Guichao Zeng

Role of tumor-associated gangliosides in cancer progression

Neuroectodermic tumors can mostly be characterized by the presence of tumor-associated glycosphingolipid antigens, such as gangliosides, defined by monoclonal antibodies. Recently, cumulative evidence indicates that gangliosides modify the biological effects of several trophic factors, in vitro and in vivo, as well as the mitogenic signaling cascade that these factors generate. The functional roles of gangliosides in tumor progression can be revisited: (i) ganglioside antigens on the cell surface, or shed from the cells, act as immunosuppressors, as typically observed for the suppression of cytotoxic T cells and dendritic cells, (ii) certain gangliosides, such as GD3 or GM2, promote tumor-associated angiogenesis, (iii) gangliosides strongly regulate cell adhesion/motility and thus initiate tumor metastasis, (iv) ganglioside antigens are directly connected with transducer molecules in microdomains to initiate adhesion coupled with signaling, and (v) ganglioside antigens and their catabolites are modulators of signal transduction through interaction with tyrosine kinases associated with growth factor receptors or other protein kinases. Given the potential importance of these sialylated gangliosides and their modulating biological behavior in vivo, further studies on the role of gangliosides are warranted.

Reduced cell migration, tumor growth and experimental metastasis of rat F-11 cells whose expression of GD3-synthase is suppressed.

We previously established a rat F‐11 cell line whose expression of ganglioside GD3 was inhibited by stable transfection of the anti‐sense vector against the GD3‐synthase gene, showing that specific inhibition of GD3‐synthase expression in tumor cells greatly reduced their growth rate in nude mice. Here, we report that down‐regulation of GD3‐synthase expression in anti‐sense–transfected F‐11 cells correlates with reduced cell migration and invasion in vitro and tumor growth and metastasis in vivo. When cultures were denuded of cells in a 1‐mm‐wide strip, the anti‐sense–transfected F‐11 cells migrated very slowly into the denuded area. Differences in migration between anti‐sense–transfected cells and control parental cells were easily apparent. In vitro invasion assay of F‐11 cells revealed a 3‐fold decrease in invasion ability from the GD3‐synthase–suppressed cells; colony formation in soft agar was not affected. Injection (i.v.) of control sense‐transfected and untransfected F‐11 cells resulted in multiple, large metastatic nodules in each of the 12 mice, whereas i.v. injection of anti‐sense–transfected F‐11 cells formed a single, small metastatic nodule in only 2 of the 8 nude mice. In addition, even if metastasis occurred, the anti‐sense–induced metastatic nodules were much smaller than the metastatic nodules formed by control F‐11 cells. These results demonstrate that suppression of GD3‐synthase expression, which results primarily in a marked decrease in the concentration of ganglioside GD3, greatly reduces cell spreading, invasion and both the incidence and growth rate of experimental metastasis of F‐11 cells. Int. J. Cancer 88:53–57, 2000.

Down-regulation of WNK1 protein kinase in neural progenitor cells suppresses cell proliferation and migration

WNK1, a Ser/Thr protein kinase, is widely expressed in many tissues. Its biological functions are largely unknown. Disruption of the WNK1 gene in mice leads to embryonic lethality at day 13, implicating a critical role of WNK1 in embryonic development. To investigate this potential function, we used antisense strategy to knock down the expression of WNK1 in a mouse neural progenitor cell line, C17.2. Down‐regulation of WNK1 in C17.2 cells greatly reduced cell growth. Addition of epidermal growth factor (EGF), a mitogen for C17.2 cells, had no effect on growth. The WNK1‐knockdown cells showed a flat and rounded morphology, characteristic of the immature and non‐differentiated phenotype of the progenitor cells; this was further demonstrated by immunostaining for the progenitor and neuronal markers. Migration of the WNK1‐knockdown C17.2 cells was reduced as tested in culture dishes or Matrigel‐covered chambers. Moreover, activation of extracellular signal‐regulated kinase (ERK1)/2 and ERK5 by EGF in the WNK1‐knockdown cells was suppressed. These results demonstrate a novel function of WNK1 in proliferation, migration, and differentiation of neural progenitor cells, likely by mechanisms involving activation of the mitogen‐activated protein (MAP) kinase ERK1/2 and/or ERK5 pathways.

Down-regulation of the expression of O-acetyl-GD3 by the O-acetylesterase cDNA in hamster melanoma cells : Effects on cellular proliferation, differentiation, and melanogenesis

Abstract : The composition of the gangliosides of hamster melanoma cells is closely related to their cellular growth and degree of differentiation, with slow‐growing, highly differentiated melanotic melanoma MI cells expressing GM3 and fast‐growing, undifferentiated amelanotic Ab melanoma cells having a preponderance of GD3 and O‐acetyl‐GD3. To study the putative function of O‐acetyl‐GD3, we established stably transfected AbC‐1 amelanotic hamster melanoma cells with O‐acetylesterase gene from influenza C virus to hydrolyze the O‐acetyl group from O‐acetyl‐GD3. The content of O‐acetyl‐GD3 in the transfected cells expressing O‐acetylesterase gene was reduced by >90%. These O‐acetyl‐GD3‐depleted cells differed from the parental ones in their cellular morphology, growth behavior, and melanogenesis activity. The absence of O‐acetyl‐GD3 in the transfected cells was accompanied by increased thick dendrite formation with an enlarged cell body, which is in striking contrast to the control cells, which were rounded and flattened, with few processes. Their growth was significantly slower than that of the control cells. They also demonstrated significantly lower tyrosinase activity and melanogenic potential. We suggest that the enhanced expression of melanoma‐associated O‐acetyl‐GD3 ganglioside may stimulate cellular growth and suppress certain differentiated phenotypes such as dendrite formation but not melanogenesis.

Cloning and transcriptional regulation of genes responsible for synthesis of gangliosides.

Ganglioside synthases are glycosyltransferases involved in the biosynthesis of glycoconjugates. A number of ganglioside synthase genes have been cloned and characterized. They are classified into different families of glycosyltransferases based on similarities of their amino acid sequences. Tissue-specific expression of these genes has been analyzed by hybridization using cDNA fragments. Enzymatic characterization with the expressed recombinant enzymes showed these enzymes differ in their donor and acceptor substrate specificities and other biochemical parameters. In vitro enzymatic analysis also showed that one linkage can be synthesized by multiple enzymes and one enzyme may be responsible for synthesis of multiple gangliosides. Following the cloning of the ganglioside synthase genes, the promoters of the key synthase genes in the ganglioside biosynthetic pathway have been cloned and analyzed. All of the promoters are TATA-less, lacking a CCAAT box but containing GC-rich boxes, characteristic of the house-keeping genes, although transcription of ganglioside synthase genes is subject to complex developmental and tissue-specific regulation. A set of cis-acting elements and transcription factors, including Sp1, AP2, and CREB, function in the proximal promoters. Negative-regulatory regions have also been defined in most of the promoters. We present here an overview of these genes and their transcriptional regulation.

Isolation and functional analysis of the promoter of the rat CMP- NeuAc: GM3 α2,8 sialyltransferase gene

A 2.1-kb 5′-flanking fragment of the rat CMP-NeuAc:GM3 α2,8 sialyltransferase (GD3-synthase) gene was cloned by the genomic walking procedure. The promoter activity of the fragment was assessed in F-11 cells by transient transfection and the locations for the basal and maximal promoter activities were defined. Primer extension analysis identified a transcription start site approximately 98 bp upstream of the ATG start codon. DNA sequence analysis of the promoter revealed a number of consensus binding sites for known transcription factors such as SP1, AP1, NFκB, C/EBP and TFIID, and a repeat GC–GT sequence motif seen for the formation of Z-type DNA. Both TATA and CCAAT boxes were not found in the promoter. Our results from deletion constructs suggested that both positive and negative cis-acting regulatory regions were present in this TATA-less promoter of the rat GD3-synthase gene.

Characterization of the promoter and the transcription factors for the mouse UDP-Gal: βGlcNAc β1,3-galactosyltransferase gene

Abstract Galβ1-3Gal-NAcβ1-4Gal(3-2αNeuAc)β1-4Glcβ1-1Cer (GM1) is one of the most extensively investigated gangliosides that plays critical roles in the development and functions of the nervous system. UDP-Gal:βGlcNAc β1,3-galactosyltransferase (Gal-T-II) is responsible for synthesis of ganglioside GM1 in the ganglioside biosynthetic pathway. To understand the transcriptional regulation of Gal-T-II gene expression, we cloned a 1448 bp 5′-flanking fragment from the mouse Gal-T-II gene. The transcriptional activity of the fragment was demonstrated in mouse Neuro-2a cells by a luciferase assay. The proximal 550 bp fragment showed the highest transcriptional activity as determined by a series of 5′-truncated constructs of the promoter. One negative regulatory region was also identified. Primer extension assay revealed a transcription initiation site approximately 242 bp upstream from the ATG translation start codon. Analysis of the promoter sequence revealed a number of potential binding sites for known transcription factors. To determine which transcription factors bind to the promoter, we carried out a systematic search for the binding proteins using the 1142 bp Gal-T-II promoter fragment containing both positive and negative regulatory regions in a combination of DNA pull-down assay and transcription factor array analysis. Twenty-seven transcription factors bound to consensus sites in the promoter region. In addition, four other factors without consensus binding sites in this region were also recruited, possibly as components of transcription factor complexes. These data indicate that the basal regulation of Gal-T-II gene transcription involves multiple transcription factors, some of which may be present in complexes.

Variations in gene expression patterns correlated with phenotype of F-11 tumor cells whose expression of GD3-synthase is suppressed

Alteration in ganglioside composition in F-11 cells by suppression of GD3-synthase gene expression resulted in greatly reduced tumor growth and metastasis when the cells were injected into nude mice. To identify genes whose expression is correlated with the decreased level of ganglioside GD3, we analyzed gene expression profiles of the GD3-suppressed F-11 cells and the control F-11 cells using DNA microarrays. We identified a set of GD3-related genes, most of which are involved in tumor growth and development. The genes that define the proliferation-transformation signature are down-regulated, such as creatine kinase-B (CKB), upstream stimulation factor 1 (USF-1), type II cAMP-dependent protein kinase regulatory subunit (RII PKA), and tyrosine hydroxylase (TH). On the other hand, the genes that define the differentiation-reverse transformation signature are up-regulated, including p160 myb-binding protein (P160), brain factor-2, insulin-like growth factor-binding protein (IGFBP), and growth/differentiation factor 11. Transcriptional levels of the genes that showed the most distinct GD3-related expression change were validated by reverse transcription-polymerase chain reaction (RT-PCR). Defining GD3-related genes may lead to identification of clinically relevant therapeutics and to understanding of the mechanism(s) by which ganglioside GD3 affects tumor growth and metastasis.