DNA Methyltransferase 1 Protein Levels Research Articles

Significance of aberrant expression of DNA methyltransferase 1 in cervical cancerization tissue and cervical cancer cell lines

Objective To explore the effects of aberrant expression of DNA methyltransferase 1 (DNMT1) in cervical cancerization tissue and cervical cancer cells.Methods Cervical tissues were collected from 80 cases with a diagnosis of invasive cervix squamous cell carcinoma (SCC),53 cases with high-grade cervical intraepithelial neoplasia (CIN Ⅱ/Ⅲ),52 cases with low-grade cervical intraepithelial neoplasia (CIN Ⅰ)and 53 cases with normal cervix (NC).Meanwhile,Caski (HPV16-positive) and C33A (HPV-negative) cells selected from cervical cancer cell lines were cultured routinely in vitro.The expression of DNMT1 protein and mRNA were examined by Western blot analysis and real-time PCR (qRT-PCR) in the tissues and cells,respectively.Results The levels of DNMT1 protein were 1.33,1.84 and 2.28,and the Ct-ratios (DNMT1/β-actin) of DNMT1 mRNA were 1.27,1.27 and 1.26 in CIN Ⅰ,CIN Ⅱ/Ⅲand SCC group,respectively.Comparing with NC group,the expression of DNMT1 protein or mRNA was elevated in deficient cervical groups,with statistical significance (F =110.57,P ＜ 0.001,F =2.68,P =0.048).The expression levels of DNMT1 protein were increased steadily according to severity of the cervix lesions (x2tend =50.80,P ＜ 0.001),however,the expression of DNMT1 mRNA was not observed the same tendency (x2tend =3.63,P ＞ 0.05).The results from experiment in vitro showed that the levels of DNMT1 protein or mRNA were both higher in Caski cell than in C33A cell,especially for DNMT1 mRNA with significantly difference (t =7.134,P =0.002).Conclusion Aberrant expression of DNMT1 protein or mRNA could link with the risk of cervical cancerization by both transcriptional and posttranscriptional mechanisms.There would be a synergistic effect between overexpression of DNMT1 and HPV16 infection in the progression of cervix carcinogenesis. Key words: DNA methyltransferase 1; Aberrant expression; Cervical cancerization; Cervical cancer cells

Increased recycling of polyamines is associated with global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts

Global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts (RASFs) contributes to their intrinsic activation. The aim of this study was to investigate whether increased polyamine metabolism is associated with a decreased level of S-adenosyl methionine (SAM), causing global DNA hypomethylation. Synovial fibroblasts were isolated from synovial tissue obtained from 12 patients with RA and from 6 patients with osteoarthritis (OA). The cells were stained for S-adenosyl methionine decarboxylase (AMD), spermidine/spermine N1-acetyltransferase (SSAT1), polyamine-modulated factor 1-binding protein 1 (PMFBP1), solute carrier family 3 member 2 (SLC3A2), DNA methyltransferase 1 (DNMT-1), α9 integrin, and β1 integrin and analyzed by flow cytometry. Nuclear 5-methylcytosine (5-MeC) was measured by flow cytometry, the expression of diacetylspermine (DASp) in cell culture supernatants and cell extracts was determined by enzyme-linked immunosorbent assay, and SAM expression in cell extracts was measured by fluorometry. The expression of SSAT1, AMD, and PMFBP1 was significantly increased in RASFs compared with OASFs. The expression of DASp in cell culture supernatants and the expression of SLC3A2 were significantly elevated in RASFs. The levels of SAM in cell culture extracts, as well as the levels of DNMT-1 protein and 5-MeC, were significantly reduced in RASFs. Parameters of polyamine metabolism were negatively correlated with the expression of SAM, DNMT-1, and 5-MeC. These data clearly show that intrinsic elevations of PMFBP1 and SSAT1 enhance the catabolism and recycling of polyamines in RASFs and suggest that high consumption of SAM via this pathway is an important factor contributing to global DNA hypomethylation in these cells.

The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation in Laryngeal carcinoma

Over-expression of DNA methyltransferase 1 (DNMT1) correlates with hypermethylation of tumor suppressor genes (TSGs) in tobacco-induced cancers. The tobacco component nitrosamine 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK) increases protein levels of the DNMT1 in human lung cancer. However, the role of DNMT1 expression induced by NNK is not clear during laryngeal carcinogenesis. We investigated DNMT1 expression levels in 101 cases of human laryngeal carcinoma specimens and 54 cases clear surgical margin specimens by reverse transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry (IHC) detection. Then, we analyzed the relationship between the DNMT1 expression and the smoking status of the patients with laryngeal carcinoma. Moreover, we investigated the effects of tobacco carcinogen NNK on DNMT1 expression in Hep-2 cells. We found that DNMT1 mRNA and protein expressions were up-regulated in laryngeal cancer tissues (p<0.05 and p<0.01, respectively). Among the 101 cases, DNMT1 protein from patients with heavier smoking habit had a significant trend of an increase with IHC scores (p<0.01). The overall survival rates of patients DNMT1-positive were significantly lower than those of patients DNMT1-negative (p<0.05). We observed that NNK increased DNMT1 protein levels, not mRNA levels, in cultured Hep-2 cells significantly in both dose- and time-dependent manner (p<0.05). These results supported the idea that NNK-induced DNMT1 expression may result from protein stabilization. Increased DNMT1 protein expression may play a critical role in the malignant progression of larynx.

Effects of DNA methyltransferase 1 inhibition on esophageal squamous cell carcinoma

To explore the role of DNA methyltransferase 1 (DNMT1) in esophageal squamous cell carcinoma (ESCC) and the potential of DNMT1-targeted small interfering RNA as ESCC therapy, we examined expression changes of DNMT1 in ESCC and investigated the effect of DNMT1 knockdown by RNA interference in a human ESCC cell line, KYSE30. DNMT1 messenger RNA was over-expressed in seven out of 12 ESCC samples, and the percentage of cells expressing DNMT1 was significantly higher in ESCC tissues compared with paired non-cancerous tissues. DNMT1 protein levels correlated with lymph node metastasis, but exhibited no correlation with sex, age, tumor site, or tumor differentiation. Knockdown of DNMT1 in KYSE30 cells using RNA interference resulted in a reduction of promoter methylation and re-expression of methyl-guanine methyl-transferase and retinoic acid receptors beta, inhibition of cell proliferation/viability and induction of cell apoptosis. These results indicate that DNMT1 over-expression is involved in ESCC and correlated with lymph node metastasis. Knockdown of DNMT1 led to promoter demethylation and re-expression of several tumor suppressor genes thereby inhibiting cell proliferation/viability and inducing cell apoptosis.

Abstract 2014: Modulation of DNA methyltransferases in leukemia cells by curcumin and its associated anti-leukemia activities

Abstract Purpose: Curcumin, a natural product of curcuma longa and a safe and widely consumed spice, has been shown to exhibit strong in-vitro and in-vivo anti-tumor activity through the inhibition of NF-κB activity, increase of reactive oxygen species (ROS) levels and decrease of the global DNA methylation levels in leukemia cells. We investigated here the molecular mechanisms of down-regulation of DNA Methyltransferases in leukemia cells by curcumin and its associated anti-leukemia activities. Method: Leukemia cell lines MV4-11, Kasumi-1 and K562 cells and primary leukemia cells from patient with AML were treated with 1-30 μM of curcumin and MV4-11 xenografted nu/nu athymic mice was given i.p. curcumin at 100 mg/kg, and the transcription and protein levels of DNMT1, DNMT3a, DNMT3b, Sp1, p15INK4B and p21 were assessed by either RT-PCR or western blot. The inhibition of NF-κB was determined using Electrophoretic Mobility Shift Assays and the disruption of Sp1-NF-κB/DNMTs promoter complex was investigated by anti-body gel supershifts. The hypomethylating activity of curcumin on the promoter of p15INK4B in MV4-11 cells in-vitro was assessed by a LC-MS/MS method for regional DNA methylation. The anti-proliferative activity of curcumin and cell cycle distribution and apoptosis were evaluated using MTS assay and flow cytometry. Results: In addition to the pre-defined direct inhibition of the enzymatic activity of DNA methyltransferase 1 (DNMT1), curcumin disrupts Sp1/NF-κB complex on DNMT1, DNMT3a and DNMT3b promoter thereby decreasing the transactivation activity of these complexes and down-regulating their mRNA and protein expression levels in AML cells in vitro and in vivo. Consistent with these findings, we showed that promoter hypermethylation of silenced TSGs i.e., p15INK4B decreased while their expression was up-regulated. This results in the increase of protein function, as we showed that in association with p15INK4B re-expression, curcumin induced cell cycle arrest in the S-phase and increase in SubG1 cell fraction, thereby resulting in significant anti-leukemia activity. Conclusion: Different from nucleoside analogs (e.g., azanucleosides), curcumin is an effective S-phase independent DNA methylation inhibitor. Further evaluation of curcumin as a single agent and in combination with other epigenetic targeting compounds in human disease is warranted. Supported by NIH-NCI-R21 CA135478 [ZL], NIH-NCI-RO1 CA102031 [GM], and the BioMedical Mass Spectrometry Laboratory [KC &amp; ZL]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2014. doi:10.1158/1538-7445.AM2011-2014

Abstract 2618: DNMT1 as a marker of differential sensitivities to epigenetic therapy of a Kras mutant and Kras wild type human non small cell lung cancer cell line

Abstract INTRODUCTION: Epigenetic therapy with a demethylating agent and an histone deacetylase inhibitor (HDACi) is now being pursued in hematological as well as solid tumor malignancies. Clinical responses have been varied with complete responses in some patients and no efficacy of the drugs in others. We investigated the effect of this therapy on two targets, DNA methyltransferase1 (DNMT1) and H3K4 modified histones, respectively. METHODS: Two human lung adenocarcinoma cell lines, A549 (Kras mutant) and H838 (Kras wild type) were pre-exposed for 72 hours with daily treatment of 500nM azacitidine alone or 72 hours of daily 500nM azacitidine followed subsequently with 10 nM, 100nM, 1000nM entinostat at 96 hours. DNMT1 and H3K4 protein levels were assayed by Western blot analysis at various time points after treatment. Cells were also re-plated after treatment to assess differential growth characteristics. RESULTS: In vitro growth of H838 cells is sensitive to azacitidine alone and in combination with entinostat whereas growth of the A549 cell line is only sensitive to azacitidine and entinostat in combination. Treatment with azacitidine, severely depleted DNMT1 protein expression in H838 cells both when given alone and in combination with most dose levels of entinostat. This decrease was transient and within 4 days, the protein was fully replenished. Azacitidine alone did not cause an appreciable decrease in DNMT1 protein expression of A549 cells but, when combined with increasing levels of entinostat, DNMT1 levels were significantly depleted and did not replenish for 7 days. Re-plating after treatment showed growth inhibition versus mock of all regimens containing azacitidine in the H838 cell line only whereas growth inhibition in the A549 cells was observed only in the combinatory therapy arms. Furthermore, in the A549 cell line, H3K4 protein levels (a marker of active gene expression) peaked at 7 days after treatment versus a peak in the first 24 hours in the H838 cell line. CONCLUSIONS: Differing sensitivities to epigenetic therapy in lung adenocarcinoma cell lines may reflect underlying differences in molecular phenotypes. Large decreases in DNMT1 expression are associated with sensitivity to azacitidine. In a cell line insensitive to azacidine alone, the addition of entinostat may accentuate DNMT1 protein depletion and delay its reexpression. This effect on DNMT1 may render this latter cancer cell line more sensitive to epigenetic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2011-2618

Phase 1 Trial of Decitabine and Bortezomib in High Risk Acute Myeloid Leukemia (AML)

AbstractAbstract 3293 Background:The hypomethylating agent decitabine has significant activity in AML. We previously demonstrated a novel epigenetic mechanism of action for the proteasome inhibitor bortezomib in AML cells (Liu, Blood 2008). Bortezomib induced hypomethylation of leukemic cells in vitro and in vivo via disruption of the Sp1/NF-kB transcriptional activation complex on the DNA methyltransferase 1 (DNMT1) gene promoter, which resulted in down-regulation of DNMT1 mRNA and protein levels, DNA hypomethylation, and re-expression of otherwise hypermethylated target genes. Based on this, we designed a phase 1 dose escalation trial of decitabine in combination with bortezomib. Methods:Adults with high risk AML who had preserved organ function and ECOG ≤2 were eligible. High risk AML included relapsed/refractory AML or age>60 years with previously untreated disease (if ineligible for or refused standard induction therapy). Patients received decitabine at 20mg/m2 IV daily for days (d) 1–10 of 28 d cycles with dose modification in subsequent cycles based on response and myelosuppression. Bortezomib (given immediately after decitabine) was gradually dose escalated in standard 3+3 fashion from 0.7mg/m2 on d 5 and 8 to the target dose of 1.3mg/m2 on d 5, 8, 12, and 15. Cycles were repeated every 28 d, regardless of count recovery. The plan was to administer 3 cycles if possible before discontinuation due to lack of response. For responding pts, therapy was continued indefinitely. Six additional pts were treated at the recommended phase 2 dose. Dose limiting toxicities (DLT) were assigned for cycle 1 of therapy. Given the high likelihood of infection in this population regardless of therapy, infection was not considered a DLT unless toxicity exceeded that expected with conventional therapy. Results:19 pts were enrolled with a median age of 69 years (range, 32–83). 12 pts were age>70. 10 pts were previously untreated. Median presenting WBC count was 3,900/uL (range, 1,300-69,200/uL); median bone marrow blast was 34%. Patients received a median of 2 cycles of therapy (range, 1–14 cycles). Two pts received combination therapy beyond 3 cycles of treatment; one received combination for 8 cycles, then decitabine alone for 6 additional cycles, and the other received combination for 4 cycles, then decitabine alone for 6 additional cycles. Dose escalation was halted once the target bortezomib dose was reached; the MTD was decitabine at 20mg/m2 d 1–10 plus bortezomib 1.3mg/m2 d 5, 8, 12, and 15. One DLT of death due to sepsis occurred in dose level 3. Febrile neutropenia and infectious complications were frequent. Death within 8 weeks occurred in 4 pts (21%). Neuropathy attributable to bortezomib, though not meeting DLT criteria, was problematic with repetitive cycles of administration. Specifically, two pts had Grade 3 neuropathy requiring discontinuation of treatment. Responses occurred in 4/10 previously untreated pts: 3 had complete remission (CR) and one had CR with incomplete blood count recovery (CRi). One 84 year old pt with complex karyotype discontinued therapy after one cycle due to fatigue with persistent disease (non-responder). Notably, the pt subsequently had complete count recovery and lived for 14 months with no additional treatment, refusing further marrow evaluation of response. Response durations for the 3 CR were as follows: 12 months, 9 months, and the last patient died in CR after 10 months response duration due to unrelated and preexisting cardiac disease. The CRi lasted 3 months before relapse. Responses occurred in 2/9 relapsed/refractory pts. Both had CRi. Response duration for one patient was only 2 months. The other had allogeneic transplant in first remission 22 months ago before recently expiring in remission with transplant-related complications. In 3/6 responders with abnormal karyotype, two achieved cytogenetic CR. Conclusions:The combination of decitabine and bortezomib was reasonably well tolerated and active in high risk AML. Neuropathy beyond cycle 1 limited prolonged exposure to both agents. Given recent data suggesting equivalent efficacy with weekly dosing of bortezomib in multidrug treatments for myeloma, modification of the bortezomib schedule may facilitate more prolonged exposure to the combination. A phase 2 study of this combination is being planned, with bortezomib modification as noted. Correlative studies are ongoing.NCI U01 CA 76576, NIH/NCI K23CA120708. Disclosures:Blum:Celgene: Research Funding. Off Label Use: decitabine and bortezomib in AML.

Effect on proliferation and apoptosis of T24 cell lines via silencing DNMT1 with RNA interference

Expression of DNA methyltransferase 1 (DNMT1), which plays an important role on aberrantly methylated CpG in the promoter regions of tumor suppressor genes (TSGs), is higher in bladder cancer cells than in normal bladder cells. Therefore, its overexpression is closely related to tumor formation. In this study, the eukaryotic vector pshRNA-DNMT1 was constructed and transfected into T24 cells. Levels of DNMT1 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Relative to the blank control at the 24th, 48th and 72nd hour after transfection of pshRNA-DNMT1, the inhibitory rates of DNMT1 mRNA levels in T24 cells were 28.44%, 52.48%, 70.91%, respectively. Those of DNMT1 proteins were 24.27%, 57.79%, and 77.74%, respectively. Proliferation and apoptosis were assayed by MTT and flow cytometry with Annexin-V-FITC/PI staining. The growth inhibition rates of pshRNA-DNMT1 at the 24th, 48th and 72nd hour after transfection of pshRNA-DNMT1 were (4.34 ± 0.76)%, (9.87 ± 1.54)% and (13.78 ± 1.93)%, respectively. There were statistically significant differences between pshRNA-DNMT1 and the control blank at each time points (P < 0.01); 24, 48 and 72 hours after T24 cells were transfected by pshRNA-DNMT1, the apoptosis rates of pshRNA-DNMT1 were (3.87 ± 0.81)%, (8.69 ± 1.23)% and (11.46 ± 1.24)%, respectively (P < 0.01 vs blank control). Based on this case, our conclusion is that the recombinant plasmid pshRNA-DNMT1 can silence the expression of gene DNMT1 mRNA and protein effectively, and to some extent, it also can inhibit the proliferation of bladder cancer cell and promote the cellular apoptosis.

Adiponectin stimulates Wnt inhibitory factor-1 expression through epigenetic regulations involving the transcription factor specificity protein 1

Adiponectin (ADN) is an adipokine possessing growth inhibitory activities against various types of cancer cells. Our previous results demonstrated that ADN could impede Wnt/beta-catenin-signaling pathways in MDA-MB-231 human breast carcinoma cells [Wang,Y. et al. (2006) Adiponectin modulates the glycogen synthase kinase-3 beta/beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice. Cancer Res., 66, 11462-11470]. Here, we extended our studies to elucidate the effects of ADN on regulating the expressions of Wnt inhibitory factor-1 (WIF1), a Wnt antagonist frequently silenced in human breast tumors. Our results showed that ADN time dependently stimulated WIF1 gene and protein expressions in MDA-MB-231 cells. Overexpression of WIF1 exerted similar inhibitory effects to those of ADN on cell proliferations, nuclear beta-catenin activities, cyclin D1 expressions and serum-induced phosphorylations of Akt and glycogen synthase kinase-3 beta. Blockage of WIF1 activities significantly attenuated the suppressive effects of ADN on MDA-MB-231 cell growth. Furthermore, our in vivo studies showed that both supplementation of recombinant ADN and adenovirus-mediated overexpression of this adipokine substantially enhanced WIF1 expressions in MDA-MB-231 tumors implanted in nude mice. More interestingly, we found that ADN could alleviate methylation of CpG islands located within the proximal promoter region of WIF1, possibly involving the specificity protein 1 (Sp1) transcription factor and its downstream target DNA methyltransferase 1 (DNMT1). Upon ADN treatment, the protein levels of both Sp1 and DNMT1 were significantly decreased. Using silencing RNA approaches, we confirmed that downregulation of Sp1 resulted in an increased expression of WIF1 and decreased methylation of WIF1 promoter. Taken together, these data suggest that ADN might elicit its antitumor activities at least partially through promoting WIF1 expressions.

Retinoblastoma Pathway Dysregulation Causes DNA Methyltransferase 1 Overexpression in Cancer via MAD2-Mediated Inhibition of the Anaphase-Promoting Complex

We have examined the mechanism of normal DNA methyltransferase 1 (DNMT1) degradation as well as its mechanism of dysregulation in cancer. We have previously reported that DNMT1 protein levels were elevated and abnormally stabilized because of defective degradation through its N-terminal destruction domain. Here, we report that DNMT1 was abnormally stabilized in several cancer cell lines and that, in cells with normal DNMT1 destruction, depletion of CDC20 or FZR1 (two substrate recognition adaptor components of the anaphase-promoting complex) resulted in stabilization of DNMT1 that was partially dependent on the N-terminal destruction domain, thus implicating this cell cycle regulator in the destruction of DNMT1. MAD2, an inhibitor of CDC20, was shown to stabilize DNMT1 levels, and overexpression of MAD2, a consequence of retinoblastoma (RB) pathway dysregulation, was shown to correlate with impaired G(1) phase DNMT1 destruction and RB inactivation by hyperphosphorylation in several normal and cancer cell lines. Furthermore, in a series of 85 cases of human breast cancer, a moderately strong, but highly significant, correlation between MAD2 and DNMT1 immunohistochemical staining was observed, yielding a Spearman rank order correlation coefficient of 0.37 (P<0.001). This suggests that RB pathway inactivation, a common dysfunction in cancer cells, may be the underlying cause of DNMT1 dysregulation.

Inhibition of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Decreases DNA Methylation in Colon Cancer Cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

Regulation of h MSH2 and h MLH1 expression in the human colon cancer cell line SW1116 by DNA methyltransferase 1

Aberrant DNA methylation is now recognized as an important epigenetic alteration occurring early in human cancer. To directly study the role of DNA methyltransferase 1 (DNMT1) in the regulation of expression of tumor-related genes in human colon cancer cells, we stably transfected expression constructs containing sense or antisense DNMT1 into the human colon cancer cell line, SW1116. The expression level of mismatch repair genes (MMR), human mut-L homologue 1 (h MLH1) and human Mut S homologue 2 (h MSH2), was monitored by real-time RT-PCR. The methylation status of h MLH1 and h MSH2 promoters was determined by bisulfite modification and methylation-specific PCR (MSP). The protein levels of DNMT1, h MSH2 and h MLH1 were determined by Western analysis. The results show that DNMT1 protein expression was increased or decreased in transfected cell lines containing sense or antisense DNMT1 constructs, respectively. In cells expressing the sense DNMT1 construct, the expression of h MLH1 and h MSH2 was down-regulated through hypermethylation of their respective promoters. Furthermore, antisense DNMT1 expression induced promoter demethylation and up-regulated transcription of h MSH2 ( P<0.05) and h MLH1 ( P=0.064) in SW1116 cells.

Increased Protein Stability Causes DNA Methyltransferase 1 Dysregulation in Breast Cancer

We report that DNA methyltransferase 1 (DNMT1) expression is dysregulated in breast cancer. The elevated protein levels are not a result of increased mRNA levels, but rather an increase in protein half-life. We found that DNMT1 protein levels were elevated in breast cancer tissues and in MCF-7 breast cancer cells relative to normal human mammary epithelial cells (HMECs) without a concomitant increase in DNMT1 mRNA or proliferative fraction. Although DNMT1 mRNA levels were properly S-phase-regulated in both cell types, DNMT1 protein levels did not follow S-phase fraction in MCF-7 cells. Rather, an increase in DNMT1 protein stability was found for MCF-7 cells relative to HMECs, and a destruction domain was mapped to the N-terminal 120 amino acids of DNMT1, which was required for its proper ubiquitination and degradation in HMECs. Furthermore, overexpression of DNMT1 with this deleted destruction domain in HMECs resulted in significantly increased genomic 5-methylcytosine levels relative to overexpression of the full-length protein. The regulation of DNMT1 destruction via this domain may be dysfunctional in cancer cells leading to subsequent cytosine hypermethylation in the genome.

Methylation and inhibition of expression of uPA by the RAS oncogene: divergence of growth control and invasion in breast cancer cells

Expression of urokinase-type plasminogen activator (uPA), a protease only expressed in highly invasive human breast cancer cells, is inhibited by DNA methylation of its promoter. We tested the hypothesis that up-regulation of DNA methyltransferase 1 (DNMT1) will lead to methylation and silencing of uPA and inhibition of the invasiveness of metastatic breast cancer cells. Since RAS was previously shown to up-regulate DNA methylation, we examined the effects of ectopic expression of constitutively active RAS on methylation and expression of uPA. Transfection of Ha-RAS into MDA-MB-231 human breast cancer cells resulted in a significantly shorter cell doubling time compared with the controls. However, expression and activity of the metastatic gene uPA and invasive capacity of the cells were significantly reduced by the oncogene RAS. Silencing of uPA by RAS is mediated by a cis modification of the uPA promoter and not through an effect on a trans-acting factor, since a transiently transfected unmethylated uPA-luicferase reporter is expressed at a similar level in RAS-transfected and control cells. We then examined the levels of DNMT1 and methylated DNA-binding protein 2 (MBD2) expressions in these cells to determine whether this reduction in uPA expression is associated with changes in the DNA methylation machinery. Our results showed that ectopic expression of RAS induced DNMT1 expression and activity and inhibited MBD2 expression. Consistent with methylation-mediated repression, uPA was partially methylated in RAS-transfected cells and uPA expression was induced upon treatment of RAS transfectants with the demethylating agent 5'-azacytidine. These results therefore imply that the RAS-DNMT1 DNA methylation pathway which promotes oncogenic growth in many cancers can exert an opposite effect on the invasive capacity of the highly invasive MDA-MB-231 cells, thus illustrating the divergence of growth and metastasis promoting pathways in cancer. This has important implications for new therapeutic approaches to metastasizing cancer.