Hypomethylation in Cervical Tissue: Is There a Correlation with Folate Status?1

We have shown previously that DNA hypomethylation is significantly associated with grade of cervical intraepitheliab neoplasia (CIN; Y. I. Kim et aL, Cancer, 74: 893-899, 1994). The objective of this study was to further describe this relationship and to investigate the role of folate in the observed association of DNA hypomethylation and CIN. Eighty-three patients with abnormal PAP smear results were referred to the Cervical Dysplasia Clinic at the University of Arizona for colposcopic examination and biopsy. Patients completed a short questionnaire and provided a nonfasting serum sample. DNA hypomethylation was assessed by incubating DNA extracted from biopsy samples with [3H]methyl-S-adenosylmethionine and Sss 1 methylase. Cervical tissue and serum folate concentrations were assessed using a microbiological assay. All folate levels were log transformed prior to statistical analysis. The histological distribution of the samples was: 7 adjacent normal, 30 CIN I, 18 CLN II, 13 CIN III, and 11 carcinoma in situ (CIS). The mean age of participants was 29.8 ± 9.6 years. DNA hypomethybation was significantly different between select histological levels. Both cervical tissue folate and serum folate levels were significantly correlated to methylation level (P = 0.0211 and P = 0.0569, respectively). Smoking, hormonal contraceptive use, parity, and human papillomavirus infection were not associated with DNA hypomethylation or folate status. The current use of vitamins was significantly associated with serum folate level but not with methylation or cervical folate levels. These data extend our earlier findings that DNA hypomethylation is an early event in cervical carcinogenesis. To conclude that the folate level is significantly related to DNA hypomethylation, further investigation of DNA hypomethylation of specific genes is required. Introduction DNA methylation status is associated with carcinogenesis of many epithelial and nonepithelial cancers. DNA methylation is an early postreplicative event that is primarily found in CpG islands. SAM3 is the primary methyl donor, and folate is essential for the synthesis of SAM and the purine nucleotides. Several studies have described the association between global methylation status and carcinomas of the colon (1, 2), liver (3), intestinal type gastric (4), ovary (5), and cervix (6). Colon adenomas and adenocarcinomas have an average reduction of 8 and 10%, respectively, of genomic 5-methylcytosine and no significant differences between the methylation level of benign and malignant tumors (1). A graduated increase in hypomethylation was observed from normal gastric mucosa to superficial gastritis and to chronic atrophic gastritis, indicating that global DNA hypomethylation is an early event in gastric carcinogenesis (4). Global methylation levels in ovarian tumors of low malignancy potential and carcinomas were 21 and 25% bower than in cystadenomas (benign neoplasms), demonstrating that alterations in DNA methylation are early events in ovarian tumorigenesis (5). In the cervix, we showed a progressive stepwise increase of DNA hypomethylation with increasing grades of dysplasia through invasive cancer (6). Both laboratory and clinical investigations have indicated that folate status influences carcinogenesis of several epithelial tissues. In animal studies, methyl donor deficiencies (e.g. , folate, B12, and/or methionine) are associated with hepatic (3, 7, 8) and colonic carcinogenesis (9). Induced methyl deficiency of laboratory animals results in DNA hypomethylation (10), structural chromosome instability (1 1-13), increased expression of the hypomethylated gene (14), DNA strand breaks (15), and oncogene activation (14, 16-18). Genomic hypomethylation and increased DNA methyltransferase activity in liver was noted after treatment of rodents with a methyl-deficient diet (3). However, an isolated folate-deficient diet did not induce global hypomethylation, rather hypomethylation of specific genes (15). Several clinical investigations indicate an association between folate status and risk of coborectal adenoma or cancer (19-24). Studies that examine the combined effect of high alcohol intake and low folate status find a significant risk of coborectal adenoma or cancer (25, 26) as high as a 4-fold increased risk of cancer (27). Two reports have described a significant association between cervical dysplasia and low folate levels (28, 29). A case-control study of mild to moderate dysplasia found a 5-fold greater risk of dysplasia among women infected with HPV type 16 when RBC fobate status was lower than normal (30). However, folate was not related to risk of invasive cervical cancer 3 The abbreviations used are: SAM, S-adenosylmethionine: HPV. human papillomavirus; CIN. cervical intraepithelial; CIS, carcinoma in situ; DY!. dithiothreon April 28, 2017. © 1998 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from 902 Hypomethylation and Folate Status in Cervical Tissue in studies using either serum (31) or dietary (32-34) measures of folate status. This lack of effect of folate with invasive disease suggests that folate status may be more important in the early stages of cervical abnormalities. Intervention trials of folate supplementation have found no significant difference in CIN regression between supplemented and unsupplemented subjects (35, 36). However, most participants in these intervention studies were individuals with CIN I at study entry, lesions that are known to have a high rate of spontaneous regression (>60%). In addition, these studies had small sample sizes, reducing the power to detect differences, and short follow-up periods. Folate status is known to modulate methylation of DNA (3, 10, 14, 15, 16, 17), although no previous studies have examined cervical tissue folate levels in relation to methylation status or CIN. The purpose of this study was to evaluate whether cervical DNA hypomethylation increases with progression of cervical dysplasia and to determine whether serum and cervical tissue folate concentrations are associated with DNA hypomethybation and CIN. This study also sought to determine whether the association between cervical DNA hypomethylation and CIN is independent of smoking, hormonal contraceptive use (either oral contraceptive or Depo-Provera), vitamin use, and HPV infection. Materials and Methods Subjects. Cervical biopsy samples were collected between 1991 and 1994 from women referred to the Cervical Dysplasia Clinic at the University of Arizona Health Sciences Center for colposcopic examination to evaluate abnormal Papanicolaou (PAP) smear results. Eligibility for study enrollment included no current pregnancy, no history of chronic disease, and no previous treatment of cervical dysplasia. Subjects completed a short interviewer-administered questionnaire that gathered information on reproductive history, vitamin use, birth control practices, and smoking behaviors. Subjects submitted a nonfasting blood sample for folate analysis. Colposcopic lesions were identified as those with an aceto-white appearance with and without characteristic vascular patterns after applying a dilute 3-5% acetic acid solution according to the international colposcopic criteria (37). A colposcopically directed biopsy of any suspected lesion was obtained and immediately frozen at -70#{176}Cfor later analysis. If the biopsy size was of sufficient size, the biopsy sample was split into two halves (one for histological categorization and the other for analysis of methylation and folate level). If the biopsy sample was too small to be split, an additional sample of the lesion immediately adjacent to the first biopsy was obtained for methylation and folate analyses. In seven patients, normal tissue distant from the lesion site was biopsied and labeled as normal. Prior to the collection of the biopsy sample, exfoliated cervical cells were collected from the cervix using a Dacron-tipped swab and stored at -70#{176}C for HPV analysis. All histological samples were reviewed by the same pathologist, who was unaware of the methylation and folate analysis results. CIN terminology was used to report results. DNA Methylation Assay. Global DNA methylation was determined by a modification of a method described previously (10, 38). Briefly, 1 p.g DNA was incubated with 2.5 p.Ci of [3H]SAM (New England Nuclear, Boston, MA; 3-10 Ci! mmol), 4 units of Sss 1 methybase (New England Biolabs, Beverly, MA), and 2.5 p.1 lOX methylation buffer [ 1 X methylation buffer is 50 mM NaCl, 10 mti Tris-HC1 (pH 7.9), 10 msi MgCb2, and 1 mM DTTJ in a total reaction volume of 25 p.1 for 3 h at 37#{176}C. The incubation mixtures were heated for 20 mm at 65#{176}C to inactivate the enzyme and were then applied to discs of Whatman DE-81 paper (Whatman, Hillsboro, OR). The discs were washed two times with 25 ml of 5% sodium phosphate dibasic (pH 7.0) and then soaked in this solution for 45 mm at room temperature. The discs were subsequently dried at 95100#{176}C for 30 mm, and the resulting radioactivity of the DNA retained in the discs was measured by scintillation counting using a commercial scintillation cocktail (Econo-Safe; Research Products International, Mount Prospect, IL). The amount of radiobabel bound to a filter from an incubation mixture lacking DNA was used as a background and subtracted from the values obtained with mixtures containing DNA. Samples lacking DNA generally contained < 1% of the label found in DNAcontaining samples. This assay quantitated the in vitro transfer of radiolabeled methyl groups from SAM to sites in DNA that were not methylated in vivo. Therefore, endogenous DNA methylation status and exogenous [3Hjmethyl incorporation are inversely related. Folate Assays. A modification of the Lactobacillus casei microbiobogical assay was used for the analysis of both serum and tissue total folate levels (39). Folates were extracted from cervical biopsy samples while on ice and protected from light. Six hundred p.1 of freshly prepared 1% ascorbate (pH 6.0) was added to the pellet, which was hand homogenized and sonicated at 2.5% for 20 s using an ultrasonic cell disrupter. One hundred p.1 of the cell homogenate were removed for the determination of protein. The remaining 500 p.1 of cell homogenate were dipped in a boiling water bath for 5 mm, cooled, and then incubated with 25 p.1 of Tris buffer (pH 7.2), along with 50 p.1 of folate-free rat plasma conjugase preparation for 60 mm at 37#{176}C. The reaction mixture was again dipped into a boiling water bath for 5 mm, cooled, and centrifuged at 750 X g for 10 mm. The supernatant was collected, filter sterilized, and stored at -70#{176}Cuntil further analysis. Folate content of the samples was then quantitatively measured using a 96-webb plate adaption of the L. casei microbiological assay (39). The reaction volumes containing 20 p.1 of sample and folate standard (15 ng!ml) were adjusted to a total reaction volume of 300 p.1 using 0. 1 M phosphate buffer (pH 8.6) containing 10 mg!ml of ascorbic acid. Six serial dilutions of sample and standard were made, resulting in folate standards in the range of 0.005 to 0.15 ng!0.3 ml assay well. One hundred fifty p.1 of L. casei medium with a concentration of 5 p.1/10 ml were added to all wells and incubated for 18 h at 37#{176}C. The contents of each well were then resuspended by repeated aspiration and flushing with a 12-channel pipetter. Bacterial growth was measured by reading the absorbance at 600 nm. HPV Analysis. Exfoliated cells were collected from the cervix and stored in standard transport medium (ViraType; Digene). HPV status was determined using the Digene Hybrid Capture System using an intermediate and high-risk probe that detects HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56. The sensitivity of this test is 1 pg of viral DNA per 100 p.1 of sample. The Digene Hybrid Capture System is a rapid and relatively inexpensive and reliable method for detecting HPV in exfoliated cells (40-43). Statistical Analysis. Intercooled STATA 4.0 for Windows 3. 10 statistical application was used for all statistical analyses. All categories of continuous variables were defined by variable distribution. Serum and tissue folate concentrations were natural log transformed to attain normal distributions for statistical analyses. One-way ANOVA and simple linear regression were used to test for differences in means and associations at the P on April 28, 2017. © 1998 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from Table I Association between selected subject characteristics and level of cervical DNA methylation, cervical tissue folate concentration, and serum 0.6