Quantitative analyses of GFRalpha-1 and GFRalpha-2 mRNAs and tyrosine hydroxylase protein in the nigrostriatal system reveal bilateral compensatory changes following unilateral 6-OHDA lesions in the rat.

Copy numbers of mRNAs for GFRalpha-1 and GFRalpha-2, the preferred receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) were determined by real-time quantitative RT-PCR (QRT-PCR). Receptor expression was assessed in striatum (ST) and substantia nigra (SN) of normal rats and rats acutely or progressively lesioned by 6-OHDA injected into the medial forebrain bundle or ST, respectively. GFRalpha-1 mRNA was clearly detected in normal ST. In normal SN, significantly higher expression of both receptors was observed. At 4 weeks after acute lesion, GFRalpha-2 mRNA was markedly decreased in SN bilaterally, whereas GFRalpha-1 mRNA in SN and ST was not affected. A progressive lesion resulted in a progressive decrease of GFRalpha1 mRNA in ST bilaterally. In SN, levels of GFRalpha-1 mRNA were not significantly affected by a progressive lesion, whereas GFRalpha-2 mRNA was markedly decreased bilaterally. Quantitative western blotting standardized against tyrosine hydroxylase (TH) protein from PC12 cells revealed the expected decrease in TH protein in lesioned SN, but also significant increases in TH protein in contralateral, unlesioned SNs at 4 weeks after both acute and progressive lesions. These data suggest that previously unrecognized compensatory changes in the nigrostriatal system occur in response to unilateral dopamine depletion. Since the changes observed in receptor expression did not always parallel loss of dopamine neurons, cells in addition to the nigral dopamine neurons appear to be affected by a 6-OHDA insult and are potential targets for the neurotrophic factors, GDNF and NTN.

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR.

Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.

Microarray analysis detects novel Pax3 downstream target genes.

Pax3 is a transcription factor that is required for the development of embryonic neural tube, neural crest, and somatic derivatives. Our previous study (Mayanil, C. S. K., George, D., Mania-Farnell, B., Bremer, C. L., McLone, D. G., and Bremer, E. G. (2000) J. Biol. Chem. 275, 23259-23266) reveals that overexpression of Pax3 in a human medulloblastoma cell line, DAOY, resulted in an up-regulation in alpha-2,8-polysialyltransferase (STX) gene expression and an increase in polysialic acid on neural cell adhesion molecule. This finding suggests that STX might be a previously undescribed downstream target of Pax3. Because Pax3 is important in diverse cellular functions during development, we are interested in the identification of additional downstream targets of Pax3. We utilized oligonucleotide arrays and RNA isolated from stable Pax3 transfectants to identify potential target genes. A total of 270 genes were altered in the Pax3 transfectants as compared with the vector control and parental cell line. An independent analysis by cDNA expression array and real-time quantitative polymerase chain reaction of several genes confirmed the changes observed by the oligonucleotide microarray data. Of the genes that displayed significant changes in expression, several contain paired and homeodomain binding motifs of Pax3 in their promoter regions. Using promoter-luciferase reporter transfection assays and electromobility shift assays, we showed at least one previously undescribed downstream target, STX, to be a biological downstream target of Pax3. Thus we report several previously undescribed candidate genes to be potential downstream targets of Pax3.

Ganglioside modulates ligand binding to the epidermal growth factor receptor.

Whereas previous investigations have shown that pharmacologic addition of gangliosides inhibits keratinocyte proliferation by downregulating epidermal growth factor receptor phosphorylation, the underlying biochemical basis and physiologic relevance are unknown. Using Scatchard and displacement plots, we have shown that supplemental purified gangliosides decrease the binding of (125)I-labeled epidermal growth factor to keratinocyte-derived SCC12 cells. Conversely, SCC12 cells transfected with sialidase and thus depleted of gangliosides show increased ligand binding to the epidermal growth factor receptor, which is consistent with their increased proliferation in response to epidermal growth factor and transforming growth factor-alpha, and increased phosphorylation of the epidermal growth factor receptor, and downstream signal transduction pathway components. The mechanism of the altered binding appears to involve primarily decreased numbers of available receptors within the intact membrane, but not altered receptor protein expression. These studies provide evidence that the effect of gangliosides on keratinocyte proliferation results, at least in part, from the direct binding of ganglioside to the receptor and disruption of the receptor-ligand interaction. Manipulation of membrane ganglioside content may be a powerful new means to alter epidermal growth factor receptor-dependent cell proliferation.

Overexpression of murine Pax3 increases NCAM polysialylation in a human medulloblastoma cell line.

Polysialic acid (PSA) is a developmentally regulated carbohydrate found primarily on neural cell adhesion molecules (NCAM) in embryonic tissues. The majority of NCAM in adult tissues lacks this unique carbohydrate, but polysialylated NCAM (PSA-NCAM) is present in adult brain regions where neural regeneration persists and in some pediatric brain tumors such as medulloblastoma, which show greater propensity for leptomeningeal spread. Pax3, a developmentally regulated paired homeodomain transcription factor, is thought to be involved in the regulation of neural cell adhesion molecules. Overexpression of murine Pax3 into a human medulloblastoma cell line (DAOY) resulted in an increase in NCAM polysialylation and a 2-4-fold increase in alpha2, 8-polysialyltransferase type II mRNA levels. No difference was observed in alpha2,8-polysialyltransferase type IV message. The addition of PSA to NCAM changed the adhesive behavior of these Pax3 transfectants. Transfectants expressing high PSA-NCAM show much less NCAM-dependent aggregation than those with less PSA-NCAM. In addition, Pax3 transfectants having high PSA-NCAM show heterophilic adhesion involving polysialic acid to heparan sulfate proteoglycan and agrin. These observations suggest that a developmentally regulated transcription factor, Pax3, could affect NCAM polysialylation and subsequently cell-cell and cell-substratum interaction.

Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431.

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.

Expression of bisecting GlcNAc in pediatric brain tumors and its association with tumor cell response to vinblastine.

Increased expression of the bisecting GlcNAc has been correlated with tumor progression in several experimental tumor models. Its expression and function in brain tumors are, however, not yet known. In this study, we investigated expression of the bisecting GlcNAc structure in a series of pediatric brain tumors and its relationship to tumor response to vinblastine. A plant lectin (E-PHA) that recognizes the bisecting GlcNAc structure was used for detection of this molecule in a total of 90 pediatric brain tumors and normal brain tissue specimens. Our results showed that, whereas E-PHA staining was undetectable in the normal brain tissue, pediatric brain tumor specimens exhibited different levels of reactivity. Lectin staining was particularly prominent in high-grade astrocytomas (73%) and ependymomas (72%). In astrocytomas, there was a positive correlation with the tumor grade, which suggests that the bisecting GlcNAc may be of particular interest as a tumor marker for diagnosis and/or prognosis. By using a human glioma cell culture model, we have found that treatment of these cells with E-PHA lectin enhances their sensitivity to vinblastine. E-PHA interacted directly with the drug transporter P-glycoprotein and inhibited its drug efflux function. In a drug-resistant glioma cell line transfected with the mdr1 gene, drug resistance was reversed by E-PHA. Our findings indicate that: (a) expression of the bisecting GlcNAc in pediatric brain tumors may have a potential relevance as a tumor marker; and (b) glioma response to chemotherapy may be modulated through the bisecting GlcNAc.

Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431.

Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.

Growth factor receptors as targets for therapy in pediatric brain tumors.

Growth factor receptors (GFRs) have been described as overexpressed in several types of brain tumors. Overexpression of these transmembrane proteins is considered to be an important part of tumorigenesis. Genetic as well as epigenetic modulation of the receptors have to be considered when trying to understand the role of GFRs in tumors or as targets for tumor therapy. GFR function can be modulated by membrane components (e.g. gangliosides) or by the change in receptor glycosylation. These types of changes and the occurrence of the expression of mutated receptor expressed in tumor cell can result in altered signaling. In this review, we have focused on GFRs, their expression and mutations in brain tumors. Recently the correlation between GFR expression and patient outcome has suggested that these tyrosine kinases and their signaling might play a decisive role in the course of patients with brain tumors. The importance of GFRs as possible targets for brain tumor therapy is also discussed.

alpha2,6-Sialyltransferase gene transfection into a human glioma cell line (U373 MG) results in decreased invasivity.

Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP-NeuAc:Galbeta1,4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated alpha3beta1 integrin. Adhesion-mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion-mediated signal transduction and block glioma cell invasivity in vitro.

alpha2,3-sialyltransferase mRNA and alpha2,3-linked glycoprotein sialylation are increased in malignant gliomas.

CMP-NeuAc: Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase (alpha2,3-ST) mRNA was expressed in human glioma specimens, human fetal astrocytes, and a panel of brain tumor cell lines. Maackia amurensis agglutinin staining revealed the presence of alpha2,3-linked sialic acids on glioma cell surfaces and extracellular matrices whereas normal human adult astrocytes were negative. Increased expression of alpha2,3-linked glycoprotein sialylation may play a role in glial tumorigenesis.

Gene transfection-mediated overexpression of beta1,4-N-acetylglucosamine bisecting oligosaccharides in glioma cell line U373 MG inhibits epidermal growth factor receptor function.

N-linked oligosaccharides appear to be important for the function of the epidermal growth factor (EGF) receptor. In a previous study (Rebbaa, A., Yamamoto, H., Moskal, J. R., and Bremer, E. G. (1996) J. Neurochem. 67, 2265-2272), we showed that binding of the erythroagglutinating phytohemagglutin lectin from Phaseolus vulgaris to the bisecting structures on the EGF receptor from U373 MG glioma cells blocked EGF binding and receptor autophosphorylation. In this study we examined the consequences of overexpression of the bisecting structure on the EGF receptor by gene transfection of U373 MG cells with the N-acetylglucosaminyltransferase III (GnT-III). This modification leads to a significant decrease in EGF binding and EGF receptor autophosphorylation. In addition, the cellular response to EGF was found to be altered. Proliferation of U373 MG cells in serum-free medium is inhibited by EGF. In contrast, proliferation of the GnT-III-transfected cells was stimulated by EGF. These data demonstrate that changes in EGF receptor glycosylation by GnT-III transfection reduces the number of the active receptors in U373 MG cells and that this change results in change in the cellular response to EGF.

Binding of erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris to the epidermal growth factor receptor inhibits receptor function in the human glioma cell line, U373 MG.

Little is known about the role of the N-linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N-linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N-linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.

Ganglioside GM3 inhibition of EGF receptor mediated signal transduction.

Ganglioside GM3 is a membrane component that has been described to modulate cell growth through inhibition of EGF receptor associated tyrosine kinase. In order to determine if the inhibition of cell growth by this ganglioside is specifically mediated through EGF receptor signaling, the effects of GM3 on key enzymes implicated in EGF signaling were determined and compared to another inhibitor of the EGF receptor kinase. Treatment of A1S cells in culture by GM3 or a tyrosine kinase inhibitor, leflunomide, led to the inhibition of MAP kinase and PI3 kinase activities. There was no detectable effect on phosphotyrosine phosphatases. In a cell free system, however, GM3 had no effect on the activity of these signaling intermediates. Leflunomide was able to directly inhibit MAP kinase activity. GM3 and leflunomide were also found to act differently on the expression of the early immediate genes. The expression of c-fos and c-jun was inhibited by both GM3 and leflunomide. The expression of c-myc, however, was only inhibited by leflunomide. These findings suggest that the action of GM3 on cell growth and signaling is specifically mediated by EGF receptor and that this ganglioside does not act directly on the intracellular intermediates of EGF receptor signaling. In addition, soluble small molecule tyrosine kinase inhibitors such as leflunomide can directly affect the activity of MAP kinases and possibly other signaling intermediates. The direct effects of leflunomide on signaling intermediates may explain the differential effects of leflunomide and GM3 on gene expression and cell growth.

Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosuppressive agent, leflunomide.

Leflunomide, a novel immunosuppressive drug, is able to prevent and reverse allograft and xenograft rejection in rodents, dogs, and monkeys. It is also effective in the treatment of several rodent models of arthritis and autoimmune disease. In vitro studies indicate that leflunomide is capable of inhibiting anti-CD3- and interleukin-2 (IL-2)-stimulated T cell proliferation. However, the biochemical mechanism for the inhibitory activity of leflunomide has not been elucidated. In this study, we characterized the inhibitory effects of leflunomide on Src family (p56lck and p59fyn)-mediated protein tyrosine phosphorylation. Leflunomide was able to inhibit p59fyn and p56lck activity in in vitro tyrosine kinase assays. The IC50 values for p59fyn (immunoprecipitated from either Jurkat or CTLL-4 cell lysate) autophosphorylation and phosphorylation of the exogenous substrate, histone 2B, were 125-175 and 22-40 microM respectively, while the IC50 values for p56lck (immunoprecipitated from Jurkat cell lysates) autophosphorylation and phosphorylation of histone 2B were 160 and 65 microM respectively. We also demonstrated the ability of leflunomide to inhibit protein tyrosine phosphorylation induced by anti-CD3 monoclonal antibody in Jurkat cells. The IC50 values for total intracellular tyrosine phosphorylation ranged from 5 to 45 microM, with the IC50 values for the zeta chain and phospholipase C isoform gamma 1 being 35 and 44 microM respectively. Leflunomide also inhibited Ca2+ mobilization in Jurkat cells stimulated by anti-CD3 antibody but not in those stimulated by ionomycin. Distal events of anti-CD3 monoclonal antibody stimulation, namely, IL-2 production and IL-2 receptor expression on human T lymphocytes, were also inhibited by leflunomide. Finally, tyrosine phosphorylation in CTLL-4 cells stimulated by IL-2 was also inhibited by leflunomide. These data collectively demonstrate the ability of leflunomide to inhibit tyrosine kinase activity in vitro, and suggest that inhibition of tyrosine phosphorylation events may be the mechanism by which leflunomide functions as an immunosuppressive agent.

Preferential binding of the epidermal growth factor receptor to ganglioside GM3 coated plates.

Ganglioside GM3 has been shown to modulate epidermal growth factor receptor function. These observations have lead to the hypothesis that GM3 may bind to the epidermal growth factor receptor. An enzyme-linked immunosorbant assay was designed to test this hypothesis. In these experiments, receptor-rich vesicle preparations were incubated with ganglioside GM1 or GM3 coated 96-well microtiter plates and the amount of bound receptor was compared. Plates coated with GM3 consistently bound more epidermal growth factor receptor than did GM1 coated plates. The binding of epidermal growth factor receptors to GM3 coated wells appeared to be specific and saturable. These results suggest that GM3 may modulate epidermal growth factor receptor function owing to a specific association of the two molecules.

Altered RNA turnover in carcinogenesis. The diagnostic potential of modified base excretion.

Excretion of urinary modified nucleosides is frequently elevated in patients with oncogenic disease. Increases of urinary pseudouridine excretion are now demonstrated in patients with a variety of brain tumors. The potential use of urinary modified base excretion as a cancer marker is discussed and possible sources of the elevated nucleosides are detailed. The specific steps in RNA metabolism that result in increased levels of RNA nucleoside excretion are poorly understood. This knowledge will be necessary to understand the molecular mechanism and the clinical significance of urinary nucleoside excretion in treatment and diagnosis of oncogenic disease.

Inhibition of the epidermal growth factor receptor tyrosine kinase activity by leflunomide.

The active metabolite of leflunomide, A77 1726 inhibits the proliferation of a variety of mammalian cell lines in culture. Epidermal growth factor (EGF)-dependent proliferation is inhibited by A77 1726 at an effective dose of 30-40 microM. A77 1726 appears to directly inhibit the EGF receptor tyrosine-specific kinase activity both in intact cells and purified EGF receptors at the same effective dose. These data suggest that leflunomide inhibits cellular proliferation by the inhibition of tyrosine-specific kinase activities.

Gangliosides inhibit platelet-derived growth factor-stimulated receptor dimerization in human glioma U-1242MG and Swiss 3T3 cells.

We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) in a dose-responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [35S]Methionine-labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross-linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti-PDGFR antibody. Using both Swiss 3T3 and human glioma U-1242MG cells, western blots with anti-PDGFR and anti-phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350-kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF-mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor-mediated cell functions.

Ganglioside GM3 inhibits the proliferation of cultured keratinocytes.

Ganglioside GM3 is the predominant ganglioside of keratinocyte membranes. It has been proposed in other cell types that GM3 may participate in the regulation of cell proliferation. To examine the role of GM3 in keratinocyte proliferation, purified GM3 was added to cultured keratinocytes from normal foreskin, from lesional skin of patients with psoriasis and ichthyosis, and to cutaneous squamous carcinoma cell lines. Supplemental GM3 inhibited the growth of all cultured keratinocytes in a dose-dependent manner at concentrations of 10-100 microM. Keratinocytes from patients with psoriasis and ichthyosis were most sensitive to the inhibitory effects of GM3, and confluent undifferentiated keratinocytes were least sensitive. No change in differentiation was noted after addition of GM3. GD3, 9-0-acetyl-GD3, and GD1b also inhibited keratinocyte proliferation. Gangliosides GM1 and GD1a and sialic acid had little effect. Addition of 50 microM 3H-GM3 to cultured keratinocytes resulted in 1.7 times the amount of cellular GM3. These data suggest that hematoside (GM3) and "b" pathway gangliosides (GD3, GD1b), generated by the preferential activation of sialyltransferase II versus N-acetylgalactosaminyltransferase, may be involved in control of keratinocyte growth but not of differentiation.