(Received for publication, May 14, 1994; and in revised form, October 14, 1994)
From the
The effect of human wild type and mutant p53 proteins on the human multidrug resistance (MDR1) promoter was studied in a p53-negative human cell line. Transient expression of MDR1 promoter-chloramphenicol acetyltransferase reporter gene constructs (MDRCAT) cotransfected with p53 expression vectors was analyzed in H358 lung carcinoma cells. Cotransfection with a wild type p53 expression vector stimulated MDRCAT activity, while cotransfection with mutant p53 expression vectors altered at amino acid positions 181, 252, 258, or 273 failed to stimulate expression. Wild type p53 stimulation of MDRCAT activity was time dependent with maximal expression occurring 24-30 h following transfection and correlating with high p53 protein levels. MDR1 promoter deletion analysis suggested that the sequences involved in wild type p53 stimulation of MDRCAT activity were contained within the region from -39 to +53 relative to the start of transcription at +1. This region contains no TATA or p53 consensus binding sequence but does contain an initiator sequence. Wild type p53 stimulation of MDRCAT expression also occurred in parental and doxorubicin-resistant SW620 colon and parental 2780 ovarian cancer cell lines, indicating that wild type p53-mediated simulation of the MDR1 promoter is not restricted to a single cell line.
The human multidrug resistance gene (MDR1) encodes P-glycoprotein, an energy-dependent membrane-bound drug efflux pump, which has been implicated in resistance to some cytotoxic drugs used for chemotherapy (reviewed in (1) and (2) ). MDR1 expression is induced in cultured cells or transient transfection systems by a variety of stresses such as chemotherapeutic drugs(3, 4, 5) , heavy metals(6) , UV light(7) , and heat shock(6, 8) . Several of these agents are also known to induce the tumor suppressor p53 (reviewed in (9) and (10) ).
Previous studies have suggested that in a transient assay, the MDR1 promoter was responsive to p53(11, 12, 13) . These studies demonstrated that cotransfection with several mutant p53 expression vectors stimulated transient MDR1 promoter-driven expression, whereas cotransfection with a wild type p53 expression vector either had no effect or repressed promoter activity. One study suggested that this effect was mediated through core promoter sequences (12) . The goal of the present study was to determine the critical MDR1 promoter element(s) responsible for the p53 effect.
The
previous studies used heterologous human/rodent systems and/or cell
lines that expressed endogenous p53. Because of concern that these
factors might complicate the results, the present study was done in a
human cell line, which was null for p53 expression with both human MDR1 promoter reporter constructs and human p53 expression
vectors. The majority of these studies were done in the H358 human
non-small cell lung cancer cell line which contains a homozygous
deletion of the p53 gene(14, 15) . To characterize the
effect of p53 on the MDR1 promoter, a series of human MDR1 promoter-chloramphenicol acetyltransferase reporter gene (MDRCAT)
constructs was transiently transfected with either wild type or mutant
p53 expression vectors into human H358 lung carcinoma cells. The
activity of the MDR1 promoter was determined by assaying CAT ()activity, and p53 levels were determined by Western blot
or RT-PCR analysis.
Figure 2:
The effect of cotransfection of MDRCAT and
p53 expression vectors. A, transient expression of
cotransfected -89/+53 MDRCAT and p53 expression vectors in
H358 lung cells. The cells were transfected with the vector (CMV
expression vector control without any p53 cDNA), p53-wt (wild type),
p53-m181 (Arg His), p53-m252 (Leu
Pro), p53-m258 (Glu
Lys), or p53-m273 (Arg
His)(20) . B,
RT-PCR analysis of p53 RNA and glyceraldehyde-3-phosphate dehydrogenase (GAP) control in transfected cells. The chemiluminescent
RT-PCR analysis was performed on 1 µg of total cellular RNA
isolated from the indicated transfected cells and untransfected H358
cells as previously described(53) . The transfections shown in A and B were done in parallel on the same day. The
cDNA made from RNA was diluted 1:10 for p53 and 1:50 for
glyceraldehyde-3-phosphate dehydrogenase, and the PCR was carried out
for 35 cycles. The p53 primers were obtained from Genset (Clifton,
NJ).
Figure 3:
Time
course following cotransfection with MDRCAT and p53 expression vectors. A, the DNA was applied to the H358 cells in a transient
transfection at t = 0. Plates of cells were harvested
at 6-h intervals from 18 to 54 h following application of the DNA. The
cells were cotransfected with -89/+53 MDRCAT and CMV
expression vector control (), p53-wt (
), or p53-m273
(
). The MDRCAT activity was determined as described under
``Experimental Procedures.'' B, Western blot
analysis was performed on cells from the indicated time points and on
untransfected H358 cells as previously described(54) . An equal
aliquot of cell lysate from the same cells analyzed in A was
loaded on to 10% SDS-polyacrylamide gels. After electrophoresis, the
gels were transferred in parallel to nitrocellulose membranes. The
membranes were prepared for chemiluminescence in parallel and exposed
together to a single piece of film to ensure equivalent exposure time. C, the film in B was digitized using an Eagle Eye II
(Stratagene). The relative density of the bands (units) from p53-wt
(
) or p53-m273 (
) samples was determined using the NIH
Image 1.55 program.
The transient expression of the MDR1 promoter reporter gene constructs was studied in the H358 lung cells. The level of MDRCAT activity in H358 cells transfected with a series of MDRCAT constructs containing various lengths of sequence 5` to the start of transcription was determined. The expression of the MDRCAT constructs in the p53 null H358 lung cell line was similar to that found in other human cell lines studied in this laboratory with deletion of the transcriptional regulatory regions containing the Y-box sequence and the Sp1 binding sequence, causing a reduction in MDRCAT activity (data not shown)(16, 21) .
To determine if the p53 expression vectors were functioning properly in the H358 lung cells, the effect of the wild type and one mutant p53 expression vector was first studied on p53 control constructs. These reporter gene constructs contain multiple copies of either a consensus p53 binding sequence for the positive control PG13-CAT or a scrambled p53 binding sequence for the negative control MG15-CAT adjacent to a minimal promoter and CAT reporter gene(19) . Fig. 1shows that the wild type p53 expression vector stimulated the p53-positive control PG13-CAT and failed to stimulate the p53-negative control MG15-CAT in the H358 lung cell line(18) . The mutant p53 expression vector p53-m273 did not stimulate either control vector. Thus, the wild type and mutant p53 expression vectors behaved as expected in an assay with p53 control reporter constructs.
Figure 1:
Transient expression of p53 control
constructs in H358 lung cancer cells. The cells were cotransfected with
PG13-CAT, a wild type p53-inducible positive control construct, or
MG15-CAT, a negative control construct (19) and p53-wt ()
or p53-m273 (&cjs2100;) as described under ``Experimental
Procedures'' for MDRCAT
cotransfections.
Wild type and several mutant p53 expression vectors were used to study the effects of p53 on MDRCAT expression(18, 20) . The -89/+53 MDRCAT construct, which contains all of the nucleotide sequences required for basal MDR1 transcription, was cotransfected with the p53 expression vectors into the H358 lung cells. The results shown in Fig. 2A indicate that the wild type p53 stimulated expression from the -89/+53 MDRCAT construct severalfold over the base-line expression produced by cotransfection with the CMV expression vector without any p53. In contrast, cotransfection with several mutant p53 expression vectors failed to stimulate and in fact slightly suppressed MDRCAT activity. These p53 mutants had altered amino acids at positions 181, 252, 258, or 273. All of the mutant p53 expression vectors examined produced high but not equivalent levels of p53 mRNA in the H358 lung cells as determined by RT-PCR analysis, indicating that the failure to stimulate MDRCAT activity was not caused by a lack of mutant p53 mRNA (Fig. 2B). Unequal quantities of some p53 mRNAs may have been caused by repression of the CMV promoter in the expression vector by some forms of p53(22) . These studies show that in H358 lung cells, wild type p53 stimulated MDRCAT activity while several different p53 mutants did not.
Because of the known instability of wild type p53 protein, a time course of the MDRCAT activity following cotransfection was performed. Fig. 3A shows that MDRCAT activity was maximally stimulated 24-30 h following cotransfection with the -89/+53 MDRCAT construct and the wild type p53 expression vector. In contrast, the MDRCAT activity was unchanged between 18 and 54 h following cotransfection with mutant p53-m273. These results illustrate the transient nature of the p53-mediated stimulation of MDRCAT activity in the H358 lung cells. It should be noted that if MDRCAT activity were examined at 48 h, a frequently used termination time for transient expression studies, minimal stimulation of expression by wild type p53 would be observed.
Because the H358 lung cells lack endogenous p53 protein, the level of p53 protein produced by the p53 expression vectors can be evaluated directly. To determine whether the transient stimulation of MDRCAT activity was due to changes in p53 protein levels, Western blot analysis shown in Fig. 3B was performed on an aliquot of the cells from the time course experiment shown in Fig. 3A. Quantitation of this blot shown in Fig. 3C indicates that the production of wild type p53 preceded the stimulation of the MDRCAT activity and then decreased with similar kinetics as the MDRCAT activity. In contrast, the level of the mutant p53 protein remained essentially constant over the entire time course. The differences in MDRCAT activity between cotransfections with wild type and mutant p53 therefore cannot be attributed to differences in p53 protein levels since the level of mutant p53 protein is equivalent or higher than the level of wild type p53 at all time points. These results show that there was a correlation between the level of MDRCAT activity and the amount of wild type p53 protein.
Previous studies from our laboratory using a series of MDRCAT deletion constructs defined two areas of the MDR1 promoter, which are required for basal transcriptional regulation. In parental and doxorubicin-resistant 2780 ovarian and SW620 colon cancer cell lines, deletion of the Y-box sequence (-82/-73) or deletion of the Sp1 binding sequence (-56/-41) resulted in a 5-10-fold decrease in basal expression(16) . These studies demonstrated that the minimal sequences required for maximal basal MDR1 expression were between -89 and +53.
To determine which MDR1 promoter sequences were involved in wild type p53-mediated stimulation of expression, a series of MDRCAT constructs with various 5`-ends was analyzed by cotransfection into H358 cells. All of these MDRCAT constructs had 3`-ends at position +53 relative to the start of transcription at +1. Fig. 4A shows the base-line expression in H358 cells of the MDRCAT constructs cotransfected with the CMV expression vector control. This analysis showed a reduction in MDRCAT activity upon removal of the two critical regulatory sequences described above. Although the effects of these deletions on MDRCAT expression in the H358 cells are not as pronounced as in the cell lines previously tested, these data suggested that the nucleotide sequences regulating MDR1 expression are identical to those previously found (16) . Cotransfection of the MDRCAT deletion constructs with wild type p53 resulted in increased levels of MDRCAT activity, compared with the control vector with all constructs tested. In these cotransfection experiments with wild type p53, deletion of the Y-box or Sp1 sequences also caused a reduction in MDRCAT expression, suggesting that these nucleotide sequences are also functional in p53-mediated stimulation.
Figure 4:
Deletion analysis of a series of MDRCAT
constructs cotransfected with p53 expression vectors. A series of
MDRCAT constructs with deletions at their 5`-ends (shown in C)
was cotransfected with p53-wt, p53-m273, or CMV expression vector
control into H358 cells, and the cells were harvested 40 h following
application of the DNA. The data were normalized to the median
luciferase value of all samples transfected with the same p53
expression vector. The results are the average of two experimental
points. A, MDRCAT activity of cells cotransfected with the
indicated MDRCAT expression construct and with either CMV expression
vector control (&cjs2108;&cjs2098;) or p53-wt (). B,
fold increase in expression of cells cotransfected with the MDRCAT
expression construct and p53-wt (
) or p53-m273 (&cjs2100;)
compared with cells cotransfected with the MDRCAT construct and the CMV
expression vector control. C, map of the MDRCAT expression
constructs. The positions of several transcription regulatory sequences
are indicated as Y-box(7, 16, 55) ,
SP1(21) , INR(17, 56) , and DSE (downstream
element)(56) .
In Fig. 4B, the cotransfection results shown above are expressed as fold increase in MDRCAT expression over the base-line expression. This method of analysis more clearly shows that deletion of nucleotide sequences from the 5`-end of the MDRCAT construct (-457 to -89) resulted in an enhancement of the stimulatory effect of wild type p53. These results suggest that there are regulatory elements within this upstream region of the MDR1 promoter that blunt the p53 stimulatory effect. Putative repressor sequences in this region of the MDR1 promoter have been found in other cell types(16, 21) . In contrast to the wild type p53-mediated stimulation of MDRCAT expression shown in Fig. 4B, cotransfection of H358 cells with a mutant p53 at amino acid 273 failed to stimulate any of the MDRCAT deletion constructs.
As shown in Fig. 4, A and B, the shortest MDRCAT construct analyzed, which had only 39 nucleotides 5` to the start of transcription, still retained responsiveness to wild type p53. These results suggested that MDR1 promoter sequences between -39 and +53 appeared to be sufficient for the stimulation by wild type p53. It is not yet known whether the p53-mediated stimulation is an activation or a derepression of the MDR1 promoter caused by the addition, removal, or modification of proteins binding to nucleotide sequences around the start of transcription. To determine if the effects of wild type and mutant p53 observed with the H358 p53 null cells were unique to that cell line, several other cell lines were tested. Fig. 5shows a transfection experiment of the -89/+53 MDRCAT construct into parental and doxorubicin-resistant SW620 colon and 2780 ovarian carcinoma cell lines in addition to the H358 lung cell line. These studies showed that wild type p53 stimulated MDRCAT activity in four of the five cell lines analyzed. In each case, cotransfection with the mutant p53-m273 failed to stimulate MDRCAT activity. Thus, most but not all human cell lines tested showed wild type p53 mediated stimulation, suggesting that stimulation of the minimal MDR1 promoter by wild type p53 may be a common phenomenon.
Figure 5:
The effect of cotransfection of multiple
cell lines with MDRCAT and p53 expression vectors. The
-89/+53 MDRCAT construct was cotransfected with pUC7
(&cjs2108;), CMV expression vector control (&cjs2108;&cjs2098;), p53-wt
(), or p53-m273 (&cjs2100;) in parental (620wt) and
doxorubicin-resistant (620A) SW620 colon
carcinoma(57, 58) , parental (2780wt) and
doxorubicin-resistant (2780A) 2780 ovarian carcinoma(59) , and
H358 lung carcinoma (H358) cell lines. For the CAT assays, the amount
of cell lysate analyzed and the incubation times were 25 µg/90 min,
25 µg/22.5 min, 25 µg/6 h, 50 µg/3 h, and 50 µg/3 h,
respectively.
The tumor suppressor gene p53 is mutated at a high frequency in a wide variety of human tumors (reviewed in (10) ). A significant percentage of tumors of many types also express MDR1 RNA (23) . These include tumors that are intrinsically drug resistant, arising from tissues that naturally express the MDR1 gene and tumors that acquire drug resistance following chemotherapeutic drug treatment. The ability of tumors from MDR1-negative tissues to acquire multidrug resistance lead to the suggestion that the genes associated with malignant transformation like p53 can activate MDR1 expression(2, 11) . Support for this correlation came from studies that showed that several mutant p53 expression vectors stimulated reporter gene activity from MDR1 promoter expression constructs while wild type p53 repressed or did not affect expression(11, 12, 13) . However, several recent studies of clinical specimens found no correlation between MDR1 expression and the presence of mutant p53(24, 25, 26) .
The studies described in this manuscript characterize the effects of p53 on the human MDR1 promoter in a p53-negative cell line. They showed that cotransfection of a wild type p53 expression vector with human MDR1 promoter CAT reporter gene constructs caused a stimulation of MDRCAT activity, while cotransfection with several different mutant p53 expression vectors slightly repressed expression. The stimulation of MDRCAT activity by wild type p53 was transient and in general correlated well with wild type p53 protein levels. Although the mutant p53 protein was expressed at a high level at all time points, no stimulation of MDRCAT expression was found. The stimulatory effect was still present with a MDR1 promoter reporter construct containing nucleotide sequences between -39 and +53 relative to the start of transcription at +1. In addition, this stimulation of MDRCAT activity by wild type p53 was not restricted to the lung cell line but was observed in multiple cell lines.
These results differ from those previously reported by other laboratories(11, 12, 13) . Although the bases for the apparently opposite results are unclear, the cell lines, p53 expression vectors, the MDR1 reporter constructs, the method of transfection, and the time of analysis in the present study were different from those used by other researchers. These considerable experimental differences make a direct comparison among the studies difficult. Some of these experimental parameters were also found to affect the ability of wild type p53 to act on the herpes simplex virus thymidine kinase promoter (27) . Comparison of the results reported here with those previously reported suggests that the effects of wild type p53 on the MDR1 promoter may depend on the cells chosen for study. In fact, one of the cell lines, the human 2780 doxorubicin-resistant ovarian carcinoma cell line, failed to show wild type p53-mediated stimulation. None of the p53 mutants examined in this study stimulated the MDR1 promoter in the H358 cells; however, given the wide spectrum of effects of different p53 mutations, it is quite possible that some p53 mutants other than the ones tested may activate the MDR1 promoter. This possibility is supported by studies in other systems. Several p53 mutants have been shown to activate transient expression from natural and artificial genes(28, 29, 30) . In addition, several p53 mutants were able to activate transcription from several synthetic consensus but not a genomic p53 binding site in a stably transfected yeast transcription assay(27) . When considered together, these studies indicate that the context in which p53 functions is critical in determining biological effects.
The p53 tumor suppressor protein is a sequence-specific DNA binding protein(31) . Genes containing a p53 consensus binding sequence are activated by wild type p53, but p53 has been shown to inhibit transcription of many genes that lack a p53 binding sequence(32, 33) . Studies have shown that wild type p53 binds to proteins that interact with the TATA sequence and inhibit transcription(34) . In addition to binding to the TATA binding protein(35) , wild type p53 also binds to other transcriptional regulatory proteins including Sp1 (36) and CCAAT binding factor(37) .
The properties of p53 and the nucleotide sequence of the human MDR1 promoter can be compared to attempt to gain some insights into the mechanism of p53 activation(17, 38, 39) . The promoter between -39 and +53 does not appear to contain a consensus binding sequence for p53 which has been shown to mediate wild type p53 activation of genes like GADD45, mdm-2, and WAF-1/CIP-1(40, 41, 42, 43, 44, 45) . Since this core region of the MDR1 promoter does not contain a classic p53 consensus binding sequence, either wild type p53 or some factor induced by it may interact with the proteins involved in transcription initiation to enhance transcription or, alternatively, p53 may interact with a negative regulatory protein to prevent repression of the gene. The MDR1 promoter also does not contain a TATA sequence that is responsible for p53-mediated transcriptional repression(34) . However, the promoter does contain an initiator or Inr sequence, which is involved in the initiation of transcription at the proper start site (reviewed in (46) ). The stimulatory effect of wild type p53 on MDRCAT activity is apparently different from the effect on two other Inr-containing promoters, proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase (TdT), which were not responsive to wild type p53(47) . However, the nucleotide sequences of these three Inr elements are similar but not identical(48, 49) . Perhaps these sequence differences or other surrounding nucleotide sequences cause these Inr-containing promoters to respond differently to wild type p53.
Additional studies will be necessary to determine the significance of these observations on the human MDR1 promoter. The plethora of proteins that interact with the tumor suppressor p53, the multiple functions of p53, and the question of their significance in tumor and normal cells have been recently discussed(50) . However, studies from other laboratories have shown that the MDR1 gene is activated by DNA-damaging agents, including cytotoxic drugs and UV light(3, 4, 5, 7) . Since these agents are known to activate wild type p53 (51, 52) , perhaps wild type p53 is the mediator through which the MDR1 gene is activated under these stress conditions.