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Vol. 95, Issue 15, 8698-8702, July 21, 1998
* Ireland Cancer Center and Contributed by Paul L. Modrich, May 21, 1998
Mutations of DNA mismatch repair genes, including the
hMLH1 gene, have been linked to human colon and other
cancers in which defective DNA repair is evidenced by the associated
instability of DNA microsatellite sequences (MSI). Germ-line
hMLH1 mutations are causally associated with inherited
MSI colon cancer, and somatic mutations are causally associated with
sporadic MSI colon cancer. Previously however, we demonstrated that in
many sporadic MSI colon cancers hMLH1 and all other DNA
mismatch repair genes are wild type. To investigate this class of
tumors further, we examined a group of MSI cancer cell lines, most of
which were documented as established from antecedent MSI-positive
malignant tumors. In five of six such cases we found that hMLH1 protein
was absent, even though hMLH1-coding sequences were wild
type. In each such case, absence of hMLH1 protein was associated with
the methylation of the hMLH1 gene promoter. Furthermore,
in each case, treatment with the demethylating agent 5-azacytidine
induced expression of the absent hMLH1 protein. Moreover, in single
cell clones, hMLH1 expression could be turned on, off,
and on again by 5-azacytidine exposure, washout, and reexposure. This
epigenetic inactivation of hMLH1 additionally accounted
for the silencing of both maternal and paternal tumor
hMLH1 alleles, both of which could be reactivated by
5-azacytidine. In summary, substantial numbers of human MSI cancers
appear to arise by hMLH1 silencing via an epigenetic
mechanism that can inactivate both of the hMLH1 alleles.
Promoter methylation is intimately associated with this epigenetic
silencing mechanism.
Germ-line defects in DNA mismatch repair (MMR) genes account for
the inherited familial cancer syndrome of hereditary nonpolyposis colon
cancers in which affected individuals show accelerated development of
cancers of the proximal colon, endometrium, and stomach (1-5). These
cancers typically demonstrate inactivation of the residual wild-type
MMR allele inherited opposite the germ-line mutant (1-6), absence of
DNA MMR activity in in vitro assays (7, 8), and acquisition
of an in vivo mutator phenotype showing up to 1,000-fold increased gene mutation rates (9, 10). Additionally, these cancers
display an associated instability of genomic microsatellite sequences
(MSI) (1-5). MSI is similarly found in approximately 15-20% of
sporadic colon cancers that arise in individuals without any family
history of colon cancer (1-5). Similarly to hereditary nonpolyposis
colon cancer-associated colon cancers, sporadic MSI colon cancers arise
predominantly in the proximal colon and show a high rate of frameshift
mutations at a mutational hotspot in the transforming growth factor- In this study we focused on further elucidating the mechanism that
results in MSI in sporadic cancers bearing apparently wild-type MMR
genes. We report here that, in five sporadic MSI cancers (four colon
and one endometrial) in which MMR genes are wild type, MSI arises via a
new epigenetic mechanism that inactivates MMR function by silencing
transcription of the wild-type hMLH1 MMR gene. Epigenetic inactivation of hMLH1 is biallelic, is associated with
methylation of the hMLH1 promoter, and is reversed by the
demethylating agent 5-azacytidine. Moreover, hMLH1
inactivation is reestablished after withdrawal of the demethylating
agent, suggesting that an underlying active process maintains the
inactivated methylated state.
Cell Culture.
Cells were cultured in MEM supplemented with
gentamycin, L-glutamine, nonessential amino acids, and
sodium selenite. AN3CA growth medium was supplemented with 10% fetal
bovine serum and sodium pyruvate. For HCT116, Vaco5, and Vaco6, growth
medium was supplemented with 8% calf serum. For SW480, RKO, Vaco432,
Vaco457 and Vaco703, growth medium was supplemented with 2% fetal
bovine serum (HyClone), 10 mg/liter bovine insulin, 2 mg/liter
human transferin, and 1 mg/liter hydrocortisone. Monolayer cultures were grown at 37°C in a 5% CO2 atmosphere. The Vaco
cell lines were established from colon cancers as described (11, 15). HCT116, AN3CA, and SW480 were obtained from the American Type Culture
Collection, and RKO was a generous gift from M. Brattain (University of
Texas Health Science Center, San Antonio, TX).
5-Azacytidine Treatment.
Cells were seeded at
105 cells per T75 flask on day 0. The cultures
were treated for 24 h on day 2 and day 5 with 5-azacytidine at
1-3 µg/ml as individually tolerated. The medium was changed 24 h after addition of the 5-azacytidine (i.e., on day 3 and day 6). On day 8 the cells were harvested for analysis of the methylation status of the hMLH1 promoter region and of production of
hMLH1 protein. Additionally, in some experiments on day 8 cells were either reseeded for continued analysis of hMLH1 expression in bulk
culture or subcloned by limiting dilution on 96-well culture plates.
Evaluation of Methylation Status of the hMLH1 Promoter
Region.
The methylation status of the hMLH1 promoter
was evaluated by a PCR-based assay according to the procedure detailed
by Kane et al. (14). Briefly, genomic DNA samples were
digested with no enzyme, HpaII, or MspI, and then
the 603-bp region upstream of the hMLH1 gene, which contains
four HpaII sites, was amplified by PCR. The amplification
products were then visualized with ethidium bromide after agarose gel
electrophoresis.
Western Analysis.
Approximately 107
cells were lysed in cell lysis buffer [50 mM Tris·HCl (pH
7.4)/1 mM EGTA/1% Nonidet P-40/0.25% sodium deoxycholate/150 mM NaCl] and were subjected to electrophoresis through an SDS polyacrylamide gel. The resultant proteins were then transferred to a
poly(vinylidene difluoride) nylon membrane, which was probed with 2 µg/ml anti-human MLH1 monoclonal antibody (PharMingen). Immune
complexes were visualized by using an enhanced chemiluminescence reagent (Amersham) after incubation with horseradish peroxidase-coupled secondary antibody (Calbiochem). The membrane was then deprobed and
reprobed with 300 ng/ml anti-human actin monoclonal antibody (Amersham).
Amplification and Sequencing of hMLH1 Genomic and
cDNA.
Genomic DNA was obtained by lysing cells in proteinase K
buffer [10 mM Tris (pH 7.4)/10 mM EDTA/0.4% SDS/150 mM
NaCl/100 µg/ml proteinase K] and extracting with
phenol/chloroform/isoamyl alcohol (25:24:1). Genomic DNA was
amplified in a thermocycler for 38 cycles by using the following
protocol: cycle 1, 95°C × 5 min; subsequent cycles, 95°C × 45 s, 55°C × 30 s, and 72°C × 30 s; final cycle, 72°C × 5 min and hold at 4°C. Each 30-µl
reaction contained 100 ng genomic DNA, 0.125 mM 2'-deoxynucleoside
5'-triphosphate, 300 ng forward primer, 300 ng reverse primer, and 2.5 units Perkin-Elmer Amplitaq Gold polymerase.
Genetics
Biallelic inactivation of
hMLH1 by epigenetic gene
silencing, a novel mechanism causing human MSI cancers
,
,,
,, and,,
Department of Medicine, Case
Western Reserve University and University Hospitals of Cleveland,
Cleveland, OH 44106; Howard Hughes Medical Institute, Suite
200, 11001 Cedar Road, Cleveland, OH 44106; § Department of
Pharmacology, Medical College of Ohio, 3000 Arlington Avenue, Toledo,
OH 43699; ¶ Department of Pathology and Laboratory Medicine,
University of Kentucky Medical Center, 800 Rose Street, Lexington, KY
40536; Biology Department, Indiana University, Third and
Hawthorne, Bloomington, IN 47405; and ** Howard Hughes Medical
Institute, Duke University Medical Center, Nanaline Duke Building,
Durham, NC 27710
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
type II receptor tumor suppressor gene (1, 11). Familial and sporadic
MSI colon cancers thus appear to share a common carcinogenic pathway.
In this regard, previous studies from our group established that MMR
gene inactivation via somatic mutation was the cause of some cases of
sporadic MSI colon cancers (12). However, unexpectedly, in many
sporadic MSI colon cancers, MMR genes were found to remain wild type
(12). MMR-coding sequences have similarly been reported to be wild type in most sporadic MSI endometrial cancers (13). Thus, a mechanism that
induces MSI independently of MMR gene mutation appears to account for a
significant number of human MSI cancers. Intriguingly, methylation of
the hMLH1 promoter region has recently been described in
some MSI tumors (14).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Loss of MLH1 Protein Expression and Functional Activity in Sporadic MSI Cancers. We initially investigated five cell lines established from sporadic MSI colon cancer tumors that we had previously reported bore only wild-type MMR genes (Vaco432, Vaco5, Vaco6, RKO, and Vaco481) (12). Fig. 1 depicts our Western blot analysis that demonstrates absence of hMLH1 protein expression in four of these five cell lines (Vaco432, Vaco5, Vaco6, and RKO). Furthermore, the western blot shows that no hMLH1 protein was expressed in a sporadic MSI endometrial cancer, AN3CA, which previously was shown to possess wild-type MMR genes but to lack hMLH1 transcript (17). Finally, hMLH1 protein was also absent in the Vaco703 colon cancer, which we previously reported carries an inactivating somatic mutation of the hMLH1 gene (12), and in Vaco457, a hereditary nonpolyposis colon cancer-derived colon cancer. Expression of hMLH1 protein was, however, readily detected in the non-MSI colon cancer SW480. Northern analysis confirmed that, in sporadic MSI cancers with wild-type hMLH1-coding sequences, those lacking hMLH1 protein expression also lacked hMLH1 transcript expression. Western blots demonstrated continued expression of hMSH2 protein in Vaco5, Vaco6, Vaco432, RKO, and AN3CA. This suggested that in all of these cases the expression of the wild-type hMLH1 alleles had been specifically silenced.
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deficiency similar to that observed
previously with cisplatin- and adriamycin-resistant ovarian tumor cells
(18). Functional repair activity could be restored by addition of
hMutL, the heterodimeric protein complex of hMLH1 and hPMS2, to each
of these extracts (19). Thus, the absence of hMLH1 protein expression
in these cancers is the direct cause of their MSI phenotype.
Loss of hMLH1 Expression Is Associated with Methylation of the hMLH1 Gene Promoter. To establish the mechanism of loss of hMLH1 gene expression in AN3CA, RKO, Vaco5, Vaco6, and Vaco432, we first sequenced the hMLH1 promoter for evidence of mutations. No mutations were found in any of these cell lines within 685 bp 5' of the hMLH1 initiator ATG. We next examined the hMLH1 promoter region in these tumors for evidence of methylation at four HpaII sites located between 341 and 567 bp 5' of the hMLH1 ATG (14). Methylation was detected by the ability to recover and amplify an intact hMLH1 promoter fragment after digestion of genomic DNA with the methylation-sensitive restriction enzyme HpaII. As shown in Fig. 2B, the five sporadic MSI cancers that displayed loss of hMLH1 expression also have methylation of the hMLH1 promoter (AN3CA, RKO, Vaco5, Vaco6, and Vaco432).
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hMLH1 Silencing Is Reversible After Treatment with the Demethylating Agent 5-Azacytidine. To determine whether hMLH1 promoter methylation could be further linked to the loss of hMLH1 expression, cancer cell lines were treated with the demethylating agent 5-azacytidine. As shown in Fig. 3, 5-azacytidine successfully restored hMLH1 protein expression in each of the five sporadic MSI cancers that lacked hMLH1 expression and that harbored a methylated hMLH1 promoter. In contrast, 5-azacytidine was ineffective in reactivating hMLH1 expression in two MSI cancers in which the hMLH1 promoter was unmethylated and in which hMLH1 inactivation was caused by somatic mutation (Vaco703) or germ-line defect (Vaco457) (Fig. 3). Thus, the presence of hMLH1 promoter methylation was tightly correlated with the ability to reactivate hMLH1 by 5-azacytidine treatment. Moreover, the 5-azacytidine induction of hMLH1 expression in five sporadic MSI cancers repeatedly demonstrated that an epigenetic mechanism must be responsible for silencing of hMLH1 expression.
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hMLH1 Expression Is Again Silenced After Withdrawal of 5-Azacytidine. To determine whether hMLH1 gene silencing is actively maintained, we asked whether hMLH1 gene reexpression would persist after washout of the 5-azacytidine-inducing agent. As shown in Fig. 4, 5-azacytidine treatments of AN3CA cells on days 2 and 5 induced abundant hMLH1 protein expression by day 8. However by day 21, 2 weeks after drug washout, hMLH1 protein expression was again undetectable. This result suggested that, after withdrawal of 5-azacytidine, an active process again resilenced expression of the hMLH1 gene.
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Epigenetic Inactivation of hMLH1 Is Biallelic. No wild-type hMLH1 protein was detected by Western analysis in the Vaco5, Vaco6, Vaco432, RKO, or AN3CA cell lines, demonstrating in these tumors that both wild-type hMLH1 alleles had been inactivated. In many cancers biallelic inactivation of suppressor genes results from mutation of one allele and then by gene deletion of the remaining allele (1). This two-step process is reflected by loss of heterozygosity of polymorphic markers linked to the suppressor gene locus (1). We looked for such loss of heterozygosity at the hMLH1 locus in three of the cases, Vaco5, Vaco6, and Vaco432, for which matched normal tissue was available. A coding region polymorphism at codon 219 (ATC to GTC) was found to be informative in two cases, Vaco5 and Vaco432 (12). Significantly, retention of both maternal and paternal alleles was detected in each of the Vaco5 and Vaco432 tumors and cell lines. Moreover, after 5-azacytidine treatment, induction of transcripts bearing both maternal and paternal polymorphisms was detected in cDNA amplified from either Vaco5 or Vaco432 (Fig. 6). Thus, in these tumors biallelic inactivation of hMLH1 expression is caused by epigenetic inactivation of both parental copies of the gene. This finding suggests that in these tumors epigenetic silencing of hMLH1 is not a rare chance event but the end result of an active and efficient mechanism that can work in trans. Furthermore, material was available to confirm that the antecedent tumor from which the Vaco432 cell line was established demonstrated both the MSI phenotype as well as methylation of the hMLH1 gene promoter. Thus, epigenetic inactivation of hMLH1 almost certainly was the initiating event in the genesis of the MMR deficiency of this individual's colonic malignancy.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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These findings establish that the epigenetic silencing of the hMLH1 gene promoter is an important event in many sporadic human colon cancers. This epigenetic silencing mechanism is closely associated with methylation of the hMLH1 gene promoter. Moreover, this mechanism was operative in a majority, four of seven, of the sporadic MSI colon cancers in our collection. Additionally, in three of these four cases, we confirmed that the MSI phenotype was present in the antecedent tumors from which the cell lines were established. Thus, the mechanism accounting for MSI in the cell lines almost certainly was present in the antecedent primary tumors. Although larger confirmatory trials will be necessary, our findings suggest that epigenetic inactivation of hMLH1 underlies the pathogenesis of many human MSI colon cancers.
Colon cancer has served as a useful paradigm for understanding the molecular mechanisms of human carcinogenesis. In many colon cancers biallelic inactivation of two tumor suppressor genes, APC and p53, is demonstrable (1), and for these genes biallelic inactivation is usually caused by mutational inactivation of one allele, accompanied by deletion or loss of the cognate wild-type allele via mechanisms that cause loss of heterozygosity (1). In contrast, we found biallelic inactivation of the hMLH1 MMR gene commonly proceeds by epigenetic inactivation of both parental alleles and is not accompanied by loss of heterozygosity. These findings suggest that in these cancers epigenetic gene inactivation is a highly efficient method for silencing hMLH1 gene expression.
Previous studies of MSI colon cancers have demonstrated that these
cancers as a class harbor increased methylation within promoter regions
of multiple genes, including increased methylation of a candidate tumor
suppressor gene, p16, as well as of a candidate tumor-promoting growth factor, the insulin-like growth factor II (20).
It is thus possible that a large subset of MSI tumors harbors a general
defect in gene regulation that induces aberrant promoter methylation at
many loci. In this instance methylation and silencing of the
hMLH1 promoter would provide a direct route that connects a
general defect in promoter regulation with the specific MSI-associated
pathway of carcinogenesis. This would be similar to the finding that
mutational inactivation of a tumor suppressor gene, the type II
transforming growth factor- receptor, links the global mutator
phenotype accompanying MSI with a defined pathway of malignant
transformation (9-11). Future studies should reveal the specific
relationship between epigenetic inactivation of the hMLH1
promoter and increased promoter methylation seen at other loci in some
MSI cancers.
In addition to showing increased methylation of endogenous promoters, MSI cancers also have an increased propensity to methylate and silence exogenous retroviral gene sequences (21). This phenotype has most clearly been demonstrated for the HCT116 MSI colon cancer in which the hMLH1 gene is inactivated by mutation and in which the hMLH1 gene promoter is unmethylated. Future studies should elucidate whether the propensity to methylate exogenous gene sequences is or is not a reflection of the same disordered mechanism that underlies methylation and gene silencing of the hMLH1 locus. Additionally, increased methylation of the estrogen receptor promoter in normal colonic tissue has been reported to be a correlate of aging (22). Again, elucidating the relationship between this form of aberrant methylation and the epigenetic inactivation of the hMLH1 gene in cancer will be an important question for future investigation.
The causal role of MMR gene inactivation in initiating the pathway of MSI colon carcinogenesis suggests that in the tumors we studied the methylation and epigenetic silencing of the hMLH1 gene were neither consequences of disordered gene regulation in cancer nor results of the establishment of cancer cell lines. Instead, the epigenetic silencing of hMLH1 was a key pathophysiologic step in the genesis of these cancers. These findings support the idea that a similarly direct role in carcinogenesis is played by other tumor suppressor genes, such as p16 and VHL, in cancers in which those genes have been noted to be methylated and to additionally be inducible by 5-azacytidine (23-26).
Methylation of the hMLH1 gene promoter in association with absent hMLH1 protein was previously noted in several MSI colon cancers and in the AN3CA endometrial cancer (14). This current study establishes that hMLH1 promoter region methylation is strongly associated with epigenetic gene silencing, that the silencing mechanism is sufficient to accomplish biallelic inactivation of the hMLH1 gene, that silencing of the hMLH1 gene is reversible, that silencing of the hMLH1 gene appears to be actively maintained, and that hMLH1 silencing is a common route for the genesis of sporadic MSI cancers. Additionally, while this study was in progress, Herman et al. (27) also noted a significant incidence of hMLH1 promoter methylation among sporadic MSI colon cancers, as well as the ability to induce hMLH1 expression with demethylating agents. The large number of tumors analyzed in these three studies provides strong and consistent support for the contention that epigenetic silencing of hMLH1 is a common route leading to the genesis of sporadic MSI colon cancers. Also in support of this contention is the recent report that hMLH1 protein expression is absent by immunohistochemical assay in 90% of unselected human MSI colon cancers (28).
A singular finding of this current study is the observation that epigenetic inactivation of hMLH1 is biallelic and that reactivation of the hMLH1 allele is rapidly followed by resilencing of the gene. These observations suggest that hMLH1 silencing is the result of an underlying active mechanism that efficiently turns off hMLH1 gene expression. Moreover, it is highly tempting to suggest that hMLH1 promoter methylation is the direct mechanism that inactivates hMLH1 gene expression. However, both hypotheses require further investigation, and final judgment will have to await the ultimate elucidation of the mechanisms mediating hMLH1 promoter methylation and hMLH1 gene silencing.
Current data do not allow us to judge whether reexpression of wild-type hMLH1 will have therapeutic utility in certain MSI colon cancers, e.g., by reversing the resistance of such tumors to treatment with certain alkylating agents (29). Any trial of such therapy will need to reflect our finding that repetitive dosing of 5-azacytidine is required to maintain hMLH1 expression. It is also tempting to consider that demethylating agents might play a role in cancer prevention in individuals who are at risk for cancer or in individuals in whom hMLH1 promoter methylation might be detected as an early neoplastic change. Our data support the suggestion that hMLH1 gene silencing accounts for a significant number of human colon cancers. Additionally, the epigenetic inactivation of hMLH1 demonstrated in the AN3CA MSI endometrial cancer may prove to be representative of MSI endometrial cancers, which as a class bear mostly wild-type MMR alleles (13). Finally, many sporadic human gastric cancers also arise via the MSI pathway (2). Elucidating the ultimate mechanism underlying the epigenetic inactivation of hMLH1 expression may thus prove relevant to the understanding of many different types of human cancer.
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ACKNOWLEDGEMENTS |
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We thank James K. V. Willson for establishing the Vaco cell lines and Bert Vogelstein for his help in determining the MSI status and the sequences of the MMR genes in the Vaco cell lines and also for many helpful discussions. We also thank James Herman and Bo Liu for helpful discussions and Jen Sedwick for help with manuscript preparation. This work was supported by Grant CA 67409 (to S.D.M.), Grant CA 72160 (to S.D.M.), Grant CA 70788 (to M.L.V.), and Grant CA 4370301 (to the Case Western Reserve University Cancer Center) from the National Institutes of Health, Grant FRA-451 (to S.D.M.) from American Cancer Society, Grant 96B084 (to W.D.S.) from American Institute for Cancer Research, and from funds from the State of Ohio Board of Regents. S.D.M. and P.M. are Investigators of the Howard Hughes Medical Institute.
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FOOTNOTES |
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To whom reprint requests should be addressed at:
U.C.R.C. #2, Room 200, 11001 Cedar Road, Cleveland, OH 44106. e-mail:
sxm10{at}po.cwru.edu.
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ABBREVIATIONS |
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MMR, mismatch repair; MSI, microsatellite instability.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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) of MSI and the status of the coding region of the
hMLH1 gene (wt, wild type; mut, mutant) are denoted
below each lane. The Western analysis was performed as described in
Materials and Methods.
