Human MeWo Research Articles

Effect of 460 nm blue light PBM on human MeWo melanoma cells.

Photobiomodulation (PBM) using 460 nm blue light has been shown to have an inhibitory effect on skin cancer cells. In this study, we used a continuous LED light source with a wavelength of 460 nm and designed various combinations of power density (ranging from 6.4 to 25.6 mW) and dose (ranging from 0.96 to 30.72 J/cm2) to conduct treatment experiments on MeWo cells to investigate the effects of blue light on MeWo melanoma cells. We are focusing on cell viability, cytotoxicity, mitochondrial function, oxidative stress, and apoptosis. We found that blue light inhibits these melanoma cells through oxidative stress and DNA damage, and this inhibition intensifies at higher irradiance levels. Although the cells initially attempt to resist the stress induced by the treatment, they eventually undergo apoptosis over time. These findings contribute to understanding melanoma's molecular response to blue light PBM, lay the groundwork for future clinical applications.

Oncolytic varicella-zoster virus engineered with ORF8 deletion and armed with drug-controllable interleukin-12

BackgroundThe varicella-zoster virus (VZV), belonging to the group of human α-herpesviruses, has yet to be developed as a platform for oncolytic virotherapy, despite indications from clinical case reports suggesting a...

Interferon Gamma Inhibits Equine Herpesvirus 1 Replication in a Cell Line-Dependent Manner.

The sole equine herpesvirus 1 (EHV-1) immediate-early protein (IEP) is essential for viral replication by transactivating viral immediate-early (IE), early (E), and late (L) genes. Here, we report that treatment of mouse MH-S, equine NBL6, and human MRC-5 cells with 20 ng/mL of IFN-γ reduced EHV-1 yield by 1122-, 631-, and 10,000-fold, respectively. However, IFN-γ reduced virus yield by only 2–4-fold in mouse MLE12, mouse L-M, and human MeWo cells compared to those of untreated cells. In luciferase assays with the promoter of the EHV-1 early regulatory EICP0 gene, IFN-γ abrogated trans-activation activity of the IEP by 96% in MH-S cells, but only by 21% in L-M cells. Similar results were obtained in assays with the early regulatory UL5 and IR4 promoter reporter plasmids. IFN-γ treatment reduced IEP protein expression by greater than 99% in MH-S cells, but only by 43% in L-M cells. The expression of IEP and UL5P suppressed by IFN-γ was restored by JAK inhibitor treatment, indicating that the inhibition of EHV-1 replication is mediated by JAK/STAT1 signaling. These results suggest that IFN-γ blocks EHV-1 replication by inhibiting the production of the IEP in a cell line-dependent manner. Affymetrix microarray analyses of IFN-γ-treated MH-S and L-M cells revealed that five antiviral ISGs (MX1, SAMHD1, IFIT2, NAMPT, TREX1, and DDX60) were upregulated 3.2–18.1-fold only in MH-S cells.

Radiochemical and radiopharmacological characterization of a 64 Cu-labeled α-MSH analog conjugated with different chelators.

Radiolabeled α-melanocyte-stimulating hormone (α-MSH) derivatives have a high potential for diagnosis and treatment of melanoma, because of high specificity and binding affinity to the melanocortin-1 receptor (MC1R). Hence, the α-MSH-derived peptide NAP-NS1 with a β-Ala linker (ε-Ahx-β-Ala-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 ) was conjugated to different chelators: either to NOTA (p-SCN-Bn-1,4,7-triazacyclononane-1,4,7-triacetic acid), to a hexadentate bispidine carbonate derivative (dimethyl-9-(((4-nitrophenoxy)carbonyl)oxy)-2,4-di(pyridin-2-yl)-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate), or to DMPTACN (p-SCN-Ph-bis(2-pyridyl-methyl)-1,4,7-triaza-cyclononane), labeled with 64 Cu, and investigated in terms of radiochemical and radiopharmacological properties. For the three 64 Cu-labeled conjugates negligible transchelation, suitable buffer and serum stability, as well as appropriate water solubility, was determined. The three conjugates exhibited high binding affinity (low nanomolar range) in murine B16F10, human MeWo, and human TXM13 cells. The Bmax values of [64 Cu]Cu-bispidine-NAP-NS1 ([64 Cu]Cu-2) and [64 Cu]Cu-DMPTACN-NAP-NS1 ([64 Cu]Cu-3) were higher than those of [64 Cu]Cu-NOTA-NAP-NS1 ([64 Cu]Cu-1), implying that different charged chelate units might have an impact on binding capacity. Preliminary in vivo biodistribution studies suggested the main excretion pathway of [64 Cu]Cu-1 and [64 Cu]Cu-3 to be renal, while that of [64 Cu]Cu-2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60minutes. All three 64 Cu-labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.

Synthesis, Characterization, and Initial Biological Evaluation of [99m Tc]Tc-Tricarbonyl-labeled DPA-α-MSH Peptide Derivatives for Potential Melanoma Imaging.

α-Melanocyte stimulating hormone (α-MSH) derivatives target the melanocortin-1 receptor (MC1R) specifically and selectively. In this study, the α-MSH-derived peptide NAP-NS1 (Nle-Asp-His-d-Phe-Arg-Trp-Gly-NH2 ) with and without linkers was conjugated with 5-(bis(pyridin-2-ylmethyl)amino)pentanoic acid (DPA-COOH) and labeled with [99m Tc]Tc-tricarbonyl by two methods. With the one-pot method the labeling was faster than with the two-pot method, while obtaining similarly high yields. Negligible trans-chelation and high stability in physiological solutions was determined for the [99m Tc]Tc-tricarbonyl-peptide conjugates. Coupling an ethylene glycol (EG)-based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [99m Tc]Tc-tricarbonyl-peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the 99m Tc-labeled peptides for in vivo imaging.

Radiopharmacological characterization of ⁶⁴Cu-labeled α-MSH analogs for potential use in imaging of malignant melanoma.

The melanocortin-1 receptor (MC1R) plays an important role in melanoma growth, angiogenesis and metastasis, and is overexpressed in melanoma cells. α-Melanocyte stimulating hormone (α-MSH) and derivatives are known to bind with high affinity at this receptor that provides the potential for selective targeting of melanoma. In this study, one linear α-MSH-derived peptide Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) without linker and with εAhx-β-Ala linker, and a cyclic α-MSH derivative, [Lys-Glu-His-D-Phe-Arg-Trp-Glu]-Arg-Pro-Val-NH2 (NAP-NS2) with εAhx-β-Ala linker were conjugated with p-SCN-Bn-NOTA and labeled with (64)Cu. Radiochemical and radiopharmacological investigations were performed with regard to transchelation, stability, lipophilicity and in vitro binding assays as well as biodistribution in healthy rats. No transchelation reactions, but high metabolic stability and water solubility were demonstrated. The linear derivatives showed higher affinity than the cyclic one. [(64)Cu]Cu-NOTA-εAhx-β-Ala-NAP-NS1 ([(64)Cu]Cu-2) displayed rapid cellular association and dissociation in murine B16F10 cell homogenate. All [(64)Cu]Cu-labeled conjugates exhibited affinities in the low nanomolar range in B16F10. [(64)Cu]Cu-2 showed also high affinity in human MeWo and TXM13 cell homogenate. In vivo studies suggested that [(64)Cu]Cu-2 was stable, with about 85 % of intact peptide in rat plasma at 2 h p.i. Biodistribution confirmed the renal pathway as the major elimination route. The uptake of [(64)Cu]Cu-2 in the kidney was 5.9 % ID/g at 5 min p.i. and decreased to 2.0 % ID/g at 60 min p.i. Due to the prospective radiochemical and radiopharmacological properties of the linear α-MSH derivative [(64)Cu]Cu-2, this conjugate is a promising candidate for tracer development in human melanoma imaging.

SOX2 modulates levels of MITF in normal human melanocytes, and melanoma lines in vitro

SOX2 modulates levels of MITF in normal human melanocytes, and melanoma lines in vitro

The FGF-2-Derived Peptide FREG Inhibits Melanoma Growth In Vitro and In Vivo

Previous data report that fibroblast growth factor-2 (FGF-2)-derived peptide FREG potently inhibits FGF-2-dependent angiogenesis in vitro and in vivo. Here, we show that FREG inhibits up to 70% in vitro growth and invasion/migration of smooth muscle and melanoma cells. Such inhibition is mediated by platelet-derived growth factor-receptor-α (PDGF-Rα); in fact, proliferation and migration were restored upon PDGF-Rα neutralization. Further experiments demonstrated that FREG interacts with PDGF-Rα both in vitro and in vivo and stimulates its phosphorylation. We have previously shown that overexpressing PDGF-Rα strongly inhibits melanoma growth in vivo; we, therefore, hypothesized that PDGF-Rα agonists may represent a novel tool to inhibit melanoma growth in vivo. To support this hypothesis, FREG was inoculated intravenously (i.v.) in a mouse melanoma model and markedly inhibited pulmonary metastases formation. Immunohistochemical analyses showed less proliferation, less angiogenesis, and more apoptosis in metastasized lungs upon FREG treatment, as compared to untreated controls. Finally, in preliminary acute toxicity studies, FREG showed no toxicity signs in healthy animals, and neither microscopic nor macroscopic toxicity at the liver, kidney, and lungs level. Altogether, these data indicate that FREG systemic treatment strongly inhibits melanoma metastases development and indicate for the first time that agonists of PDGF-Rα may control melanoma both in vitro and in vivo.

The DNA-Mismatch Repair Enzyme hMSH2 Modulates UV-B-Induced Cell Cycle Arrest and Apoptosis in Melanoma Cells

The mechanisms by which the post-replicative DNA mismatch repair (MMR) enzyme MSH2 is involved in the complex response mechanisms to UV damage are yet to be clarified. Here, we show increased levels of MSH2 mRNA in malignant melanoma, metastases of melanoma, and melanoma cell (MeWo) lines as compared with melanocytic nevi or primary cultured benign melanocytes. UV-B treatment modulated MSH2 expression and silencing of MSH2 gene expression using small interfering RNA technology regulated UV-B-induced cell cycle arrest and apoptosis in human MeWo. We show that MSH2-deficient non-malignant mouse fibroblasts (MEF-/-) are partially resistant against UV-B-induced apoptosis and show reduced S-Phase accumulation. In addition, we show that an Msh2 point mutation (MEFGA) that affects MMR does not affect UV-B-induced apoptosis. In conclusion, we demonstrate that MSH2 modulates in human melanocytes both UV-B-induced cell cycle regulation and apoptosis, most likely via independent, uncoupled mechanisms.

The genotoxic potential of electric and magnetic fields: an update.

We review 23 studies on the potential genotoxicity of electric and magnetic fields that have appeared in the published literature since our 1993 review of 55 published studies (McCann et al., Mutat. Res. 297 (1993) 61-95) and six additional studies published prior to 1993, which were not previously reviewed. As in our previous review, internal electric fields present in media (for in vitro experiments) and in the torso (for in vivo experiments) were estimated. Individual experiments are evaluated using basic data quality criteria. The potential for genotoxicity of electric and magnetic fields is discussed in light of the significant body of genotoxicity data that now exists. Three unsuccessful attempts to replicate previously reported positive results have appeared since our previous review. We conclude that, in spite of the 34 studies reviewed in this and our previous publication that report positive genotoxic effects, none satisfy all of three basic conditions: independent reproducibility, consistency with the scientific knowledge base, and completeness according to basic data quality criteria. As we discuss, these criteria are satisfied for several groups of negative studies in several exposure categories (ELF magnetic fields, 150 microT-5 mT, combined ELF electric and ELF magnetic fields, approx. 0.2 mT, 240 mV/m, and static magnetic fields, 1-3.7 T). The evidence reviewed here strengthens the conclusion of our previous review, that the preponderance of evidence suggests that ELF electric or magnetic fields do not have genotoxic potential. Nevertheless, a pool of positive results remains, which have not yet been tested by independent replication. Among the 12 studies reviewed here, which report statistically significant or suggestive positive results, we point particularly to results from five laboratories [J. Miyakoshi, N. Yamagishi, S. Ohtsu, K. Mohri, H. Takebe, Increase in hypoxanthine-guanine phosphoribosyl transferase gene mutations by exposure to high-density 50-Hz magnetic fields, Mutat. Res. 349 (1996) 109-114; J. Miyakoshi, K. Kitagawa, H. Takebe, Mutation induction by high-density, 50-Hz magnetic fields in human MeWo cells exposed in the DNA synthesis phase, Int. J. Radiat. Biol. 71 (1997) 75-79; H. Lai. N.P. Singh, Acute exposure to a 60-Hz magnetic field increases DNA strand breaks in rat brain cells, Bioelectromagnetics, 18 (1997) 156-165; H. Lai, N.P. Singh, Melatonin and N-tert-butyl-alpha-phenylnitrone block 60-Hz magnetic field-induced DNA single and double strand breaks in rat brain cells, J. Pineal Res. 22 (1997) 152-162; T. Koana, M. Ikehata, M. Nakagawa, Estimation of genetic effects of a static magnetic field by a somatic cell test using mutagen-sensitive mutants of Drosophila melanogaster, Bioelectrochem. Bioenergetics 36 (1995) 95-100; F.L. Tabrah, H.F. Mower, S. Batkin, P.B. Greenwood, Enhanced mutagenic effect of a 60-Hz time-varying magnetic field on numbers of azide-induced TA100 revertant colonies, Bioelectromagnetics 15 (1994) 85-93; S. Tofani, A. Ferrara, L. Anglesio, G. Gilli, Evidence for genotoxic effects of resonant ELF magnetic fields, Bioelectrochem. Bioenergetics, 36 (1995) 9-13], which satisfy most basic data quality criteria and may be of interest.

Stimulation of the protein tyrosine kinase c-Yes but not c-Src by neurotrophins in human brain-metastatic melanoma cells.

The c-Yes proto-oncogene (pp62c-Yes) encodes a non-receptor-type protein tyrosine kinase (NRPTK) of the Src family. c-Yes activities and protein levels are elevated in human melanoma and melanocyte cell lines. Because the neurotrophins (NT) are important in the progression of melanoma to the brain-metastatic phenotype, we determined whether NT stimulate c-Yes activity in human MeWo melanoma cells and two variant sublines with opposite metastatic capabilities, 3 S 5 and 70W. The highly brain-metastatic 70W subline had an intrinsically higher c-Yes activity than parental MeWo or poorly metastatic 3 S 5 cells. c-Yes kinase was further induced by the prototypic human NT, nerve growth factor (NGF) in a dose and time-dependent manner. In contrast, c-Src activity (pp60-Src) was similar in all these cells and unaffected by NGF exposure. Additionally, human NGF and neurotrophin-3 stimulated c-Yes in brain-metastatic 70W cells. The magnitude of c-Yes activation correlated with the degree of invasion of 70 W cells following incubation of these neurotrophins. To further examine NT stimulation of c-Yes in melanoma cells, three additional cell lines were examined. Metastatic TXM-13 and TXM-18 increased c-Yes activity in response to NGF. In contrast, no increase was observed in low-metastatic TXM-40 cells. Together, these data suggest that altered c-Yes expression may play a role in the malignant progression of the human melanocyte towards the brain-metastatic phenotype and that NT enhance the activity of c-Yes in signaling penetration into the matrix of NT-rich stromal microenvironments such as the brain.

Mutation induction by high-density, 50-Hz magnetic fields in human MeWo cells exposed in the DNA synthesis phase

. Exposure of cultured human MeWo cells to highdensity (400 mT at 50 Hz) extremely low frequency magnetic fields (ELF-MF) induced mutations in the hypoxanthineguanine phosphoribosyl transferase gene. Mutation induced by the ELF-MF increased during the DNA-synthesis phase in synchronously growing cells. DNA replication errors and/or disturbance of the mismatch repair systems caused by exposure to ELF-MF may be involved in the mutagenic effect.

Regulation of melanoma cell adhesion stabilization to fibronectin.

Tumour cell arrest and the formation of stable adhesive interactions between tumour cells and endothelial cells or underlying matrix in the microvasculature are crucial steps in the metastatic process. We have developed a sensitive hydrodynamic adhesion assay to investigate the regulation of melanoma cell adhesion stabilization to the extracellular matrix protein fibronectin. Modulators of human MeWo melanoma Ca2+ concentration and stores, including ionomycin, thapsigargin, dantrolene and caffeine, inhibited cell adhesion stabilization to fibronectin; however, removal of Ca2+ from the extracellular medium did not affect stabilization. The calmodulin inhibitor W-7 and the protein kinase C inhibitor chelerythrine also blocked MeWo adhesion stabilization to fibronectin, as did the tyrosine kinase inhibitor genistein and the cytoskeletal inhibitor cytochalasin D. Manipulation of MeWo cell intracellular CAMP levels had no effect of adhesion stabilization to fibronectin, nor did treatment of cells with phorbol ester, pertussis toxin or cholera toxin. Drug treatments that inhibited adhesion stabilization also had significant effects on the actin cytoskeleton organization of the melanoma cells. This study suggests a role for calcium, calmodulin, protein kinase C and tyrosine kinases in the intracellular regulation of MeWo adhesive stabilization.

Specific Induction of Cell Motility on Laminin by α7 Integrin

Laminin, the major glycoprotein of basement membranes, actively supports cell migration in development, tissue repair, tumor growth, metastasis, and other pathological processes. Previously we have shown that the locomotion of murine skeletal myoblasts is specifically and significantly enhanced on laminin but not on other matrix proteins. One of the major laminin receptors of myoblasts is the alpha 7 beta 1 integrin, which was first described in human MeWo melanoma cells and Rugli glioblastoma cells. In order to investigate and directly test the role of the alpha 7 integrin in cell migration on laminin, we expressed the murine alpha 7B splice variant in human 293 kidney cells and 530 melanoma cells which cannot migrate on laminin and are devoid of endogenous alpha 7. Northern blotting of the transfected cells showed that the alpha 7 mRNA was expressed efficiently, and the protein was detected on the cell surface by immunofluorescence and fluorescence-activated cell sorter analysis. Cell motility measurements by computer-assisted time-lapse videomicroscopy of the alpha 7-transfected cells revealed an 8-10-fold increase in motility on laminin-1 and its E8 fragment, but not on fibronectin. Mock-transfected cells did not migrate significantly of alpha 7-transfected 293 cells through laminin-coated filters in a Boyden chamber assay was significantly enhanced in comparison to mock transfected cells. These findings prove that alpha 7 integrin expression confers a gain of function-motile phenotype to immobile cells and may be responsible for transduction of the laminin-induced cell motility.

Mediation of NGF-stimulated extracellular matrix invasion by the human melanoma low-affinity p75 neurotrophin receptor: melanoma p75 functions independently of trkA.

Although overexpression of the low-affinity p75 neurotrophin receptor (p75NTR) is frequently associated with advanced stages of human melanoma progression, the functional significance of this finding is unknown. We examined whether the degree of cell surface expression of p75NTR in human melanoma cell variants determines their extent of invasion stimulated by nerve growth factor (NGF). Treatment of MeWo melanoma cells or a metastatic spontaneous wheat germ agglutinin-resistant variant subline (70W) of MeWo cells with 2.5S NGF resulted in a dose-dependent enhancement of invasion through a reconstituted basement membrane. This effect was most pronounced with the 70W subline that exhibits brain-metastasizing potential in nude mice but was not found with a poorly metastatic MeWo variant subline (3S5). The expression of p75NTR as determined by Northern blotting and immunoprecipitation analysis of 125I-labeled cell surface proteins correlated with NGF-stimulated invasion. The MeWo melanoma sublines used in this study did not express p140proto-trkA mRNA or any p140proto-trkA variant transcripts including p70trkA as determined by Northern analysis and RT-PCR analysis. Thus, these melanoma cells would not be expected to form functional p75-p140 heterodimers or p140-p140 homodimers capable of transducing an NGF-generated signal to p140proto-trkA cytoplasmic substrates. These cells did express authentic p145trkC transcripts. However, NGF did not catalytically activate p145trkC receptors via increased tyrosine phosphorylation as would be expected if p145trkC participated in the signaling established by NGF. Furthermore, a NGF-stimulated purine-analogue-sensitive kinase activity was found to coimmunoprecipitate with p75NTR. This p75NTR-associated kinase may coordinate initial signaling events evoked by p75NTR ligand interaction. Addition of 2.5S NGF, at concentrations that should saturate cell surface p75NTR, to matrix-adherent cultures of human MeWo and 70W but not 3S5 melanoma cells suppressed the expression of 92-kDa type IV collagenase and stimulated the production of 72-kDa type IV collagenase in its fully active 68-kDa form. In the absence of p140proto-trkA, the matrix-dependent effects of NGF on metalloproteinase expression of brain-metastatic 70W melanoma cells suggest a signaling role for the low-affinity melanoma p75NTR receptor and its associated purine-analogue-sensitive kinase in signaling enhanced matrix penetration of NGF-rich stromal microenvironments such as the brain.

Inhibition of N-linked oligosaccharide processing in tumor cells is associated with enhanced tissue inhibitor of metalloproteinases (TIMP) gene expression.

Previous studies on tumor-cell glycosylation mutants and drugs which inhibit oligosaccharide processing suggest that expression of sialylated and highly branched complex-type N-linked oligosaccharides is required for efficient tumor-cell metastasis. These observations prompted the present investigation, in order to determine whether loss of sialylated and highly branched complex-type oligosaccharide in cellular glycoproteins might affect the expression of genes, particularly of genes which can influence the malignant phenotypes. Swainsonine, an inhibitor of Golgi alpha-mannosidase II, has previously been shown to inhibit invasion in vitro and reduces solid tumor in vivo. Metastatic sub-lines of the SP1 murine mammary carcinoma cells cultured in the presence of swainsonine for 48 hr showed approximately 3-fold enhancement of TIMP mRNA levels, while urokinase (uPA) transcripts remained unchanged. To determine whether swainsonine's effect on TIMP mRNA levels was related to inhibition of oligosaccharide processing, we examined somatic glycosylation mutants with processing defects which attenuate metastatic potential. The Golgi UDP-Gal transport defect in murine MDAY-D2 lymphoma cells, Chinese hamster ovary cells (CHO) and human MeWo melanoma cells (i.e., D35W25, Lec8, 3S5 cell lines, respectively) was associated with increased TIMP mRNA levels. A revertant of Lec8 showed a return to the wild-type levels of TIMP mRNA, consistent with a causal relationship between the glycosylation mutation and TIMP gene expression. Similarly, CHO and MDAY-D2 mutants defective in GlcNAc-TV (i.e., Lec4 and KBL-1 respectively), which also reduces metastatic potential, showed increases in TIMP transcript levels. Nuclear run-on assays showed that transcription of the TIMP gene was increased in cells where N-linked oligosaccharide processing was inhibited either by swainsonine or by a glycosylation mutation. The results suggest that cell-specific patterns of glycoprotein glycosylation in human, murine and hamster cell lines affects the transcription of select genes, including TIMP, which may influence the invasive phenotype.

DNA (cytosine) methylation in murine and human tumor cell lines treated with S-adenosylhomocysteine hydrolase inhibitors

The effects of periodate-oxidized adenosine, 3-deaza-adenosine and 3-deaza-(±)aristeromycin, potent inhibitors of S-adenosylhomocysteine hydrolase activity, on DNA methylation and the intracellular ratio of S-adenosylhomocysteine and S-adenosylmethionine have been examined in a series of murine and human tumor cell lines. A 24-h exposure of murine LC3, TA3 and B16 cells and human MeWo and K562 cells to 1–10 μM periodate-oxidized adenosine had a very slight inhibitory effect upon DNA methylation. 3-Deaza-(±)aristeromycin and 3-deaza-adenosine (50 μM) had virtually no effect upon DNA methylation in LC3 and B16 cells. In LC3 cells, periodate-oxidized adenosine, 3-deaza-adenosine and 3-deaza-(±)aristeromycin reduced the intracellular ratio of S-adenosylmethionine/ S-adenosylhomocysteine approximately 20-, 6- and 16-fold, respectively. In murine B16 melanoma cells, periodate-oxidised adenosine, 3-deaza-adenosine and 3-deaza-(±)aristeromycin reduced the intracellular ratio of S-adenosylmethionine/ S-adenosylhomocysteine approximately 17-, 13- and 32-fold, respectively. These observations indicate that the cytosine methylation of DNA appears to be relatively insensitive to changes in the intracellular ratio of S-adenosylmethionine/ S-adenosylhomocysteine and that such metabolic disturbances are not likely to be the major biochemical alteration responsible for the reduced level of DNA 5-methylcytosine found within transformed and malignant cells.