Báo cáo khoa học: Ras oncogene induces b-galactoside a2,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter pptx

Ras oncogene induces b-galactoside a2,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter Martin Dalziel 1 , Fabio Dall’Olio 2 , Arron Mungul 3 ,Ve ´ ronique Piller 1 and Friedrich Piller 1 1 Centre de Biophysique Mole ´ culaire, CNRS UPR 4301 affiliated with the University of Orle ´ ans and INSERM, Orle ´ ans, France; 2 Dipartimento di Patologia Sperimentale, Universita ` di Bologna, Italy; 3 Cancer Research UK, Breast Cancer Biology Group, Guy’s Hospital, London, UK Several oncogenic proteins are known to influence cellular glycosylation. In particular, transfection o f codon 12 point mutated H-Ras increases CMP-Neu5Ac: Galb1,4GlcNAc a2,6-sialyltransferase I (ST6Gal I) activity in rodent fibroblasts. Given that Ras mediates its effects through at least three secondary effector pa thways (Raf, RalGEFs and PI3K) a nd that transcriptional c ontrol of m ouse ST6Gal I is achieved by the selective use of multiple promoters, we attempted t o i dentify which of these parameters a re i nvolved in linking the R as signal to ST6Gal I gene transcription in mouse fibroblasts. Transformation by human K-Ras o r H-Ras ( S12 a nd V12 point mutations, respectively) results in a 1 0-fold increase in ST6Gal I mRNA, but no alter ation in the expression of related sialyltran sferases. Using an indu- cible H-Ras V12 expression system, a direct causal link be- tween activated H-Ras expression and elevated ST6Gal I mRNA was demonstrated. The accumulation of the ST6Gal I transcript in response to activated Ras was accompanied b y a n i ncrease o f a2 ,6-sialylt ransferase activity and of Neu5Aca2,6Gal at the cell s urface. Results obtained with H-Ras V12 partial loss of function mutants H -Ras V12S35 (Raf signal only), H-Ras V12C40 (PI3-kinase s ignal only) and H-Ras V12G37 (RalGEFs signal only) suggest that the H-Ras induction of the m ouse ST6Gal I gene (Siat1) transcription is primarily routed through RalGEFs. 5¢-Rapid amplifica- tion of cDNA ends analysis demonstrated that the increase in ST6Gal I mRNA upon H-Ras V12 or K-Ras S12 transfec- tion is mediated by the Siat1 housekeeping promoter P3-associated 5¢ untranslated exons. Keywords: oncogenic Ras; sialyltransferase; RalGEF; housekeeping p romoter. The human Ras gene family is composed of H-Ras, K-Ras and N-Ras [1] encoding three related p21 Ras proteins that function as small GTPases bound to the plasma membrane through lipid anchors. They effectually link extracellular, ligand-generated s ignals to cytoplasmic signalling cascades through their ability to bind in a GTP- dependent manner various effector proteins and thus altering their localization, protein–protein interaction and activity. Mutations in the Ras genes at codons 12, 13 and 61 render the Ras proteins constitutively active in their GTP-bound form [2]. Those mutations lead to oncogenic Ras and are found in approximately 30% of all human cancers [3], though the frequency varies with different cancer types. The presence of constitutively activated H-Ras V12 in primary cell cultures o f human tumours has been linked t o an increase in N-glycan branching a nd sialylation [4,5]. Subsequent studies with H-Ras V12 -transfected rat fibro- blasts identified the enh anced activities of the CMP- Neu5Ac:Galb1,4GlcNAc a2,6-sialyltransferase (ST6Gal I, EC 2.4.99.1) [6–9], a nd N-acetylglucosaminyl transferase V [10] as the two likeliest effectors of these glycosylation changes. Increased levels of ST6Gal I have been identified in breast [11], c olon [12–14], cervical [15] and prostate cancer [16]. Elevated ST6Gal I activity has also been linked to markers of poor prognosis in breast cancer patients [11], to the d ifferentiation state of t he tumour in prostate [16] and colon [17] cancer, to secondary local colon tumour reoccurrence [18] and finally to metastasis in both cervical [19] and colon cancer [20]. Moreover, ST6Gal I over-expression and inhibition experiments have shown Correspondence to F. Piller, Centre de Biophysique Mole ´ culaire, rue Charles Sadron, F45071 Orle ´ ans Ce ´ dex 02, France. Fax: +33 238 631517, Tel.: +33 238 257643, E-mail: piller@cnrs-orleans.fr Abbreviations: FACS, fluorescence activated cel l so rt er; FB S, fe tal bovine serum; FITC, fluorescein isothiocyanate; Gal, galactose; Glc- NAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; MAA, Maackia amurensis agglut i nin; M ES, 2 -morpholino ethanesulfonic acid; Neu5Ac, N-acetylneuraminic acid; RACE, rapid amplification of cDNA ends; SNA, Sambucus nigra agglutinin; Siat1, Mouse ST6Gal I gene; ST3Gal I, CMP-Neu5Ac:Galb1,3GalNAc a2,3-sialyltransferase I; ST3Gal II, CMP-Neu5Ac:Galb1,3GalNAc a2,3- sialyltransferase II; ST3Gal III, CMP-Neu5Ac:Galb1,(3)4GlcNAc a2,3-sialyltransferase; ST3Gal IV, CMP-Neu5Ac:Galb1,3GalNAc/ Galb1,4GlcNAc a2,3-sialyltransferase; ST6Gal I, CMP- Neu5Ac:Galb1,4GlcNAc a2,6-sialyltransferase I; ST6Gal II, CMP- Neu5Ac:Galb1,4GlcNAc a2,6-sialyltransferase II; ST6GalNAc I, CMP-Neu5Ac:GalNAc a2,6-sialyltransferase I; Tc, tetracycline; UT, untranslated. Enzymes: CMP-Neu5Ac:Galb1,4Glc NAc a2,6-si alyltran sfera se (ST6GalI, EC 2.4.99.1); CMP-Neu5Ac:GalNAc a2,6-sialyltrans ferase I, (ST6GalNAcI, EC 2.4.99.3); CMP-Neu5Ac:Galb1,3GalNAc a2,3- sialyltransferase I (ST3Gal I , EC 2.4.99.6). (Received 1 2 May 20 04, revised 2 July 2004, accepted 12 J uly 2004) Eur. J. Biochem. 271, 3623–3634 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04284.x that ST6Gal I exhibits a profound influence on the meta- static potential of tumour cells in vitro [21,22]. Interestingly, increased cell surface a2,6-sialylation, identical t o those reported to be the result o f activated Ras expression, also influences parameters thought to be important for c ellular metastatic ability, such as motility [9] and b1- integrin activity [23]. ST6Gal I catalyses the biosynthetic transfer of Neu5Ac from CMP-Neu5Ac to the nonreducing end of type II N-acetyllactosamine structures to form Neu5Aca2,6Gal- b1,4GlcNAc-R on glycoproteins and glycolipids [24]. Although a second member of the ST6Gal family, ST6Gal II, has recently been identified [25], the combina- tion of weak and t issue restrictive expression, primarily in adult b rain [26], leaves S T6Gal I as the dominant ST6Gal enzyme in all adult tissues. This is consistent with gene kno ckout experiments in mice, where loss of ST6Gal I results in an almost complete absence of Neu5Aca2,6Galb1,4GlcNAc-R in adult and fetal tissue [27]. Transcriptional control of the mouse ST6Gal I gene Siat1 is regulated during development a nd differentiation by the s elective usage of multiple promoter regions. Differential utilization of these promoters results in mature transcripts that are identical except for their untranslated 5¢ (5¢UT) leader se quences. A t l east four Siat1 promoters a re known: P1 controls ST6Gal I expression in live r [28], P2 i n B-lymphocytes [29], P3 is used to achieve multi-tissue housekeeping expression [29] and finally P4 which is active in the mammary gland during lactation [30]. In this report, we have studied the molecular events that take place between codon 12 mutated H-Ras or K-Ras expression and elevated ST6Gal I activity in NIH3T3 cells. We present data i ndicating that the R as signal, mediated b y RalGEFs, leads directly to an accumulation of ST6Gal I mRNA, transcribed from the Siat1 h ousekeeping promoter P3, ultimately resulting in enhanced ST6Gal I enzyme activity and cell surface a2,6-sialylation. Experimental procedures Materials [ 32 P]dCTP[aP] (3000 CiÆmmol )1 ), CMP- [ 14 C]Neu5Ac (286 mCiÆmmol )1 ), Random primer based radiolabelling Mega prime kit, MicroSpin G-50 DNA purification columns and positively charged nylon membrane were purchased from Amersham (Saclay, France). Plasmid pCR2.1, pcDNA3.1, Escherichia coli TOP10F strain, Trizol and M13 reverse primer were from Invitrogen (Cergy-Pontoise, France). Marathon rapid amplification of cDNA ends (RACE) kit and human leukaemia cell line K562 poly(A) + RNA were from Clontech (Palo Alto,CA,USA).TheDNAgelextractionkitwasfrom Qiagen (Coutaboeuf, France). Wizard DNA miniprep kits were from Promega (Charbonnie ` res-les-Bains, France). Biotinylated Sambucus nigra agglutinin (SNA) and Maackia amurensis (MAA) lectins were from Vector Laboratories (Burlingame, CA, USA). Galb1,4GlcNA cb- OCH 2 Ph was a generous gift of C. LeNarvor and C. Auge ´ ,Universite ´ Paris-Sud, O rsay, France. DMEM and fetal bovine serum (FBS) were from BioWest (Paris, France). cDNA probes and DNA constructs Mouse ST6Gal I exon II (750 bp PstI fragment) genomic DNA and ST3Gal I, II, III and IV PCR fragments were provided by J. Lau (Roswell Park Cancer Institute, Buffalo, NY, USA). Mouse ST3Gal I, II, III and IV probes were amplified using the following primer pairs. ST3Gal I (product size 241 bp): p125 (sense), 5 ¢-ACCTCACCTTCT TCCTGCTCTTC-3¢ and p128 (antisense), 5¢-AGCGTTG TGGACTGTCAGCA-3¢;ST3GalII(921bp):pPE1 (sense), 5¢-GGCTATTCAGAATTCCAGCGCCTCGGC AAGGA-3¢ and pPE2 (anti sense), 5¢-TGCCAGAC CCTCGAGTGACTGGTTCTGAAGGCGCTCAGG-3¢; ST3Gal III (633 bp): p138 (sense), 5¢-CCCTCTGCCT CTTCCTGGTC-3¢ and p139 (antisense), 5¢-TCGTTCAT ATTGCTCAGGTCG-3¢; S T3Gal IV (449 bp): p136 (sense), 5¢-CCTGGCTCTGGTCCTTGTTGT-3 ¢ and p137 (antisense), 5¢-AGCCCACATCTCCCTCGTAGC -3¢. ST3Gal I, II, III and IV PCR products were cloned into pCR2.1, propagated in E. coli TOP10F, confirmed using M13 reverse primer (M13r) sequencing (MWG Biotech, Courtaboeuf, France) and released by EcoRI digestion for use as northern probes. Mouse ST6GalNAc I cDNA was obtained from S . Tsuji, The Glycoscience I nstitute, T okyo, Japan. A 1.1 kb ST6GalNAc I probe was r eleased by HindIII/EcoRV digestion. A 604 nt ST6Gal II cDNA probe, spanning the first predicted coding exon in the mouse chromosome 17 sequence E NSMUSG00000024172 (22918721–23318720), downloaded from the Wellcome Trust Sanger Institute mouse genome server (http:// www.ensembl.org/Mus_musculus), was amplified by PCR from mouse genomic DNA using the primer pair mst172 (5¢-GGATGGCACCGGCAGACATG-3¢) and mst171 (5¢-CACAGAAATGGGATCAGGCC-3¢). Both human H-Ras and K-Ras cDNA were obtained from Cancer Research UK. Human H-Ras cDNA probe was isolated from an EcoRI digestion of H-Ras V12 cDNA cloned into the EcoRI site of pcDNA3.1. The 0.4 kb human K-Ras cDNA probe (encoding the 3¢ region of the ORF) was isolated by EcoRI digestion of a 1.1 k b human K-Ras S12 cDNA cloned into the EcoRI site of pcEXV-3. The human V12 H -Ras partial loss of function mutants H-Ras V12S35 , H-Ras V12C40 (bothinpcDNA3.1)andH-Ras V12G37 (in pSG5) were obtained from A. Scibetta (Cancer Research UK, Guy’s Hospital, London, UK) [31,32]. The H-Ras V12G37 cDNA was r ecloned into p cDNA3.1 using EcoRI. Finally, mouse 18S cDNA was purchased from Ambion (Huntingdon, UK). Identification and cloning of mouse ST6Gal II Using the Wellcome Trust Sanger Institute mouse genome server database a second ST6Gal family member, ST6Gal II was identified on chromosome 17 (ENS- MUSG00000024172) as presenting significant homology to the ST6Gal I gene (Siat1) on chromosome 16. Subsequently, a 604 nt genomic probe spanning the first coding exon was generated by PCR. Of the five potential ST6Gal II coding exons, exon I contains the least homology to Siat1 . When t his probe was hybridized to multiple tissue total RNA extracted from a single male C57Bl6 mouse (stomach, whole brain, spleen, kidney, 3624 M. Dalziel et al. (Eur. J. Biochem. 271) Ó FEBS 2004 testis, large intestine, small intestine and liver), only the whole brain sample showed any d etectable signal (data not shown). A full-length cDNA ( 1.6 kb) was then amplified by PCR using Pfu polymerase from whole mouse brain cDNA, cloned into pCR2.1 and sequenced. The sequence was consistent with the predicted gene structure contained within the ENSMUSG00000024172 genomic sequence. In brief, five exons encoding a 524 amino acid sialyltransferase (containing L, S and VS motifs) with 32% overall primary sequence homology to mouse ST6Gal I (48% within the catalytic C-terminal and 18% within the N-terminal halves of t he protein). This cDNA was re-cloned into pcDNA3.1-Flag and transiently expressed in Chinese hamster ovary cells, which then exhibit high amounts of a2,6-linked Neu5Ac on cell surface N -glycans, as revealed by SNA staining (data not shown). Although the exact acceptor substrate specificity was not determined, these observations were deemed suffi cient for the validation of both the identity of the ENSMUSG00000024172 sequence as the mouse ST6Gal II sequence (thus named Siat2) and the u se of the 604 nt PCR f ragment as a specific ST6Gal II probe in subsequent experiments. While this work was in progress, the sequences of the human and mouse ST6Gal II cDNAs were reported and confirmed our results [25,26]. Cell lines All cells were grown at 3 7 °C in a humidified atmosphere of 5% (v/v) CO 2 , in DMEM (Gibco)/10% (v/v) FBS containing 100 UÆmL )1 penicillin, 100 lgÆmL )1 glutamate, 100 lgÆmL )1 streptomycin and 1.25 lgÆmL )1 amphoteri- cin B. Mouse cell lines 3T3 and K-Ras S12 ,whichare NIH3T3 parental and NIH3T3 transfected with activated human Ras cDNA, respectively, were obtained from Cancer Research UK. H-Ras V12 , which is NIH3T3 transfected with codon 12 position 2 point mutation GGC to GTC (Gly fi Val), activated mutant of human H-Ras, and mock transfected control line 3T3pB322 were obtained from E. He ´ bert (CBM, Orle ´ ans, France) [33]. The presence of oncogenic Ras and the R as variant as well as nature of the mutation were controlled by RT- PCR and nucleotid e sequencing. The 410.4 cell line (mouse mammary gland carcinoma cell line) was provi- ded by B. Miller (Michigan Cancer Foundation, Detroit, MI, USA) [34]. Th e NIH3T3 cell line s transfected w ith tetracycline (Tc) repressed H-Ras V12 construct (mib125), constitutive H-Ras V12 (mib128) and the parental NIH3T3 line (mib35) [35], were obtained from B. Willumsen (University of Copenhagen, Denmark). Stable transfections Plasmid DNA (V12-S35, V12-C40 and V12-S35, all in pcDNA3.1) used for transfections was purified using a Wizard Plus DNA purification system (Promega) and linearized with PvuI. Transfection of mouse NIH3T3 cells was performed by electroporation using a G ene P ulser electroporator (Bio-Rad, Hercules, CA, U SA) a t 960 lFD, 100 W, 0.25 V . Stable transfectants were selected with 0.5–0.75 mgÆmL )1 geneticin (G418). Extraction of total RNA from cell lines All buffers and solutions used in the preparation and analysis of RNA were prepared using DEPC treated water. Total RNA was extracted from cell pellets collected at 80–90% confluence by the Trizol method according to the manufacturer’s instructions. RNA pellets were air dried before dissolving in 20–50 lL DEPC-treated sterile water. RNA was then quantified using spectrophotometric meas- urement a t 260/280 nm and quality checked on a 1% ( w/v) agarose gel in Tris/Borate/EDTA buffer stained with ethidium bromide. 5¢-RACE analysis Twenty-five micrograms of total RNA were annealed to the primer mST1-p1 (5¢-GATGATGGCAAAC AGGAG AA-3¢) and reverse transcribed. The primer mST1-p1 is complementary to a region in exon II between nucleotides +50 (5¢)to+69(3¢) relative to the adenosine of the first ATG codon. Thus, mST1-p1 will only bind Siat1 transcripts that contain the exon II ATG translation start site and authentic reverse transcription events of Siat1 mRNA must span at least the exon I–exon II boundary. The resultant cDNA was then ligated overnight at 16 °Ctothe50 nucleotide Marathon adaptor sequence (Clontech) as per instructions and s ubjected to PCR amplification, using the TOUCHDOWN program recommended by Clontech (94 °C for 1 min, five cycles of 94 °Cfor30s,72°Cfor4min,five cycles of 94 °Cfor30s,70°C for 4 min and finally 25 cycles of 94 °C for 20 s, 68 °C for 4 m in), using the Marathon adaptor anchor sense primer AP1 (5¢-CCATCC TAATACGACTCACTATAGGGC-3¢) and either the Siat1 exon I antisense primer md11 (5¢-CTGCTTCTG GCTAATCTTCTGGGGTTGG -3¢)ortheexonOanti- sense primer O2 (5¢-CTCAGCATCCGGCTGGAAAGTG GGTACCACG-3¢). PCR amplification of ST6Gal I sequence from contaminating genomic DNA is not possible as the Siat1 gene does not contain sequences that will specifically anneal the anchor primer. The PCR products were isolated from a 3% (w/v) agarose gel (0.5 lgÆmL )1 ethidium bromide), purified and then ligated into the plasmid vector pCR2.1, cloned in TOP10 competent cells, isolated by miniprep, digested with EcoRI to check for insert, and finally sequenced, using an M13rev primer. RT-PCR of H-Ras and K-Ras mRNA Using 30 lg of total RNA, c DNA was synthesized as described in the 5¢-RACE section save that the initial primer used was the poly(A) + primer supplied in the Marathon RACE kit. Using a 1 : 100 dilution of cDNA, PCR was then performed using primers located in exon 1 and 2 to ensure that only cDNA derived from mRNA could be amplified (at the expected size of 250 bp). PCR conditions used: 94 °Cfor1min,50°Cfor1min,72°C for 1 min, 25 cycles. P rime rs for human H-Ras and K-Ras were as described [36]. H-Ras exon 1 sense: 5 ¢-CTGAG GAGCGATGACGGAAT-3¢, H-Ras exon 2 antisense: 5¢-ACACACACAGGAAGCCCTCC-3¢,K-Rasexon1 sense: 5¢-CCTGCTGAAAATGACTGAAT-3 ¢,K-Ras exon 2 antisense: 5¢-ATACACAAAGAAAGCCCTCC-3 ¢. Ó FEBS 2004 Oncogenic Ras signalling and ST6Gal I activation (Eur. J. Biochem. 271) 3625 The PCR products were isolated from a 3% (w/v) agarose gel (0.5 lgÆmL )1 ethidium bromide), purified on Qiaex resin (Qiagen) and t hen ligated into the plasmid vector pCR2.1, cloned in TOP10F c ompetent cells, isolated on m ini-Wizard columns (Promega), digested with EcoRI to check for insert, and finally sequenced using m 13rev. Northern analysis Total RNA was run on 1% (w/v) agarose/formaldehyde gels and transferred onto positively charged nylon filter (Amersham) using 20 · NaCl/Cit (diethyl pyrocarbonate- treated) capillary transfer. Filters were then prehybridized in a hybridization oven with 10 mL of hybridization buffer [ 0.5 M sodium phosphate (pH 7 .0)/1 m M EDTA/ 7% (w/v) SDS] for 1 h at 65 °C, then hybridized in 5 m L of hybridization buffer containing 32 P-labelled DNA probes [25 ng of DNA labelled with a random prime DNA labelling kit (Promega) and [ 32 P]dCTP[aP], overnight at 65 °C. Next day, blots were washed for 1 h in buffer A [0.04 M sodium phosphate, pH 7.0/1 m M EDTA/5% (w/v) BSA/5% (w/v) SDS], then twice with buffer B [0.04 M sodium phosphate, pH 7.0/1 m M EDTA/1% (w/v) SDS] at 65 °C and exposed to a Kodak phosphor screen and visualized using a Molecular Dynamics (STORM) scanner. The bands were quantified by the IMAGEQUANT software (Molecular Dynamics, Sunnyvale, CA, USA). Sialyltransferase enzyme assay ST6Gal I was assayed as previously described [37]. The reaction mixture contained in a total volume o f 25 lL 10 lL of total cell lysate ( 100 lgprotein),2m M Galb1,4GlcNAcb-OCH 2 Ph, 50 l M CMP-[ 14 C]Neu5Ac (90 000 cpmÆnmol )1 ), 50 m M Mes pH 6.0, 5 m M MgCl 2 and 0.2% (v/v) Triton CF-54. After 1 h at 37 °Cthe reaction was stopped with ice-cold 0.1 M NH 4 HCO 3, the radiolabelled product isolated on reverse phase C-18 cartridges and quantified by liquid scintillation counting. Fluorescence activated cell sorter (FACS) analysis Cells were trypsinized, washed with N aCl/P i and incubated at 2 · 10 7 cellsÆmL )1 in 25 lL of biotinylated SNA or MAA (10 lgÆmL )1 )inNaCl/P i containing 5% (v/v) FBS and 0.1% (w /v) s odium azide (NaCl/P i /FBS). Cells were left on ice for 30 min, washed three times an d streptavidin-FITC (10 lgÆmL )1 ) was added to resuspended cells (25 lLÆwell )1 ). After 30 min on ice, the cells were washed with NaCl/P i / FBS, resuspended in 200 lLofNaCl/P i /FBS, fixed in 1.5% (v/v) f ormaldehyde and analyzed on a B ecton Dickson FACscan flow cytometer. Genomic sequence of Siat1 Genomic sequence covering Siat1 on chromosome 16 (ENSMUSG00000022885) was downloaded from the Wellcome Trust Sanger Institute mouse genome server spanning chromosome 16 region 22918721–23318720 (400 000 nucleotides). Results Effect of activated human Ras on the expression of different members of the mouse sialyltransferase family in 3T3 cells Using the 0.75 kb Siat1 exon II probe, ST6Gal I mRNA was found to be approximately 10-fold increased in the 3T3K-Ras S12 and 3T3H-Ras V12 cell lines relati ve to 3T3 and 3T3pB322 (Fig. 1). In contrast, ST6Gal II transcripts were not detected in any of the cell lines, whilst ST6Gal- NAc I was present only as a weak band in the 3T3H-Ras V12 sample (Fig. 1). Strong ST6Gal II and ST6GalNAc I signals w ere observed in t he positive controls, m ouse whole brain and cell line 410.4 RNA, respectively. ST3Gal I mRNA was detected i n all four lines, and was slightly decreased in the 3T3K-Ras S12 cells. Expression of ST3- Gal I I was weak in all cell lines, with little difference between Ras transfected an d control lines. ST3Gal III le vels were equally high in both 3T3 and 3T3K-Ras S12 lines and no difference between the two cell lines could be observed Fig. 1. Altered ST6Gal I transcript expression is the predominant change within the sialyltransferase family in response to a ctivated Ras. Northern hybridization with cDNA probes o f ST6Gal I , ST6Gal II, ST6GalNAc I, ST3Gal I, S T3Gal II, ST3Gal III, ST3Gal IV sialyl- transferases and H-Ras V12 or K-Ras S12 (indicated on the side). For all panels, 30 lg of total RNA were loaded in the following order: lane 1: 3T3; lane 2: 3T3K-Ras S12 ; l ane 3: 3T3pB322; lane 4: 3T3H-Ras V12 ; and lane 5: positive controls (whole mouse brain or mouse breast tumour cell line 4 01.4 RNA for ST6Gal II and ST6GalNAc I, respectively). Loading c on trol: 18S RNA. 3626 M. Dalziel et al. (Eur. J. Biochem. 271) Ó FEBS 2004 also at shorter exposure times. The mRNA levels were somewhat lower in the 3T3pB322 and in the 3T3H-Ras V12 lines. ST3Gal IV, however, was expressed a t s lightly higher levels in both Ras transformed cell lines than in the parental controls. These data demonstrate that activated Ras has a high positive effect only on the expression of ST6Gal I a nd no or very little effect on other members of the sialyl- transferase family. Expression of oncogenic Ras induces an increase of both cellular ST6Gal I activity and cell surface a2,6- sialylation FACS analysis (Fig. 2) w ith t he Neu5 Aca2,3Gal-specific lectin from MAA found no significant differences between Ras transformed and control fibroblasts. Furthermore, MAA staining was high in both cell lines, consistent with the observation of relative high amounts of ST3Gal I, III and IV mRNA already in t he nontransformed cells (Fig. 1) and only slightly stronger in the Ras transformed cell line. However, we were not able to correlate the higher amounts of cell surface Neu5Aca2,3Gal in the transformed cells to increased a2,3-sialyltransferase activity ([7] a nd data not shown), and they may therefore be due not to an augmentation in transferase activity but to an increase in precursor s tructures a s would be expected in Ras trans- formed cells where branching of N-glycans has been shown to be more abundant [10,38]. On the other hand, a2,6- sialyltransferase activity toward the acceptor Galb1,4Glc- NAcb-OCH 2 Ph was elevated approximately sixfold in H-Ras V12 and K -Ras S12 expressing cell lines relative to mock transfected 3T3 cells (Table 1 ). In addition, FACS analysis using the Neu5Aca2,6Gal-specific lectin SNA found a sixfold higher mean fluorescence intensity on the 3T3K-Ras S12 cells than on the parental line 3T3 (Fig. 2). These data confirm e arlier work linking increased ST6Gal I activity and cell surface SNA staining to the expression of oncogenic Ras in rodent fibroblasts [6–9]. Conditional transient expression of activated Ras induces ST6Gal I mRNA accumulation Both cell lines 3T3K-Ras S12 and 3T3H-Ras V12 had been selected on the basis of colony formation in s oft a gar a s a n indicator for a malignant phenotype. However, during the lengthy selection process o ther genetic changes may o ccur and may contribute to t he increased expression o f the ST6Gal I gene. Therefore we obtained a cell line transfected with a plasmid carrying the neomycine resistance gene under the control of a strong constitutive promoter and the H -Ras V12 gene under the control of t he tetracycline repressor. Stable transfectants were selected with G418 whilst H-Ras V12 was repressed with tetracycline during the selection process [35]. T hese cells (mib125 +Tc ) show the same low l evel of ST6Gal I mRNA (Fig. 3, lanes 1 and 3 ) and corresponding enzyme activity (not shown) as the nontransfected parent cells. Upon de-rep ression of R as expression by removing tetracycline from the medium (mib125 –Tc ) both H-Ras V12 and ST6Gal I mRNAs increased to the same levels as those observed in the cell line constitutively expressing H-Ras V12 (Fig. 3, lanes 2 and Fig. 2. Increas ed cell surface a2,6-sialylation in cells expressing oncogenic Ras. FACS analysis of lectin-labelled c ells: left panels, SNA staining; right panels, MAA staining; upper panels contro l 3T3 cells, lower panels 3T3K -Ras S12 transformed cells as indicated. Narrow lines FITC-streptavidin controls, bold lines over grey background biotinylated le ctin/FITC-streptavidin staining. Ó FEBS 2004 Oncogenic Ras signalling and ST6Gal I activation (Eur. J. Biochem. 271) 3627 4). T he increase of mRNA was concomitant t o the increase in enzyme activity and SNA staining (not shown). When the Ras gene was again repressed, the ST6Gal I mRNA decreased to normal levels within 72 h of tetracycline treatment (Fig. 3, lanes 5 and 6). Again, enzyme activity and SNA staining followed the same trend. These results demonstrate that, like malignant phenotype and focus formation [35], the increase i n ST6Gal I is directly depend- ent on the expression of activated Ras. H-Ras signals to Siat1 primarily through the RalGEF pathway To investigate t he contribution o f i ndividual Ras signalling pathways on the expression of ST6Gal I, th ree effector domain mutants of oncogenic H-Ras were transfected into 3T3 fibroblasts and after selection of stable transfectants the total RNA w as extracted from several clones for each transfection and analyzed by northern hybridization (Fig. 4 shows one represen tative northern f or each transfection experiment). Only clones from cells transfected with the H-Ras V12G37 which allows binding of only the RalGEFs exhibited the same high levels of ST6Gal I mRNA and ST6Gal I activity toward the disaccharide Galb1,4Glc- NAcb-OCH 2 Ph as H-Ras V12 transformed cells (Table 1). The mutant H-Ras V12S35 activating only the Raf k inase pathway h ad no effect on ST6Gal I expression whereas the Ras mutants H-Ras V12C40 which bind only to PI3-kinase could, in some of the c lones analyzed, produce an increase of ST6Gal I mRNA and ST6Gal I enzyme activity. Only the results from the clone with the highest ST6Gal I mRNA level are shown (Fig. 4, Table 1). Oncogene expression was confirmed by H-Ras specific RT-PCR as p reviously described for 3T3H-Ras V12 . H-Ras and K-Ras induce Siat1 transcription via the housekeeping promoter P3 To obtain information on the nature o f t he 5 ¢-UTRs of the ST6Gal I transcripts expressed by Ras transfected and control 3T3 cells , RNA from each of the cell lines 3T3, 3T3K-Ras S12 ,3T3H-Ras V12 , mib125 –Tc and mib128 was subjected to 5 ¢-RACE analysis (md11/AP1 primer pair). The re sults are summarized in Table 2. In all the cell line s studied, the ST6Gal I 5¢UT sequences ob tained were composed of three k inds of sequences: (a) the sequence usually transcribed from the P3 promoter (exon Q and O immediately 5¢ of the untranslated c onserved exon I); (b) a previously unidentified 5¢UT sequence v ery close to that obtained f rom t he P3 promoter (a novel exon, n amed exon R, 5¢ of exons O and I), and (c) a probably t runcated sequence containing only part o f the sequence transcribed from the P 3 promoter ( partial exon O immediately 5¢ of the untranslated con served exon I). The dominant form in every Fig. 3. Transient expr ession of H-Ras V12 coincides with ST6Gal I expression. Northern hybridization analysis of NIH3T3 line mib125 in which H-Ras V12 expression is repressed by Tc. For all panels, 30 lgof total RNA were loaded in the following order: lane 1: mi b35 parental 3T3 cells; lane 2: mib128 expressi ng H-Ras V12 constitutively; lane 3: mib 125 + Tc; lane 4: mib125 –Tc f or 72 h; lane 5: mib125 –Tc for 120 h ; lane 6: mib125 kept w/o Tc for 72 h then Tc was added for 72 h. Labelled cDNA prob es were as indicated to the left. Loading control: 18S R NA. Fig. 4. H-Ras V12 signals to Siat1 primarily through the RalGEFs signal transduction pa thway. Northern analysis of total RNA from NIH3T3 cells transfected w ith p artial lo ss of function H-Ras V12 mutants S 35, G37 and C40 with cDNA probes of ST6Gal I and H -Ras as indicate d to the left. For all pan els, 30 lg of total RNA were loaded in the following order: lane 1: 3T3; lane 2: mock transfected 3T3; lane 3 : 3T3H-Ras V12C40 ;lane4:3T3H-Ras V12S35 ;lane5:3T3H-Ras V12G37 ; and lane 6: 3T3H-Ras V12 . Loading co ntrol: 18S RNA. Table 1. ST6Gal I activities in Ras transformed and control NIH3T3 cells. Enzyme activities were measured in total cell lysates as described under Experimental procedures. The values are the mean of three independent experiments. Cell lines ST6Gal I activity (pmolÆh )1 Æmg protein )1 ) 3T3 4.8 ± 0.4 3T3pB322 11.7 ± 1.8 3T3K-Ras S12 74.2 ± 2.6 3T3H-Ras V12 77.4 ± 4.7 3T3Neo 11.1 ± 0.9 3T3H-Ras V12–S35 8.1 ± 0.4 3T3H-Ras V12–C40 32.3 ± 1.7 3T3H-Ras V12–G37 70.1 ± 3.1 3628 M. Dalziel et al. (Eur. J. Biochem. 271) Ó FEBS 2004 sample studied was the ÔtruncatedÕ P3 form (ranging from 74 to 95% of the total number of clones). As these truncated P3 sequences could be representative of either partial 5 ¢-RACE cDNA synthesis or actual t ranscription initiation within exon O, a second 5¢-RACE experiment w as carried out using an antisense primer located at the extreme 3¢ tip of exon O (primer O2) a nd the M arathon AP1. When the 3T3pB322 and 3 T3H-Ras V12 samples were subjected to this modified 5¢-RACE, only clones representing the common (Q-O-I) and novel (R-O-I) P3 isoforms were obtained (Table 2). These data indicate that the truncated P3 sequences generated from the AP1/md11 5¢-RACE experi- ment are derived from incomplete cDNA synthesis within exon O itself, probably as the result of seco ndary mRNA structure. Furthermore, we can eliminate the possibility that R is merely an unprocessed sequence between Q and O as the O2 RACE could cover the entire Q form, whilst still giving rise to clones containing the shorter R-O sequence, strongly suggesting that both Q and R are separate and distinct 5¢-termini. Contribution of Q and R forms of P3 to Ras signal Although no obvious association with either the classical o r alternative P3 f orms could b e seen w ith any of the R as expressing cell lines when 5¢-RACE was carried out using AP1/md11, t here was a bias toward the Q form in 3T3H- Ras V12 and the R f orm in 3T3pB322 cells using AP1/O 2, as seen by ethidium bromide-stained gel analysis of the products (not shown) and subsequent sequencing (Table 2), although the number of clones analyzed was small. Thus PCR probes for exons Q and R were amplified from 3T3H- Ras V12 cDNA and used as probes to detect the expression of each isoform in the three activated H-Ras expressing cell lines 3T3H-Ras V12 ,mib125 –Tc and mib128, relative to the control lines, 3T3pB322, mib125 +Tc and mib35, r espect- ively. The exon Q probe gave a detectable hybridization signal in all three of the Ras cell lines but very little or no signal in the respective controls (Fig. 5) Similarly, the e xon R p robe resulted in a detectable signal in all t hree Ras lines but none in the respective c ontrols (Fig. 5). High ST6Gal I mRNA levels in the three Ras samples was confirmed using the exon II probe as before (Fig. 5). The signal obtained with the Q probe was much greater than that of R in the 3T3H-Ras V12 sample (consistent with the O2 RACE results) whereas the R signal was greater than Q in the m ib125 –Tc and mib128 samples. T he data presented in Fig. 5 confirm the presence o f two independent transcription start sites in the P3 promoter region and indicate that transcription from both sites is up-regulated in response to transformation with oncogenic Ras. Mapping of exon R relative to exons Q and O within the mouse Siat1 gene Making use o f the online public access E nsembl mouse genome server (http://www.ensembl.org/Mus_musculus) the entire Siat1 gene was mapped (Ensembl gene ID: ENSMUSG00000022885, chromosome 16 nucleotides 22918721–23318720), and exon R subsequently located between exons Q and O, with an 820 bp intron between Q and R (Fig. 6B). The common splice sequence s een in the RACE clones allowed the exact definition of the 3¢ termination of both exons Q and R within the Siat1 genomic sequence, as well as the 5 ¢ of exon O (Fig. 6A). All three exons contain the splice donor sequence GT imme- diately 3¢ of the exon. Further, exon O has a splice acceptor AG immediately 5¢ of it. Using this information, a complete schematic representation o f the complete mouse Siat1 gene, including the Ras induced P3 mRNA isoforms, was constructed (Fig. 7). A nalysis of the 5¢ sequences upstream of exons Q and R b y the MatInspector database [39] failed to find TATA or CAAT boxes and identified several Table 2. 5¢UT sequences o f S T6Gal I mRNA in Ras t ransformed and control cell lines. The n um bers o f clo nes with 5 ¢ sequences begin ning within exons Q, R or O are given. For e xon nomenclature a nd the overall o rganization of t he Siat1 gene see Fig. 7 . Cell lines Number of clones starting in exons QRO 5¢-RACE results using primer pair md11 (Siat1 exon I) and AP1 (marathon adaptor) 3T3 5 1 41 3T3K-Ras S12 5734 3T3H-Ras V12 4250 mib125 –Tc 1231 mib128 0 2 37 5¢-RACE results using primer pair O2 (extreme 3¢ of exon O) and AP1 (marathon adaptor) 3T3pB322 0 3 0 3T3H-Ras V12 91 0 Fig. 5. Contribution of e xons Q and R to the 5¢UT region of ST6Gal I mRNA from Ra s tr ansfe cted an d c ontr ol 3T 3 ce lls. Northern hybrid- ization of 50 lg total RNA from Ras transfected and control lines using the PCR generated probes for 5¢UTexonsQandRandthefirst coding exon II as indicated to the left. Lane 1: 3T3pB322, lane 2: 3T3H-Ras V12 ; l ane 3: mib35; lane 4: mib125 +Tc ; l ane 5: mib125 –Tc ; lane 6: m ib128. Loading control: 18S R NA. Ó FEBS 2004 Oncogenic Ras signalling and ST6Gal I activation (Eur. J. Biochem. 271) 3629 transcription factor binding sites t ypical of housekeeping promoters. Among these, double GC boxes and Ras- responsive element binding protein-1 sites are located immediately 5¢ of bo th exons Q and R (Fig. 6B). Discussion Aberrant glycosylation occurs in essentially all types of experimental and human cancers [40]. A long-standing debate is how aberrant glycosylation is related to cancer and whether it is the result of initial oncogenic transformation. Studies on R as transformed r odent fibroblasts indicated that the expression of oncogenic H-Ras V12 leads to changes in the N-glycan structure of cell surface g lycoproteins. The principal modifications on N-glycans observed were increased complexity of N-glycan branching and changes in N-glycan sialylation from a3- to a6-linked Neu5Ac sialylation [4–10]. However, these studies were carr ied out on single clones of H-Ras V12 transformed fibroblasts which had been selected over prolonged periods of time for an increased growth rate and for the a bility to form colonies in soft agar. During this lengthy selection process unidentified genetic changes may h ave occurred which could have contributed to the m odification i n N-glycan b iosynthesis. Such changes did occur as a few clones could be selected which did not show altered N-glycan structures or increased sialylation [9]. In order to address t he q uestion whether H-Ras V12 was directly or indirectly involved, via ST6Gal I, in the augmentation of N-glycan sialylation, we measured ST6Ga- l I in NIH3T3 fibroblasts that express activated Ras conditionally. In these cells the transformed phenotype and the ability to form foci in soft agar are directly dependent on the expression of Ras and are c ompletely reversible [35]. In the absence of H-Ras V12 these cells exhibit the same low levels of ST6Gal I as the non transformed Fig. 6. The P 3 promoter region of Siat1: 5¢- RACE data deri ved from Q and R containing clones allowed the precise definition of b oth the 3¢ ends of exons Q and R as we ll as the 5¢ end of exon O. (A) Proposed conserved splice acceptor lo cation at th e 5¢ endofexonO, utilized by both Q and R. (B) Exon sequences Q and R in u pper case, introns in lower case, exon–intron boundaries underlined. The d is- tance between Q a nd R is l ess than 820 nucleotides. Putative tran scription factor binding sites: GC box (shaded); Ras-respon- sive element b inding protein-1 (double underlined). 3630 M. Dalziel et al. (Eur. J. Biochem. 271) Ó FEBS 2004 fibroblasts and only when t he expression of H-Ras V12 is induced do these cells show the same high levels of ST6Gal I mRNA as the constitutively H-Ras V12 transformed cell lines. This enhancement of ST6Gal I expression is reversible at the level of mRNA as well as at the level of cell s urface expression of the Neu5Aca2,6Galb4GlcNAc epitope (not shown). These results clearly indicate that the presence of activated Ras alone and n o other genetic or e pigenetic events are responsible for the elevated expression of ST6Gal I i n Ras transformed murine fibroblasts. Among the members of the Ras gene family we found that both K-Ras S12 and H-Ras V12 promote the same large increase in ST6Gal I mRNA in fibroblasts. The former is of particular relevance as it is the predominantly mutated RAS gene in human cancer [41]. Although N-Ras was not included in our study, it i s known that expression of normal N-Ras has a positive influence on both cellular sialylation and ST6Gal I activity [42]. The influence o f H- a nd K-Ras transformation appears t o be restricted t o the Sia t1 gene, at least amongst its immediate family members, a s none of the other sialyltransferases a nalyzed showed notable changes in their transcript levels. In several human cancers where activating Ras mutations a re common, it may b e significant that high ST6Gal I activity is the most frequent alteration to the expression pattern of the sialyltransferase family. As Ras signals directly to the Siat 1 gene we wanted to know through which pathway the signal may be delivered. Ras signals mainly through t hree pathways: t he Raf-MEK- ERK signalling cascade which promotes proliferation through the activation of transcription f actors; the PI3 kinase pathway where lipid kinases generate second mes- sengers which have diverse effects o n cellular physiology and t he RalGEF signalling cascade whic h i nvolves a whole family of RalGTPases but most of the downstream activators are still not identified. For each of the three pathways, partial loss of function mutants of activated Ras proteins have been created [31,32] which can selectively bind to one of the e ffectors and thus signal through one pathway only. H-Ras V12S35 binds only to R af and is unable to activate the two other signalling cascades whereas H-Ras V12C40 binds exclusively to PI3 kinases and the H-Ras V12G37 mutant specifically activates the RalGEF pathway. When the t hree constructs coding the mutant Ras proteins were transfected into 3T3 fibroblasts only the H-Ras V12G37 mutant was able to induce the increased expression of ST6Gal I similar to the wild type oncogenic H-Ras V12 . Interestingly, the PI3 kinase pathway may also contribute to the activation of the ST6Gal I gene but at a much lower level and not all of t he clones with high levels of H-Ras V12SC40 showed increased amounts o f ST6Gal I mRNA. These results indicate that Ras signals to the ST6Gal I gene principally through the RalGEF signalling pathway. The rise in ST6Gal I mRNA was a lways accom- panied by a concomitant increase i n ST6Gal I enzyme activity. The RalGEF pathway is the least well documen ted of the three major sign alling pathways and mo st of the physiolo- gical consequences of RalGEF activation are still outstand- ing issues. H owever its i mportance has recently come into focus with the mounting evidence that it is the principal pathway used by Ras to transform human cells [43]. One recent study links RalGEF activation i n rodent fibroblasts to the development of highly invasive metastases when those cells are administered subcutaneously to nude mice [44]. The formation of aggressive tumours may be correlated to the increased expression of ST6Gal I as clones of Ras trans- formed rat fibroblasts which had lost the e xpression of the Neu5Aca2,6Galb4GlcNAc epitope synthesized by ST6Gal I were f ound to be much less metastatic than the clones which still possessed this glycan structure [9]. Tissue-specific expression levels of ST6Gal I are regulated by the use o f tissue specific splice forms of its mRNA derived f rom selective t ranscription of multiple promoters. In order t o localize the promoter region targeted by Ras we wanted to identify the 5¢UT isoform, which is ind uced by the R alGEF s ignal. In all cell lines studied that expre ss activated K-Ras S12 or H-Ras V12 , the ST6Gal I transcripts found represent the isoform transcribed from the P3 Fig. 7. Mapping o f P3 w ithin the com plete Siat1 genomic structure. Schematic r epresen- tation of the mouse ST6Gal I gene Siat1.This gene spans over 130 kb on chromosome 16. The transcription start s ites at the four major promotors are indicated by a rrows. The open reading frame is encoded by exons II through VI. Exon I is an invariant 5¢UT exon found in all Siat1 m RNA. The two l oc ations of the presumed transcriptional start sites used by Siat1 in Ras expressing cells are indicated by big arrows (P3a and P3b). The resulting mature transcripts both contain the 5¢UT exons O and I preceded by either exon Q or exon R. Transcription start sites from tissue specific promoters are indicated by s mall arrows:P1,liver;P2a-c,Bcells,P4,lactating mammary gland. Ó FEBS 2004 Oncogenic Ras signalling and ST6Gal I activation (Eur. J. Biochem. 271) 3631 housekeeping p romoter. Although enhanced steady state transcription is the most obvious explanation for the accumulation of ST6Gal I mRNA in the presence of K-Ras S12 or H-Ras V12 it cannot be excluded that increased mRNA stability may also contribute to the high levels of ST6Gal I mRNA in Ras transformed cells. However, previous work has shown that quantitative c hanges of a particular class o f ST6Gal I 5¢UT transcripts ( including P3) are primarily the result of t ranscriptional a ctivity a t the matching promoter [45–47]. In the adult mouse, P3 is normally active in most tissues and gives rise to a mature ST6Gal I mRNA leading with the 5¢UT sequences encoded b y exons Q, O a nd I (Fig. 7). We detected these same transcripts in ST6Gal I mRNA derived from Ras transformed cells, but alongside a previously unreported variant where exon Q is replaced by a s equence named exon R. As both exons Q and R make use of a conserved splice junction at exon O, are located within the same region on the Siat1 gene (less t han 8 20 bp apart) and are c oexpressed, there are two possible explanations for the presence of these a lternative 5¢UT leader s equences. Both Q and R isoforms could be derived from a single promoter with a certain degree of initiation site variability. Although it is quite common for housekeeping promoters to have several transcription initiation sites, these are u sually foun d much closer together than those f or exons Q and R, normally less than a hundred and usually within 30 nucleotides of each other [48]. O n t he oth er h and, Q and R may represent transcription initiation sites of two r elated and overlapping housekeeping promoter regions within the Siat1 gene. However, neither Q nor R appears to be favoured by the activation through th e RalGEF pathway although s ome differences could be observed b etween cell lines. A preliminary analysis of the sequences directly upstream of the two tr anscription start sites i dentified several putative consensus sequences for transcription factors t ypical of housekeeping promoters. As both of these regions are equally responsive to Ras, shared consensus sequences for transcription f actors could be a key to understand their regulation. It is therefore interesting to note that two sequences recognized by Ras- responsive element b inding protein-1 are present within a few hundred nucleotides of each transcription start site. An additional ob servation suggests that the transcription is initiated a t a true housekeeping promoter. Although t he amount of ST6Gal I m RNA p roduced in Ras t ransformed cells is close to the levels found in liver [49] the s pecific enzymatic activity of ST6Gal I in the transformed fibro- blasts is much lower [27,37]. This is consistent with observations that some transcripts derived from housekeep- ing promoters have a low translation rate i n large part due to stable secondary structures at their 5¢UT region [50]. This strong secondary structure could possibly account for the high frequency of truncated sequences in the 5¢-R ACE experiments described in this study. The key effect of Ras on growth is to overcome c ontact inhibition between cells [35]. T he rise of a6-linked sialic acid on N-glycans of cell surface glycoproteins as a direct result of oncogenic Ras expression may contribute to the repression of contact inhibition. Contact-mediated inhibition of cell migration and cell proliferation is co-ordinately regulated by integrins and their receptors. Recently it has been reported that b1 integrin activity is dependent on the sialylation of its N-glycans a nd that the R as induced change from a3-linked to a6-linked sialic acid alters the binding to some of its ligands [23]. In addition, RalGEF activation through Ras induces aggressive behaviour in tumours that may als o be related to the increase in ST6Gal I activity. Together these two examples indicate that shifts in sialyltransferase expression patterns may be an important contribution of oncogenic Ras to the m etastatic potential of tumours. Acknowledgements MD is the recip ient of a postdoctoral fellowship from Le STU DIUMÒ (Orle ´ ans, France). This wo rk was supported by grants f rom the Ligue Nationale contre le Cancer (comite ´ sde ´ partementaux du L oiret et du Loir et Ch er), the Centre National d e la Recherche Scientifique: Prote ´ omique et Ge ´ nie des Prote ´ ines, by the Groupement de Recherche: Ge ´ nomique et Ge ´ nie des Glycosyltransfe ´ rases and by t he Orle ´ ans chapter of the Lions Club. FD acknowledges grants from MURST and the Universita ` di Bologna. We are gra teful to Dr J. La u (Ros well Park Cancer Institute, Bu ffalo, NY, USA), P rof E. 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Higai, K & Matsumoto, K (2000) Transcriptional regulation of human b-galactoside a2 ,6-sialyltransferase (hST6Gal I) gene during differentiation of the HL-60 cell line Glycobiology 10, 623–628 Ó FEBS 2004 48 Azizkhan, J.C., Jensen, D.E., Pierce, A. J & Wade, M (1993) Transcription from TATA-less promoters: dihydrofolate reductase as a model Crit Rev Eukaryot Gene Expr 3, 229–254 49 Dalziel, M., Lemaire, S., . Ras oncogene induces b-galactoside a2 ,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter Martin Dalziel 1 , Fabio. antisense: 5¢-ACACACACAGGAAGCCCTCC-3¢,K-Rasexon1 sense: 5¢-CCTGCTGAAAATGACTGAAT-3 ¢,K -Ras exon 2 antisense: 5¢-ATACACAAAGAAAGCCCTCC-3 ¢. Ó FEBS 2004 Oncogenic Ras signalling and ST6Gal I activation (Eur. J.