although these transcription
factors are capable of activating E1a- and cyclic AMP (cAMP)-inducible
promoters(46, 47, 48, 49) ,
different promoters respond very differently to these inducers. Thus,
whether mdr1b enhancer can be modulated by E1a or by cAMP
remains to be determined. However, it may be relevant to note that
cAMP-dependent protein kinase regulated sensitivity of mammalian cells
to multiple drugs has been reported(50) . Site B contains two
tetranucleotide direct repeats 5`-GTATGTAAATGTCTGAGG (Fig. 5)
and a potential HNF-1 binding site
((G/A)TTAATN(A/T)T(T/C)AG)(51) . In addition, one copy of p53
binding site (AAGACAAGTCT
) (52) is located between sites A and B of the mdr1b enhancer. However, it has been reported that a single copy of the
recognition sequence was insufficient for transcriptional activation by
p53(52, 53) . In this context, mdr1b enhancer
may not be sensitive to the function of p53, although previous studies
have demonstrated that the promoters of human MDR1(38) and Chinese hamster Pgp1(55) (both
are homologs of murine mdr1a) are modulated differentially by
wild-type and mutant p53. In any event, the identities of interacting
protein factors that confer cell type-specific enhancer function remain
to be demonstrated.
DNase I footprinting and gel mobility
retardation assays apparently suggested that protein factors
recognizing specific binding sites in the mdr1b enhancer are
present in both hepatoma and non-hepatoma cells. This does not preclude
the possibility that these proteins have a role in the regulation of mdr1b expression, specifically in hepatoma cells. For example,
ATBF1, a multiple homeodomain zinc finger protein that selectively
down-regulates hepatoma cell-specific enhancer of human
-fetoprotein gene, is present in both hepatoma and nonhepatoma
cells(56, 57) . At this time, we cannot exclude the
possibility of differential posttranscriptional modification of these
proteins (e.g. phosphorylation, poly(ADP-ribosylation), etc.)
resulting in transcriptional activation of mdr1b gene in liver
tumors. Alternatively, these enhancer binding proteins may interact
with other non-DNA binding factors to confer tissue specificity in the
similar manner as the recently identified novel B cell-derived
coactivator (OCA-B), which potentiates the activation of immunoglobulin
promoters by octamer-binding transcription factors(58) .
Several such coactivators have also been implicated in the
transcriptional regulation of liver-specific gene
expression(59, 60) . Molecular cloning of genes
encoding these mdr1b enhancer binding proteins should
facilitate understanding of the complex control mechanism of mdr1b gene expression.
Levels of mdr1b mRNA are elevated during hepatocarcinogenesis. Studies of mdr1b expression in this system are hampered by the lack of culture cell systems that mimic the in vivo situation. Culturing primary hepatocytes in vitro for several hours shows spontaneous activation of mdr1b expression(61, 62, 63) . Whether the enhancer described here is involved in the mdr1b expression in vivo remains to be determined. Investigations using transgenic animals and/or targeting gene delivery to HCC (54, 64) may allow us to address these issues. These experiments are currently under way in our laboratory.
In summary, we have characterized a hepatoma-specific enhancer in cultured cells. The identification of this enhancer may serve as a molecular basis for future studies of the regulation of mdr1b expression during hepatocarcinogenesis. These studies may eventually increase our understanding of how mdr gene expression in HCC is controlled and hence facilitate the development of approaches to control intrinsic drug resistance in this disease.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M57524[GenBank].