Restricting tumor lactic acid metabolism using dichloroacetate improves T cell functions.

BACKGROUND: Lactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism. In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells. RESULT: We found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis. CONCLUSION: Taken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells.

Recent Advances in Studies of Skin Color and Skin Cancer.

The relationship between skin color and skin cancer is well established: the less melanin in one's skin the greater the risk for developing skin cancer. This review is in two parts. First, we summarize the current understanding of the cutaneous pigmentary system and trace melanin from its synthesis in the pigment cell melanosomes through its transfer to keratinocytes. We also present new methods for reducing melanin content in hyper-pigmented areas of skin such as solar lentigenes, melasma, and post-inflammatory hyperpigmentation. Second, we present evidence that at least one mechanism for the development of metastatic melanoma and other solid tumors is fusion and hybridization of leucocytes such as macrophages with primary tumor cells. In this scenario, hybrid cells express both the chemotactic motility of the leucocyte and the de-regulated cell division of the tumor cell, causing the cells to migrate a deadly journey to lymph nodes, distant organs, and tissues.

Curcumin: A new candidate for melanoma therapy?

Melanoma remains among the most lethal cancers and, in spite of great attempts that have been made to increase the life span of patients with metastatic disease, durable and complete remissions are rare. Plants and plant extracts have long been used to treat a variety of human conditions; however, in many cases, effective doses of herbal remedies are associated with serious adverse effects. Curcumin is a natural polyphenol that shows a variety of pharmacological activities including anti-cancer effects, and only minimal adverse effects have been reported for this phytochemical. The anti-cancer effects of curcumin are the result of its anti-angiogenic, pro-apoptotic and immunomodulatory properties. At the molecular and cellular level, curcumin can blunt epithelial-to-mesenchymal transition and affect many targets that are involved in melanoma initiation and progression (e.g., BCl2, MAPKS, p21 and some microRNAs). However, curcumin has a low oral bioavailability that may limit its maximal benefits. The emergence of tailored formulations of curcumin and new delivery systems such as nanoparticles, liposomes, micelles and phospholipid complexes has led to the enhancement of curcumin bioavailability. Although in vitro and in vivo studies have demonstrated that curcumin and its analogues can be used as novel therapeutic agents in melanoma, curcumin has not yet been tested against melanoma in clinical practice. In this review, we summarized reported anti-melanoma effects of curcumin as well as studies on new curcumin formulations and delivery systems that show increased bioavailability. Such tailored delivery systems could pave the way for enhancement of the anti-melanoma effects of curcumin.

Evidence for glycosylation as a regulator of the pigmentary system: key roles of sialyl(α2-6)gal/GalNAc-terminated glycans in melanin synthesis and transfer.

UNLABELLED: The major regulators of melanogenesis are glycoproteins, however no role for glycosylation in the pathway has yet been described. We stained skin biopsies and melanocyte-keratinocyte co-cultures with a panel of 20 lectins as oligosaccharide markers. Notably, the Elderberry Bark Lectin (EBL/SNA) stained melanocytes in both systems. EBL binds the sequence Neu5Ac(α(2-6)Gal/GalNAc)- at the termini of some oligosaccharide antennae. We used inhibitors of synthesis and/or binding of this sequence to assess effects on pigmentation. METHODS: Cell culture, lectin histochemistry, siRNA transfection, and assays for dopa oxidase and melanin were carried out by standard techniques. RESULTS: 6'-sialyllactose, a short homolog of the sequence in question, anti-sialyltransferase 6 (ST6) siRNA, and cytidine, a sialyltransferase (ST) inhibitor, each inhibited EBL binding, melanogenesis and melanosome transfer. Unexpectedly, 3'-sialyllactose and siRNA for ST3, chosen as a negative controls, also inhibited these processes. Though strong inhibitors of melanization, none of the agents affected tyrosinase/dopa oxidase activity, indicating previously unrecognized post-tyrosinase regulation of melanization. CONCLUSIONS: We report for the first time that Neu5Ac (α(2-6)Gal/GalNAc)- and possibly Neu5Ac(α(2-3)Gal/GalNAc)-terminated oligosaccharides play multiple roles in melanin synthesis and transfer.

Fusion of bone marrow-derived cells with cancer cells: metastasis as a secondary disease in cancer.

This perspective article highlights the leukocyte-cancer cell hybrid theory as a mechanism for cancer metastasis. Beginning from the first proposal of the theory more than a century ago and continuing today with the first proof for this theory in a human cancer, the hybrid theory offers a unifying explanation for metastasis. In this scenario, leukocyte fusion with a cancer cell is a secondary disease superimposed upon the early tumor, giving birth to a new, malignant cell with a leukocyte-cancer cell hybrid epigenome.

Leukocyte-cancer cell fusion: initiator of the warburg effect in malignancy?

The causes of metastasis remain unknown, however it has been proposed for nearly a century that metastatic cells are generated by fusion of tumor cells with tumor-associated leukocytes such as macrophages. Indeed, regardless of cell or tissue origin, when cancer cells in the original in situ tumor transform to malignant, invasive cells, they generally become aneuploid and begin to express molecules and traits characteristic of activated macrophages. This includes two key features of malignancy: chemotactic motility and the use of aerobic glycolysis as a metabolic energy source (the Warburg effect). Here we review evidence that these phenomena can be well-explained by macrophage-cancer cell fusion, as evidenced by studies of experimental macrophage-melanoma hybrids generated in vitro and spontaneous host-tumor hybrids in animals and more recently humans. A key finding to emerge is that experimental and spontaneous cancer cell hybrids alike displayed a high degree of constitutive autophagy, a macrophage trait that is expressed under hypoxia and nutrient deprivation as part of the Warburg effect. Subsequent surveys of 21 different human cancers from nearly 2,000 cases recently revealed that the vast majority (~85%) exhibited autophagy and that this was associated with tumor proliferation and metastasis. While much work needs to be done, we posit that these findings with human cancers could be a reflection of widespread leukocyte-cancer cell fusion as an initiator of metastasis. Such fusions would generate hybrids that express the macrophage capabilities for motility and survival under adverse conditions of hypoxia and nutrient deprivation, while at the same time maintaining the deregulated mitotic cycle of the cancer cell fusion partner.

Chimeric antigen receptor T-cell therapy for melanoma.

INTRODUCTION: In recent years, chimeric antigen receptor (CAR) T cell therapy has emerged as a cancer treatment. After initial therapeutic success for hematologic malignancies, this approach has been extended for the treatment of solid tumors including melanoma. AREAS COVERED: T cells need to be reprogramed to recognize specific antigens expressed only in tumor cells, a difficult problem since cancer cells are simply transformed normal cells. Tumor antigens, namely, CSPG4, CD70, and GD2 have been targeted by CAR-T cells for melanoma. Moreover, different co-stimulatory signaling domains need to be selected to direct T cell fate. In this review, various approaches for the treatment of melanoma and their effectiveness are comprehensively reviewed and the current status, challenges, and future perspective of CAR-T cell therapy for melanoma are discussed. Literature search was accomplished in three databases (PubMed, Google scholar, and Clinicaltrials.gov). Published papers and clinical trials were screened and relevant documents were included by checking pre-defined eligibility criteria. EXPERT OPINION: Despite obstacles and the risk of adverse events, CAR T cell therapy could be used for patients with treatment-resistant cancer. Clinical trials are underway to determine the efficacy of this approach for the treatment of melanoma.

A melanoma patient with macrophage-cancer cell hybrids in the primary tumor, a lymph node metastasis and a brain metastasis.

In 1911 it was proposed that cancer might result from fusion and hybridization between macrophages and cancer cells. Using immunohistochemistry it was determined that essentially all solid tumors expressed macrophage-like molecules on their cell surface. More recently we have used forensic (STR) genetics that allows one to detect DNA from more than one individual in the same sample. By studying biopsies from individuals receiving allogeneic stem cell transplants and later developed solid tumor metastases, we were able to detect both donor and patient DNA sequences suggesting that hybrids were present. Previously we found hybrids in biopsies of a renal cell carcinoma, a melanoma in a brain metastasis and a melanoma in a primary tumor with lymph node metastases. Here we have traced hybrids from a primary melanoma to an axillary lymph node to a brain metastasis. This is the first time that the entire metastatic process has been documented.

A metabolic switch to memory CAR T cells: Implications for cancer treatment.

Therapeutic efficacy of chimeric antigen receptor (CAR) T cells is associated with their expansion, persistence and effector function. Although CAR T cell therapy has shown remarkable therapeutic effects in hematological malignancies, its therapeutic efficacy has been limited in some types of cancers - in particular, solid tumors - partially due to the cells' inability to persist and the acquisition of T cell dysfunction within a harsh immunosuppressive tumor microenvironment. Therefore, it would be expected that generation of CAR T cells with intrinsic properties for functional longevity, such as the cells with early-memory phenotypes, could beneficially enhance antitumor immunity. Furthermore, because the metabolic pathways of CAR T cells help determine cellular differentiation and lifespan, therapies targeting such pathways like glycolysis and oxidative phosphorylation, can alter CAR T cell fate and durability within tumors. Here we discuss how reprogramming of CAR T cell metabolism and metabolic switch to memory CAR T cells influences their antitumor activity. We also offer potential strategies for targeting these metabolic circuits in the setting of adoptive CAR T cell therapy, aiming to better unleash the potential of adoptive CAR T cell therapy in the clinic.

International Scientific Collaboration Is Needed to Bridge Science to Society: USERN2020 Consensus Statement.

Scientific collaboration has been a critical aspect of the development of all fields of science, particularly clinical medicine. It is well understood that myriads of benefits can be yielded by interdisciplinary and international collaboration. For instance, our rapidly growing knowledge on COVID-19 and vaccine development could not be attained without expanded collaborative activities. However, achieving fruitful results requires mastering specific tactics in collaborative efforts. These activities can enhance our knowledge, which ultimately benefits society. In addition to tackling the issue of the invisible border between different countries, institutes, and disciplines, the border between the scientific community and society needs to be addressed as well. International and transdisciplinary approaches can potentially be the best solution for bridging science and society. The Universal Scientific Education and Research Network (USERN) is a non-governmental, non-profit organization and network to promote professional, scientific research and education worldwide. The fifth annual congress of USERN was held in Tehran, Iran, in a hybrid manner on November 7-10, 2020, with key aims of bridging science to society and facilitating borderless science. Among speakers of the congress, a group of top scientists unanimously agreed on The USERN 2020 consensus, which is drafted with the goal of connecting society with scientific scholars and facilitating international and interdisciplinary scientific activities in all fields, including clinical medicine.

Autophagy in cutaneous malignant melanoma.

We show that malignant melanoma cells display high levels of autophagy, a cytoplasmic process of protein and organelle digestion that provides an energy source in times of nutrient deprivation. In a panel of 12 cases of cutaneous malignant melanoma of the superficial spreading type, cells in florid melanoma in situ (MIS) and invasive cells in the dermis appeared to be undergoing autophagy. Autophagosomes were detected through immunohistochemistry using the marker LC3B (microtubule-associated light chain 3B), and by electron microscopy. Some autophagosomes contained melanized melanosomes, accounting for the phenomenon of 'coarse melanin' in malignant melanoma. Autophagosomes also contained the Golgi 58k protein, a structural component of the Golgi apparatus, and beta1,6-branched oligosaccharides, indicating that at least some of the autophagosomal proteins were glycosylated with these structures. The findings suggest that autophagy could be a constitutive metabolic state for invasive and metastatic melanoma cells. Interestingly, a similar phenotype was also expressed by tumor-associated melanophages. The findings are consistent with previous reports that endoplasmic reticulum (ER) stress drives melanoma progression, since ER stress is known to trigger autophagy. The results suggest that therapies inhibiting autophagy may be effective for the treatment of malignant melanoma by depriving cells of an important energy source.

Roles of cell fusion, hybridization and polyploid cell formation in cancer metastasis.

Cell-cell fusion is a normal biological process playing essential roles in organ formation and tissue differentiation, repair and regeneration. Through cell fusion somatic cells undergo rapid nuclear reprogramming and epigenetic modifications to form hybrid cells with new genetic and phenotypic properties at a rate exceeding that achievable by random mutations. Factors that stimulate cell fusion are inflammation and hypoxia. Fusion of cancer cells with non-neoplastic cells facilitates several malignancy-related cell phenotypes, e.g., reprogramming of somatic cell into induced pluripotent stem cells and epithelial to mesenchymal transition. There is now considerable in vitro, in vivo and clinical evidence that fusion of cancer cells with motile leucocytes such as macrophages plays a major role in cancer metastasis. Of the many changes in cancer cells after hybridizing with leucocytes, it is notable that hybrids acquire resistance to chemo- and radiation therapy. One phenomenon that has been largely overlooked yet plays a role in these processes is polyploidization. Regardless of the mechanism of polyploid cell formation, it happens in response to genotoxic stresses and enhances a cancer cell's ability to survive. Here we summarize the recent progress in research of cell fusion and with a focus on an important role for polyploid cells in cancer metastasis. In addition, we discuss the clinical evidence and the importance of cell fusion and polyploidization in solid tumors.

The cancer cell--leukocyte fusion theory of metastasis.

The cause of metastasis remains elusive despite vast information on cancer cells. We posit that cancer cell fusion with macrophages or other migratory bone marrow-derived cells (BMDCs) provides an explanation. BMDCs fused with tumor cells were present in animal tumor xenografts where they were associated with metastases. In myeloma patients, transcriptionally active myeloma nuclei were incorporated into osteoclasts through fusion. In patients with renal cell carcinoma arising poststem cell transplant, donor genes were incorporated in recipient cancer cell nuclei, most likely through fusion, and showed tumor distribution patterns characteristic of cancer stem cells. Melanoma-macrophage hybrids generated in vitro contained chromosomes from both parental partners, showed increased ploidy, and transcribed and translated genes from both parents. They exhibited chemotactic migration in vitro toward fibronectin and exhibited high frequencies of metastasis when implanted in mice. They produced macromolecules that are characteristic of macrophages and known indicators of metastasis (c-Met, SPARC, MCR1, GnT-V, and the integrin subunits alpha(3), alpha(5), alpha(6), alpha(v), beta(1), beta(3)). They also produced high levels of beta1,6-branched oligosaccharides-predictors of poor survival in patients with melanoma or carcinomas of the breast, lung, and colon. We thus hypothesize that such gene expression patterns in cancer are generated through fusion. Tumor hybrids also showed active autophagy, a characteristic of both metastatic cancers and macrophages. BMDC-tumor cell fusion explains epidermal-mesenchymal transition in cancer since BMDCs express mesodermal traits and epithelial-mesenchymal transition regulators (Twist, SPARC, and others). If BMDC-tumor cell fusion underlies invasion and metastasis in human cancer, new approaches for therapeutic intervention would be mandated.

Why do melanomas get so dark?

Cutaneous malignant melanomas often exhibit pigmented regions that are darker than the surrounding skin. While melanoma cells are the original source of the melanin, keratinocytes and melanophages also contribute to the tumor colour because they contain melanin obtained from melanoma cells. However, little is known of the origin of darkly pigmented melanoma cells or of the molecular pathways regulating their melanin production. Here we discuss observations that dark melanoma cells emerge from within populations of melanoma in situ and that, in addition to producing abundant dark pigment, they appear to be undergoing autophagy. Moreover, autophagy appears to be a common trait of invasive melanoma cells in the dermis. The underlying cause of this phenomenon may stem from aberrant production of glycosylation structures known as beta1,6-branched oligosaccharides. Our studies of dark cutaneous melanomas were prompted by analyses of experimental mouse macrophage-melanoma hybrids fused in the laboratory. Like melanoma cells in cutaneous malignant melanoma, experimental hybrids also displayed abundant dark pigment and autophagy, and had high levels of beta1,6-branched oligosaccharides. Whether or not darkly pigmented malignant melanoma cells originate from fusion with macrophages in vivo remains to be determined. In any event, pigmentation in melanoma, long considered as a secondary aspect of the malignancy, may be a visible warning that the cells have gained competence for invasion and metastasis.

Clinical evaluation of the lightening effect of cytidine on hyperpigmented skin.

BACKGROUND: Melanocytes, which reside in the basal layer of the epidermis, produce the pigment melanin in cytoplasmic organelles known as melanosomes. Melanosomes are transferred to keratinocytes which provide the color in our skin. Recently, Diwakar et  al reported the crucial roles of protein glycosylation in both melanogenesis and melanosome transfer to keratinocytes, and each was inhibited by the nucleotide cytidine. OBJECTIVE: The main objective of this study was to determine the clinical effects of topical application of cytidine to the hyperpigmented regions of the face in a group of human volunteers. METHODS: A randomized, vehicle-controlled study was conducted for 12 weeks on healthy Korean female subjects. Cytidine was formulated into the lotion at concentrations of 2%, 3%, and 4% (w/w) and compared to the vehicle control formulation. The clinical outcomes were evaluated by performing visual assessment grading, measuring melanin index, skin brightness, and skin color parameters. In vitro skin penetration studies were conducted using Franz cell chambers for the 2% cytidine test formulation. RESULTS: The test group showed significant improvements in the visual assessment scores, melanin index, skin brightness, and skin color compared to the control group. Although significant dose-dependent improvements were seen in the clinical study, the in vitro Franz cell studies indicated that the clinical efficacy and potency of cytidine might be further enhanced by formulating a better topical delivery system, which will be the goal of our future studies. CONCLUSIONS: This randomized, double-blind, 12-week clinical study successfully demonstrated the efficacy of cytidine on skin depigmentation in a dose-dependent manner.

Leukocyte⁻Cancer Cell Fusion-Genesis of a Deadly Journey.

According to estimates from the International Agency for Research on Cancer, by the year 2030 there will be 22 million new cancer cases and 13 million deaths per year. The main cause of cancer mortality is not the primary tumor itself but metastasis to distant organs and tissues, yet the mechanisms of this process remain poorly understood. Leukocyte⁻cancer cell fusion and hybrid formation as an initiator of metastasis was proposed more than a century ago by the German pathologist Prof. Otto Aichel. This proposal has since been confirmed in more than 50 animal models and more recently in one patient with renal cell carcinoma and two patients with malignant melanoma. Leukocyte⁻tumor cell fusion provides a unifying explanation for metastasis. While primary tumors arise in a wide variety of tissues representing not a single disease but many different diseases, metastatic cancer may be only one disease arising from a common, nonmutational event: Fusion of primary tumor cells with leukocytes. From the findings to date, it would appear that such hybrid formation is a major pathway for metastasis. Studies on the mechanisms involved could uncover new targets for therapeutic intervention.

Cytidine decreases melanin content in a reconstituted three-dimensional human epidermal model.

The process of melanin biosynthesis and its distribution throughout the skin is regulated by complex processes involving several enzymes in melanocytes. Recently, Diwakar et al. demonstrated that cytidine-a sialyltransferase inhibitor, 6'-sialyllactose (6'-SL) and 3'-sialyllactose (3'-SL) inhibited melanogenesis and melanosome transfer process. In this study, we have furthered this research, considering cytidine as a commercially viable and safe option over 6'-SL and 3'-SL. The efficacy of 2% w/v cytidine was studied in MelanoDerm™ skin equivalents in comparison with the positive control 1% w/v kojic acid and the vehicle control. Both the positive control and cytidine demonstrated a significant reduction in melanin content relative to the vehicle control. These experiments conclude that cytidine can effectively reduce melanin content in a skin equivalence assay and suggests that cytidine may be a good candidate for a skin lightening agent for human skin.

Beta1,6-branched oligosaccharides regulate melanin content and motility in macrophage-melanoma fusion hybrids.

In previous studies, fusion of peritoneal macrophages or blood monocytes with mouse melanoma cells produced hybrids with upregulated expression of the glycosyltransferase beta1,6-N-acetylglucosaminyltransferase V (GnT-V) and its enzymatic product, beta1,6-branched oligosaccharides. This correlated with marked increases in motility, metastatic potential and, surprisingly, melanin content. This study was designed to establish direct roles for beta1,6-branched oligosaccharides in melanogenesis and motility. The levels of beta1,6-branched oligosaccharides were lowered by transfecting beta1,4-N-acetylglucosaminyltransferase III, a competitive inhibitor of GnT-V. beta1,4-N-acetylglucosaminyltransferase III transfection virtually eliminated melanin production and markedly decreased chemotactic motility. This implied that the metastatic and melanogenic phenotypes in hybrids were each upregulated by beta1,6-branched oligosaccharides. Although roles for beta1,6-branched oligosaccharides in motility and metastasis have been reported previously, this is the first study to directly implicate these structures in melanogenesis. Although drawn from experimental models, the findings might explain the well known hypermelanotic regions of human cutaneous malignant melanoma as hypermelanotic cutaneous malignant melanoma cells are rich in beta1,6-branched oligosaccharides. They might also explain why melanogenesis pathways differ between malignant and normal melanocytes as GnT-V is a myeloid-associated enzyme that is aberrantly expressed in melanoma cells but not in normal melanocytes.

A Melanoma Lymph Node Metastasis with a Donor-Patient Hybrid Genome following Bone Marrow Transplantation: A Second Case of Leucocyte-Tumor Cell Hybridization in Cancer Metastasis.

BACKGROUND: Metastatic disease is the principal cause of mortality in cancer, yet the underlying mechanisms are not fully understood. Macrophage-cancer cell fusion as a cause of metastasis was proposed more than a century ago by German pathologist Prof. Otto Aichel. Since then this theory has been confirmed in numerous animal studies and recently in a patient with metastatic melanoma. METHODS: Here we analyzed tumor DNA from a 51-year-old man who, 8 years following an allogeneic BMT from his brother for treatment of chronic myelogenous leukemia (CML), developed a nodular malignant melanoma on the upper back with spread to an axillary sentinal lymph node. We used laser microdissection to isolate FFPE tumor cells free of leucocytes. They were genotyped using forensic short tandem repeat (STR) length-polymorphisms to distinguish donor and patient genomes. Tumor and pre-transplant blood lymphocyte DNAs were analyzed for donor and patient alleles at 15 autosomal STR loci and the sex chromosomes. RESULTS: DNA analysis of the primary melanoma and the nodal metastasis exhibit alleles at each STR locus that are consistent with both the patient and donor. The doses vary between these samples indicative of the relative amounts of genomic DNA derived from the patient and donor. CONCLUSION: The evidence supports fusion and hybridization between donor and patient cells as the initiator of metastasis in this patient. That this phenomenon has now been seen in a second case suggests that fusion is likely to play a significant role for melanoma and other solid tumor metastasis, perhaps leading to new avenues of treatment for this most problematic disease.

GnT-V expression and metastatic phenotypes in macrophage-melanoma fusion hybrids is down-regulated by 5-Aza-dC: evidence for methylation sensitive, extragenic regulation of GnT-V transcription.

Beta1,6-acetylglucosaminyltransferase V (GnT-V) forms beta1,6 branching on the trimannosyl terminus of N-glycans, allowing for the production of beta1,6Glc-NAc-bearing oligosaccharides. These are used by healthy myeloid cells and cancer cells alike for systemic migration. GnT-V has multiple glycoprotein substrates and thereby exerts global effects on cancer progression, characteristic of a master regulator of metastasis. Yet little is known of the regulation of GnT-V expression by tumor cells. It was previously reported that fusion of macrophages with Cloudman S91 mouse melanoma cells produced macrophage-melanoma hybrids with up-regulated GnT-V expression regarding mRNA and enzymatic activity. Majority of these hybrids showed increased chemotactic motility in vitro and elevated metastatic potential in vivo. Here we attempted to understand this at the molecular genetic level focusing on DNA hypermethylation as a potentially key step. Treatment of cells with 5-Aza-dC, an inhibitor of DNA methylation, resulted in decreased expression of GnT-V mRNA and beta1,6-branched oligosaccharides along with reduced glycosylation of LAMP-1, a major substrate for GnT-V. This was accompanied by reduced chemotactic motility of the cells. The results suggested that DNA hypermethylation in some fashion stimulated GnT-V expression. We thus investigated the promoter region of the GnT-V gene for hypermethylation of CpG islands, comparing macrophage-melanoma hybrids of low and high metastatic potential with the parental melanoma cell line. Genomic DNA after bisulfite modification amplified from this region showed identical sequences between the cell lines. The findings indicated that differential methylation of the promoter region of GnT-V gene was not responsible for its transcriptional control, rather, appeared to be controlled through a negative regulator, nm23, whose own expression was regulated by hypermethylation. Although our studies involved a highly experimental system, the results further suggest that by whatever mechanism, reduction of GnT-V activity through 5-Aza-dC treatment might provide a new approach towards prevention of metastatic progression.