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Experimental Therapeutics |
Cancer Research Campaign Department of Medical Oncology, University of Glasgow, Cancer Research Campaign Beatson Laboratories, Glasgow G61 1BD, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In ovarian cancer, a higher frequency of hMLH1 promoter methylation is observed in postchemotherapy tumors compared with prechemotherapy tumors (12) . We have reported that the majority of cisplatin-resistant derivatives of the ovarian tumor cell line A2780 lack MLH1 expression because of methylation of the hMLH1 gene promoter (12) . Re-expression of MLH1 by chromosome 3 transfer or by treatment with the demethylating agent 5-azacytidine results in sensitization of resistant variants to cisplatin in vitro (12 , 13) . Thus, our observation that demethylation of the hMLH1 gene promoter results in drug sensitization in vitro raised the exciting possibility that MMR-related drug resistance could be overcome clinically. 5-Azacytidine and DAC have been used widely as demethylating agents in cell lines in vitro, and both are used clinically in the treatment of acute myeloid leukemia and myelodysplastic syndromes (14 , 15) . Of the two, DAC is the most potent in terms of DNA demethylation and gene activation and is the least carcinogenic (16) . Although the antitumour activity of DAC has been evaluated in non-small cell lung cancer (17) , the properties of DAC as a demethylating agent have not been addressed specifically in clinical trials. We have, therefore, investigated the effect of DAC treatment on MLH1 expression and the drug sensitivity of human tumor xenografts that lack MLH1 expression because of gene promoter methylation. Our results show clearly that DAC can be used in vivo at nontoxic doses to induce MLH1 expression. In addition, we show that DAC treatment sensitizes drug-resistant ovarian and colon human tumor xenografts to a number of clinically important cytotoxic drugs, raising the possibility that drug resistance mediated by methylation of hMLH1 could be overcome.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Cell Lines.
The human ovarian carcinoma cell line A2780 and a cisplatin-resistant
subline, A2780/cp70, were originally obtained from Dr. R. F. Ozols
(Fox Chase Cancer Center, Pennsylvania, PA). A2780 is MMR proficient
and expresses MLH1, whereas A2780/cp70 is MMR deficient and does not
express MLH1 protein because of hypermethylation of the
hMLH1 gene promoter (12)
. CP70-ch3 is a
derivative of A2780/cp70 that has chromosome 3 introduced by
microcell-mediated chromosome transfer (13)
. CP70-ch3
contains a wild-type copy of the hMLH1 gene, which restores
MMR proficiency and MLH1 expression. Cells were maintained in RPMI 1640
containing glutamine (2 mM) and FCS (10%), and
the chromosome transfer lines were grown in the presence of hygromycin
B (200 units/ml).
The MMR-deficient human colon tumor cell lines SW48 and HCT116 were obtained from American Type Culture Collection (Rockville, MD). SW48 lacks MLH1 expression because of hypermethylation of the hMLH1 gene promoter (1) . The gene promoter is unmethylated in HCT116, but it lacks MLH1 expression because of a mutation in the hMLH1 gene (18) . Cells were maintained in DMEM medium containing glutamine (2 mM) and FCS (10%).
Human Tumor Xenografts.
Monolayer cultures were harvested with trypsin/EDTA (0.25%/1
mM in PBS) and resuspended in PBS. About
107 cells were injected s.c. into the right flank
of athymic female nude mice (MF1 nu/nu mice from Harlan
Olac). After 10–15 days when the mean tumor diameter was at least 0.5
cm, animals were randomized into groups of six for experiments.
Cytotoxic drugs were administered on day 0 either i.p. or i.v. via a
tail vein as specified. Where specified, mice were pretreated with DAC
6 days before the cytotoxic drug, when tumors were just visible. DAC (5
mg/kg) was administered i.p. at 10:00, 13:00, and 16:00 (total dose, 15
mg/kg/mouse). For the combination studies with DAC, all cytotoxic drugs
were used at lower than the maximum tolerated dose to identify possible
interactions in the CP70-ch3 xenografts that could not be explained by
MLH1 expression. Mice were weighed daily, and tumor volumes were
estimated by caliper measurements assuming spherical geometry
(volume = d3
x 
/6). Tumor doubling times were estimated as the time taken
for the tumor volume to reach twice the initial volume. Significant
differences between groups were identified by ANOVA, and the
significance level of individual differences was determined by
Student’s t test.
Immunohistochemistry.
At specified times, mice were killed, and tumors were removed and fixed
in 10% neutral buffered formalin. Tissue was embedded in paraffin, and
5-µm sections were cut. Sections were dewaxed and rehydrated.
Endogenous peroxidase activity was blocked by incubation for 30 min in
hydrogen peroxide (0.5% in methanol). The slides were washed in water
and placed in a pressure cooker containing boiling citrate buffer (0.01
M, pH 6) and brought to full pressure for 90 s. They
were then washed in water and then in Tris-buffered saline containing
Tween 20 (0.05%, TBST). Sections were blocked with TBS containing
normal rabbit serum (1%) for 30 min and then incubated overnight at
4°C with monoclonal anti-MLH1 mouse IgG (Cambridge Bioscience) at a
dilution of 1:200 in TBS containing BSA (0.1%) and sodium azide
(0.01%). Slides were washed three times in TBST (5 min/wash). They
were then processed with streptavidin and biotin reagents, according to
the manufacturer’s instructions (StreptABC; Dako, Cambridge, United
Kingdom). Slides were counterstained with hematoxylin, dehydrated,
cleared, and mounted.
hMLH1 Gene Promoter Methylation.
The methylation status of the hMLH1 gene promoter was
determined by Southern blotting. Tumor tissue was frozen in liquid
nitrogen immediately after removal from the mouse. For DNA extraction,
the frozen tissue was crushed with a pestle and mortar and then
powdered in a Mikro-dismembrator II (Braun). It was then added to 10 ml
of lysis buffer [0.3 M sodium acetate (pH 8.0),
0.5% SDS, 5 mM EDTA, and 50 µg/ml proteinase
K] and shaken at 37°C overnight. Samples were then extracted with
phenol and chloroform:isoamyl alcohol (24:1) and then precipitated in
one tenth volume of 8 M sodium acetate and two
volumes of ethanol.
To allow methylation status of the hMLH1 gene promoter to be determined, 10 µg of genomic DNA was first digested overnight with EcoRV and XbaI (Life Technologies) at 37°C to release an 884-bp fragment of the hMLH1 promoter. The digested DNA was then further digested with either HpaII or MspI (Life Technologies) overnight at 37°C. Blotting of the digested DNA and hybridization with a probe specific for the hMLH1 promoter was carried out as described before (12) .
Global DNA Methylation.
Mice were killed, and blood was removed by cardiac puncture and placed
in tubes containing heparin anticoagulant. The Wizard Genomic DNA
Purification kit (Promega UK Ltd.) was used to isolated DNA from mouse
blood. RNA was removed by treatment of the samples with RNase A (Roche
Diagnostics Ltd.). DNA from 600 µl of blood was rehydrated with 100
µl of distilled water at 4°C overnight. It was then denatured by
heating at 95°C for 5 min and then cooled on ice to prevent
religation. DNA was then digested with P1 nuclease (5 u/sample;
Pharmacia Biotech) in the presence of alkaline phosphatase (4
units/sample) at 37°C for 24 h. Two volumes of ethanol were
added, and samples were centrifuged for 15 min at 13,500 rpm (Eppendorf
centrifuge) to pellet the proteins, and the supernatant was dried in a
Speedivac.
Deoxynucleotides were separated and quantified by HPLC. The system consisted of a Hypersil ODS column (Jones Chromatography) with a µBondapak C18 GuardPak precolumn and a photodiode array detector set at 254 nm (Waters). The mobile phase contained 50 mM sodium dihydrogen phosphate at pH 4 and 2.5% methanol, and the flow rate was 1 ml/min. Retention times were 4.5 min for deoxycytosine, 9 min for methyldeoxycytosine, 14 min for deoxyguanosine, 17 min for deoxythymidine, and 31 min for deoxyadenosine.
Global DNA methylation is quantified as the amount of methyldeoxycytosine expressed as a percentage of the total deoxycytosine present (deoxycytosine + methyldeoxycytosine).
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. Thus, xenografts of the parental MMR-proficient ovarian cell line
A2780 show a growth delay in response to cisplatin treatment (doubling
time of control, 2.17 days, and of 8 mg/kg cisplatin treated for 5.52
days; P < 0.001) and the growth delay is
dose dependent (Fig. 1A)
. In contrast, xenografts of the
cisplatin-selected, MMR-deficient derivative A2780/cp70, which is
methylated at the hMLH1 promoter, are resistant to the
maximum tolerated dose of cisplatin (8 mg/kg; Fig. 1B
).
Human chromosome 3 has been reintroduced into the A2780/cp70 line,
leading to re-expression of MLH1, restoration of MMR activity, and
sensitization in vitro to cisplatin and doxorubicin
(13)
. Re-expression of MLH1 in the resistant A2780/cp70
cells by chromosome 3 transfer (CP70-ch3) is sustained in xenografts
(results not shown), and these show a growth delay in response to
cisplatin (Fig. 1C
; doubling time of control, 2.1 days, and
of cisplatin treated, 4.8 days; P < 0.001).
Sensitization of A2780/cp70 by introduction of hMLH1
demonstrates that MMR is directly involved in drug sensitivity rather
than loss of MMR, causing higher mutation rates at drug-resistant
genes, because in the latter case, reintroduction of MLH1 will not lead
to sensitization. Although A2780 and A2780/cp70 are matched lines, it
might be anticipated that during drug selection and growth, these lines
may have diverged and may differ in a number of mechanisms that affect
cisplatin sensitivity. However, the effect of cisplatin on the growth
of xenografts of CP70-ch3 is comparable with that obtained for the
parental A2780 cell line, which suggests that MLH1 is a major
determinant of the resistance of A2780/cp70 to cisplatin in
vivo, although we cannot exclude effects of other genes present on
chromosome 3.
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, compare A with B). At this dose, there is no
weight loss or any other sign of toxicity. Lower doses or a single
bolus dose failed to induce MLH1 expression. MLH1 expression is
apparent after 3 days and is maximal by 9 days after treatment with DAC
(5 mg/kg x 3; Fig. 2C
). Southern blot
analysis of DNA from the tumors demonstrates that DAC treatment results
in the appearance of MLH1 hybridizing bands at 349 and 569 bp after
HpaII digestion, which indicates reduced hMLH1
gene promoter methylation in the tumors (Fig. 3A and B)
. It has been shown that total
demethylation of gene promoters is not essential for gene re-expression
because transcriptional repression depends on CpG methylation density
rather than complete hypomethylation (19)
. The time course
of promoter demethylation closely reflects re-expression of MLH1
(compare Fig. 3B
with Fig. 2C
), although MLH1
expression is sustained for longer than hypomethylation of the
promoter. This may be explained by the half life of the MLH1 protein.
Between 60 and 90% of cytosines in CpG dinucleotides of normal cells
are methylated (20)
, and we were able to demonstrate a
significant decrease in global DNA methylation (P < 0.05) in peripheral mononuclear cells in blood removed from
mice treated with DAC (Fig. 3C)
. Doses of DAC that do not
induce MLH1 expression have no effect on global DNA methylation
(results not shown). Thus, DAC treatment clearly induces MLH1
expression in vivo, and there is a dose response to DAC both
in terms of MLH1 expression and global DNA demethylation. These
observations suggest that measurement of global DNA methylation in
blood samples could be used in a clinical trial as a surrogate marker
of demethylation, where direct access to tumor DNA is not possible.
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. Although A2780/cp70 xenografts were resistant to the maximum
tolerated dose of cisplatin (8 mg/kg), treatment with cisplatin at a
lower dose (6 mg/kg), either 6 or 12 days after treatment with DAC (5
mg/kg x 3), results in a clear growth delay (Fig. 4A
and Table 1
; P < 0.001). The
drug combination is ineffective if the sequence is reversed and DAC is
administered 6 days after cisplatin (Fig. 4B)
. Sensitization
of A2780/cp70 by DAC was not limited to cisplatin. We also observe
sensitization of A2780/cp70 xenografts to carboplatin (80 mg/kg i.p.),
temozolomide (200 mg/kg i.p.), and epirubicin (10 mg/kg i.v.; Fig. 4
),
and MMR is known to be involved in sensitivity to these agents
(21)
. Thus, growth of the xenografts was unaffected by
treatment with carboplatin or temozolomide alone, but a clear growth
delay was apparent when mice were treated with DAC 6 days before
treatment with the cytotoxic drug (Table 1)
. Interestingly, although
xenografts of A2780/cp70 were sensitive to treatment with epirubicin
alone (Fig. 4
and Table 1
; P < 0.005),
pretreatment with DAC was still able to sensitize the tumors to this
drug. Sensitization of A2780/cp70 xenografts to the cytotoxic drugs is
comparable with that achieved by reintroduction of the hMLH1
gene by chromosome transfer (Table 1)
, although the level of protein
expression as detected by immunohistochemistry is low (
6% of
cells). Immunohistochemistry is relatively insensitive and may not
detect low level re-expression. However, this level of gene
reactivation is comparable with that reported for the FMR1
gene in cells treated with DAC in vitro (22)
.
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and Table 1
;
P < 0.01), but this is a very small effect
compared with the sensitization observed in A2780/cp70. DAC did not
sensitize MMR-proficient CP70-ch3 xenografts to the other three
cytotoxic drugs examined, carboplatin, temozolomide, and epirubicin
(Table 1)
. This suggests that the small sensitization that we observe
for cisplatin is attributable to a specific interaction between these
two agents, which may be related to the synergy reported in cell lines
in vitro (23)
. A2780/cp70 and CP70-ch3
xenografts are equally sensitive to Taxol (15 mg/kg i.v.), and DAC has
no effect on sensitivity to this drug (Fig. 4F
and Table 1
).
Because MMR is not thought to play a direct role in sensitivity to
Taxol (21)
, these results support our hypothesis that DAC
acts through re-expression of MLH1.
We have also confirmed our observations in a colon tumor xenograft
model. The colon tumor cell line SW48 lacks MLH1 expression because of
hMLH1 gene promoter methylation (1)
. These
tumors have a much longer volume doubling time (7.4 days) than the
ovarian tumors, and this allowed us to treat with the cytotoxic drug on
day 0 and again on day 7. Xenografts of this cell line are resistant to
cisplatin, carboplatin, and temozolomide. However, when mice are
pretreated with DAC (5 mg/kg x 3 on day -6), the
xenografts are sensitized to all three cytotoxic drugs and furthermore
show a second response when retreated with the drug 7 days later (Fig. 5)
. Although the ovarian tumors show a growth delay in response to
treatment, the colon tumors actually show an initial reduction in tumor
volume after treatment with the cytotoxic drug. DAC is metabolized to
5aza-deoxycytidinetriphosphate and then incorporated into DNA,
where it forms a covalent adduct with DNA methyltransferase
(24)
. It could be hypothesized that sensitization is
attributable to differences in the incorporation of DAC into DNA in
MMR-proficient and -deficient cells. However, DAC does not sensitize
xenografts of cell line HCT116 (Fig. 5)
, which lacks MLH1 expression
because of a mutation in the hMLH1 gene (18)
.
This demonstrates that sensitization by DAC is not attributable to MMR
deficiency per se but is restricted to tumors in which
hMLH1 is inactivated by promoter hypermethylation and can
thus be reactivated by inhibition of DNA methyltransferase activity.
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Induction of MLH1 expression in tumors could have a considerable impact on the efficacy of chemotherapy. We have shown that chemotherapy for breast cancer results in a significant reduction in MLH1 expression, which strongly associates with poor disease-free survival (10) . Similarly, in ovarian cancer low MLH1 expression is associated with poor survival (27) . In an analysis of the hMLH1 gene promoter of ovarian tumor samples, we reported hypermethylation of the promoter in 9% of untreated tumors but increasing to 50% of tumors that had been exposed to chemotherapy (12) . Expression of MLH1 was lost in the samples that exhibited promoter methylation while still being clearly detectable in the tumors without promoter hypermethylation.
There is one reported clinical trial that has evaluated the combination of DAC and cisplatin, which followed from an observation of synergy in vitro, although the mechanism was not understood (28) . DAC was given on 3 successive days as a 30-min infusion with cisplatin given on day 4. According to our results, this is probably too soon to give the cytotoxic drug and the DAC treatment schedule is suboptimal, because we showed that a single bolus i.p. dose of DAC in mice was insufficient to induce expression of MLH1. The maximum tolerated dose of DAC in the combination was 50 mg/m2, which gave peak plasma levels of 2 µM, which is more than adequate for demethylation based on our studies in vitro (12) . Thus, DAC could have a role in increasing the efficacy of various forms of chemotherapy for patients with a wide range of tumors that lack MLH1 expression because of hMLH1 promoter methylation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Cancer Research Campaign
(United Kingdom). 
2 To whom requests for reprints should be
addressed, at CRC Department of Medical Oncology, University of
Glasgow, CRC Beatson Laboratories, Garscube Estate, Bearsden, Glasgow
G61 1BD, United Kingdom. Phone: 141-330-4212; Fax: 141-330-4127;
E-mail: Jane.Plumb{at}beatson.gla.ac.uk
3 The abbreviations used are: MMR, mismatch
repair; DAC, 2'-deoxy-5-azacytidine.
Received 3/24/00. Accepted 8/31/00.
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), 6 mg/kg (
), and 8 mg/kg (
) on day 0 when the mean
tumor diameter was 
) on day 0. In
B, the drug sequence is reversed. Mice were treated on
days 0 and 6 with PBS (•); on day 0 with DAC (5 mg/kg x 3) and on day 6 with cisplatin (6 mg/kg i.p., 
). Results are the means of six mice;
bars, SE.