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
Trang 1Ras oncogene induces b-galactoside a2,6-sialyltransferase (ST6Gal I) via a RalGEF-mediated signal to its housekeeping promoter
Martin Dalziel1, Fabio Dall’Olio2, Arron Mungul3, Ve´ronique Piller1and Friedrich Piller1
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;3Cancer Research UK, Breast Cancer Biology Group, Guy’s Hospital, London, UK
Several oncogenic proteins are known to influence cellular
glycosylation In particular, transfection of 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 pathways (Raf, RalGEFs and
PI3K) and that transcriptional control of mouse ST6Gal I is
achieved by the selective use of multiple promoters, we
attempted to identify which of these parameters are involved
in linking the Ras signal to ST6Gal I gene transcription in
mouse fibroblasts Transformation by human K-Ras or
H-Ras (S12 and V12 point mutations, respectively) results in
a 10-fold increase in ST6Gal I mRNA, but no alteration in
the expression of related sialyltransferases Using an
indu-cible H-RasV12expression 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 by an increase of a2,6-sialyltransferase activity and of Neu5Aca2,6Gal at the cell surface Results obtained with H-RasV12partial loss of function mutants H-RasV12S35 (Raf signal only), H-RasV12C40(PI3-kinase signal only) and H-RasV12G37(RalGEFs signal only) suggest that the H-Ras induction of the mouse 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-RasV12or K-RasS12 transfec-tion is mediated by the Siat1 housekeeping promoter P3-associated 5¢ untranslated exons
Keywords: oncogenic Ras; sialyltransferase; RalGEF; housekeeping promoter
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 signals 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-RasV12 in primary cell cultures of human tumours has been linked to
an increase in N-glycan branching and sialylation [4,5] Subsequent studies with H-RasV12-transfected rat fibro-blasts identified the enhanced activities of the CMP-Neu5Ac:Galb1,4GlcNAc a2,6-sialyltransferase (ST6Gal I,
EC 2.4.99.1) [6–9], and 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], colon [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 differentiation state of the 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 cell sorter; FBS, fetal
bovine serum; FITC, fluorescein isothiocyanate; Gal, galactose;
Glc-NAc, N-acetylglucosamine; GalGlc-NAc, N-acetylgalactosamine; MAA,
Maackia amurensis agglutinin; MES, 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,
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:Galb1,4GlcNAc a2,6-sialyltransferase
(ST6GalI, EC 2.4.99.1); CMP-Neu5Ac:GalNAc a2,6-sialyltransferase
I, (ST6GalNAcI, EC 2.4.99.3); CMP-Neu5Ac:Galb1,3GalNAc
a2,3-sialyltransferase I (ST3Gal I, EC 2.4.99.6).
(Received 12 May 2004, revised 2 July 2004, accepted 12 July 2004)
Trang 2that 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 to those
reported to be the result of activated Ras expression, also
influences parameters thought to be important for cellular
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 tissue restrictive expression, primarily in
adult brain [26], leaves ST6Gal I as the dominant ST6Gal
enzyme in all adult tissues This is consistent with
gene knockout 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
Siat1is regulated during development and differentiation by
the selective 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 sequences At least four Siat1 promoters are known:
P1 controls ST6Gal I expression in liver [28], P2 in
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 indicating that the Ras signal, mediated by
RalGEFs, leads directly to an accumulation of ST6Gal I
mRNA, transcribed from the Siat1 housekeeping promoter
P3, ultimately resulting in enhanced ST6Gal I enzyme
activity and cell surface a2,6-sialylation
Experimental procedures
Materials
[32P]dCTP[aP] (3000 CiÆmmol)1), CMP-[14C]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) The DNA gel extraction kit was from
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)
Galb1,4GlcNAcb-OCH2Ph was a generous gift of C LeNarvor and
C Auge´, Universite´ Paris-Sud, Orsay, 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
(sense), 5¢-GGCTATTCAGAATTCCAGCGCCTCGGC
CCTCGAGTGACTGGTTCTGAAGGCGCTCAGG-3¢; ST3Gal III (633 bp): p138 (sense), 5¢-CCCTCTGCCT CTTCCTGGTC-3¢ and p139 (antisense), 5¢-TCGTTCAT
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 Institute, Tokyo, Japan A 1.1 kb ST6GalNAc I probe was released by HindIII/EcoRV digestion A 604 nt ST6Gal II cDNA probe, spanning the first predicted coding exon in the mouse chromosome 17 sequence ENSMUSG00000024172 (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¢-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-RasV12cDNA 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 kb human K-RasS12 cDNA cloned into the EcoRI site of pcEXV-3 The human V12 H-Ras partial loss of function mutants H-RasV12S35, H-RasV12C40 (both in pcDNA3.1) and H-RasV12G37 (in pSG5) were obtained from A Scibetta (Cancer Research
UK, Guy’s Hospital, London, UK) [31,32] The H-RasV12G37 cDNA was recloned into pcDNA3.1 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 this probe was hybridized to multiple tissue total RNA extracted from a single male C57Bl6 mouse (stomach, whole brain, spleen, kidney,
Trang 3testis, large intestine, small intestine and liver), only the
whole brain sample showed any detectable 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 the 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 sufficient for the validation of both the identity
of the ENSMUSG00000024172 sequence as the mouse
ST6Gal II sequence (thus named Siat2) and the use of
the 604 nt PCR fragment 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 37C in a humidified atmosphere
of 5% (v/v) CO2, in DMEM (Gibco)/10% (v/v) FBS
containing 100 UÆmL)1penicillin, 100 lgÆmL)1glutamate,
100 lgÆmL)1 streptomycin and 1.25 lgÆmL)1
amphoteri-cin B Mouse cell lines 3T3 and K-RasS12, which are
NIH3T3 parental and NIH3T3 transfected with activated
human Ras cDNA, respectively, were obtained from
Cancer Research UK H-RasV12, which is NIH3T3
transfected with codon 12 position 2 point mutation
GGC to GTC (Glyfi 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 Ras variant as
well as nature of the mutation were controlled by
RT-PCR and nucleotide 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] The NIH3T3 cell lines transfected with
tetracycline (Tc) repressed H-RasV12 construct (mib125),
constitutive H-RasV12(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 Gene Pulser
electroporator (Bio-Rad, Hercules, CA, USA) at 960 lFD,
100W, 0.25 V Stable transfectants were selected with
0.5–0.75 mgÆmL)1geneticin (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 at 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¢-GATGATGGCAAACAGGAG 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 16C to the 50 nucleotide Marathon adaptor sequence (Clontech) as per instructions and subjected to PCR amplification, using the TOUCHDOWN program recommended by Clontech (94C for 1 min, five cycles of 94C for 30 s, 72 C for 4 min, five cycles of 94C for 30 s, 70 C for 4 min and finally 25 cycles of 94C for 20 s, 68 C for 4 min), 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¢) or the exon O anti-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, cDNA 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: 94C for 1 min, 50 C for 1 min, 72 C for 1 min, 25 cycles Primers 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-Ras exon 1
exon 2 antisense: 5¢-ATACACAAAGAAAGCCCTCC-3¢
Trang 4The PCR products were isolated from a 3% (w/v) agarose
gel (0.5 lgÆmL)1ethidium bromide), purified on Qiaex resin
(Qiagen) and then ligated into the plasmid vector pCR2.1,
cloned in TOP10F competent cells, isolated on mini-Wizard
columns (Promega), digested with EcoRI to check for
insert, and finally sequenced using m13rev
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 [0.5M sodium phosphate (pH 7.0)/1 mM EDTA/
7% (w/v) SDS] for 1 h at 65C, then hybridized in
5 mL of hybridization buffer containing 32P-labelled
DNA probes [25 ng of DNA labelled with a random
prime DNA labelling kit (Promega) and [32P]dCTP[aP],
overnight at 65C Next day, blots were washed for 1 h
in buffer A [0.04M sodium phosphate, pH 7.0/1 mM
EDTA/5% (w/v) BSA/5% (w/v) SDS], then twice with
buffer B [0.04M sodium phosphate, pH 7.0/1 mM
EDTA/1% (w/v) SDS] at 65C 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 of 25 lL
10 lL of total cell lysate ( 100 lg protein), 2 mM
Galb1,4GlcNAcb-OCH2Ph, 50 lM CMP-[14C]Neu5Ac
(90 000 cpmÆnmol)1), 50 mM Mes pH 6.0, 5 mM MgCl2
and 0.2% (v/v) Triton CF-54 After 1 h at 37C the
reaction was stopped with ice-cold 0.1M NH4HCO3, 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 NaCl/Piand incubated
at 2· 107cellsÆmL)1in 25 lL of biotinylated SNA or MAA
(10 lgÆmL)1) in NaCl/Pi containing 5% (v/v) FBS and
0.1% (w/v) sodium azide (NaCl/Pi/FBS) Cells were left on
ice for 30 min, washed three times and streptavidin-FITC
(10 lgÆmL)1) was added to resuspended cells (25 lLÆwell)1)
After 30 min on ice, the cells were washed with NaCl/Pi/
FBS, resuspended in 200 lL of NaCl/Pi/FBS, fixed in 1.5%
(v/v) formaldehyde and analyzed on a Becton Dickson
FACscan flow cytometer
Genomic sequence ofSiat1
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-RasS12 and 3T3H-RasV12cell lines relative 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-RasV12 sample (Fig 1) Strong ST6Gal II and ST6GalNAc I signals were observed in the positive controls, mouse whole brain and cell line 410.4 RNA, respectively ST3Gal I mRNA was detected in all four lines, and was slightly decreased in the 3T3K-RasS12 cells Expression of ST3-Gal II was weak in all cell lines, with little difference between Ras transfected and control lines ST3Gal III levels were equally high in both 3T3 and 3T3K-RasS12lines 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 activated Ras Northern hybridization with cDNA probes of ST6Gal I, ST6Gal II, ST6GalNAc I, ST3Gal I, ST3Gal 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-RasS12; lane 3: 3T3pB322; lane 4: 3T3H-RasV12; and lane 5: positive controls (whole mouse brain or mouse breast tumour cell line 401.4 RNA for ST6Gal II and ST6GalNAc I, respectively) Loading control: 18S RNA.
Trang 5also at shorter exposure times The mRNA levels were
somewhat lower in the 3T3pB322 and in the 3T3H-RasV12
lines ST3Gal IV, however, was expressed at slightly 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 and
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) with the Neu5Aca2,3Gal-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 the 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] and data not
shown), and they may therefore be due not to an
augmentation in transferase activity but to an increase in
precursor structures as 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-OCH2Ph was elevated approximately sixfold in
H-RasV12 and K-RasS12 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-RasS12cells than on the parental line 3T3 (Fig 2) These data confirm earlier 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-RasS12 and 3T3H-RasV12had been selected on the basis of colony formation in soft agar as an indicator for a malignant phenotype However, during the lengthy selection process other genetic changes may occur and may contribute to the increased expression of 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-RasV12 gene under the control of the tetracycline repressor Stable transfectants were selected with G418 whilst H-RasV12was repressed with tetracycline during the selection process [35] These cells (mib125+Tc) show the same low level of ST6Gal I mRNA (Fig 3, lanes 1 and 3) and corresponding enzyme activity (not shown) as the nontransfected parent cells Upon de-repression of Ras expression by removing tetracycline from the medium (mib125–Tc) both H-RasV12 and ST6Gal I mRNAs increased to the same levels as those observed in the cell line constitutively expressing H-RasV12(Fig 3, lanes 2 and
Fig 2 Increased cell surface a2,6-sialylation in cells expressing oncogenic Ras FACS analysis of lectin-labelled cells: left panels, SNA staining; right panels, MAA staining; upper panels control 3T3 cells, lower panels 3T3K-Ras S12 transformed cells as indicated Narrow lines FITC-streptavidin controls, bold lines over grey background biotinylated lectin/FITC-streptavidin staining.
Trang 64) The increase of mRNA was concomitant to 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 in ST6Gal I is directly
depend-ent on the expression of activated Ras
H-Ras signals to Siat1 primarily through the RalGEF
pathway
To investigate the contribution of individual Ras signalling
pathways on the expression of ST6Gal I, three effector
domain mutants of oncogenic H-Ras were transfected into
3T3 fibroblasts and after selection of stable transfectants the total RNA was extracted from several clones for each transfection and analyzed by northern hybridization (Fig 4 shows one representative northern for each transfection experiment) Only clones from cells transfected with the H-RasV12G37which 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-OCH2Ph as H-RasV12transformed cells (Table 1) The mutant H-RasV12S35 activating only the Raf kinase pathway had no effect on ST6Gal I expression whereas the Ras mutants H-RasV12C40which bind only to PI3-kinase could, in some of the clones 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 previously described for 3T3H-RasV12
H-Ras and K-Ras induceSiat1 transcription via the housekeeping promoter P3
To obtain information on the nature of the 5¢-UTRs of the ST6Gal I transcripts expressed by Ras transfected and control 3T3 cells, RNA from each of the cell lines 3T3, 3T3K-RasS12, 3T3H-RasV12, mib125–Tc and mib128 was subjected to 5¢-RACE analysis (md11/AP1 primer pair) The results are summarized in Table 2 In all the cell lines studied, the ST6Gal I 5¢UT sequences obtained were composed of three kinds of sequences: (a) the sequence usually transcribed from the P3 promoter (exon Q and O immediately 5¢ of the untranslated conserved exon I); (b) a previously unidentified 5¢UT sequence very close to that obtained from the P3 promoter (a novel exon, named exon
R, 5¢ of exons O and I), and (c) a probably truncated sequence containing only part of the sequence transcribed from the P3 promoter (partial exon O immediately 5¢ of the untranslated conserved exon I) The dominant form in every
Fig 3 Transient expression of H-RasV12 coincides with ST6Gal I
expression Northern hybridization analysis of NIH3T3 line mib125 in
which H-RasV12expression is repressed by Tc For all panels, 30 lg of
total RNA were loaded in the following order: lane 1: mib35 parental
3T3 cells; lane 2: mib128 expressing H-RasV12constitutively; lane 3:
mib 125 + Tc; lane 4: mib125 –Tc for 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 probes were as indicated to the left Loading control:
18S RNA.
Fig 4 H-Ras V12 signals to Siat1 primarily through the RalGEFs signal transduction pathway Northern analysis of total RNA from NIH3T3 cells transfected with partial loss of function H-Ras V12 mutants S35, G37 and C40 with cDNA probes of ST6Gal I and H-Ras as indicated
to the left For all panels, 30 lg of total RNA were loaded in the following order: lane 1: 3T3; lane 2: mock transfected 3T3; lane 3: 3T3H-RasV12C40; lane 4: 3T3H-RasV12S35; lane 5: 3T3H-RasV12G37; and lane 6: 3T3H-RasV12 Loading control: 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)1Æmg protein)1)
3T3pB322 11.7 ± 1.8
3T3K-Ras S12 74.2 ± 2.6
3T3H-RasV12 77.4 ± 4.7
3T3Neo 11.1 ± 0.9
3T3H-Ras V12–S35 8.1 ± 0.4
3T3H-RasV12–C40 32.3 ± 1.7
3T3H-RasV12–G37 70.1 ± 3.1
Trang 7sample studied was the truncated 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 transcription initiation within
exon O, a second 5¢-RACE experiment was carried out
using an antisense primer located at the extreme 3¢ tip of
exon O (primer O2) and the Marathon AP1 When the
3T3pB322 and 3T3H-RasV12samples 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 secondary 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 or
alternative P3 forms could be seen with any of the Ras
expressing cell lines when 5¢-RACE was carried out using
AP1/md11, there was a bias toward the Q form in
3T3H-RasV12and the R form in 3T3pB322 cells using AP1/O2, 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-RasV12cDNA and used as probes to detect the expression of
each isoform in the three activated H-Ras expressing cell
lines 3T3H-RasV12, mib125–Tcand mib128, relative to the
control lines, 3T3pB322, mib125+Tc and mib35,
respect-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 exon
R probe resulted in a detectable signal in all three Ras lines
but none in the respective controls (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-RasV12sample (consistent with the O2 RACE results) whereas the R signal was greater than Q in the mib125–Tc and mib128 samples The data presented in Fig 5 confirm the presence of 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 mouseSiat1 gene
Making use of the online public access Ensembl 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 seen 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 of the complete mouse Siat1 gene, including the Ras induced P3 mRNA isoforms, was constructed (Fig 7) Analysis of the 5¢ sequences upstream
of exons Q and R by the MatInspector database [39] failed
to find TATA or CAAT boxes and identified several
Table 2 5¢UT sequences of ST6Gal I mRNA in Ras transformed and
control cell lines The numbers of clones with 5¢ sequences beginning
within exons Q, R or O are given For exon nomenclature and the
overall organization of the Siat1 gene see Fig 7.
Cell lines
Number of clones starting in exons
5¢-RACE results using primer pair md11 (Siat1 exon I) and AP1
(marathon adaptor)
5¢-RACE results using primer pair O2 (extreme 3¢ of exon O) and
AP1 (marathon adaptor)
Fig 5 Contribution of exons Q and R to the 5¢UT region of ST6Gal I mRNA from Ras transfected and control 3T3 cells Northern hybrid-ization of 50 lg total RNA from Ras transfected and control lines using the PCR generated probes for 5¢UT exons Q and R and the first coding exon II as indicated to the left Lane 1: 3T3pB322, lane 2: 3T3H-Ras V12 ; lane 3: mib35; lane 4: mib125 +Tc ; lane 5: mib125 –Tc ; lane 6: mib128 Loading control: 18S RNA.
Trang 8transcription factor binding sites typical of housekeeping
promoters Among these, double GC boxes and
Ras-responsive element binding protein-1 sites are located
immediately 5¢ of both 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 Ras transformed rodent fibroblasts indicated
that the expression of oncogenic H-RasV12leads to changes
in the N-glycan structure of cell surface glycoproteins 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 carried out on single clones
of H-RasV12 transformed fibroblasts which had been selected over prolonged periods of time for an increased growth rate and for the ability to form colonies in soft agar During this lengthy selection process unidentified genetic changes may have occurred which could have contributed
to the modification in N-glycan biosynthesis 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 the question whether H-RasV12was directly or indirectly involved, via ST6Gal I, in the augmentation of N-glycan sialylation, we measured
ST6Ga-l I in NIH3T3 fibrobST6Ga-lasts 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 completely reversible [35] In the absence of H-RasV12these cells exhibit the same low levels of ST6Gal I as the non transformed
Fig 6 The P3 promoter region of Siat1: 5¢-RACE data derived from Q and R containing clones allowed the precise definition of both the 3¢ ends of exons Q and R as well as the 5¢ end
of exon O (A) Proposed conserved splice acceptor location at the 5¢ end of exon O, utilized by both Q and R (B) Exon sequences
Q and R in upper case, introns in lower case, exon–intron boundaries underlined The dis-tance between Q and R is less than 820 nucleotides Putative transcription factor binding sites: GC box (shaded); Ras-respon-sive element binding protein-1 (double underlined).
Trang 9fibroblasts and only when the expression of H-RasV12is
induced do these cells show the same high levels of ST6Gal I
mRNA as the constitutively H-RasV12 transformed cell
lines This enhancement of ST6Gal I expression is reversible
at the level of mRNA as well as at the level of cell surface
expression of the Neu5Aca2,6Galb4GlcNAc epitope (not
shown) These results clearly indicate that the presence of
activated Ras alone and no other genetic or epigenetic
events are responsible for the elevated expression of
ST6Gal I in Ras transformed murine fibroblasts
Among the members of the Ras gene family we found
that both K-RasS12and H-RasV12promote 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 is known that expression of normal
N-Ras has a positive influence on both cellular sialylation
and ST6Gal I activity [42] The influence of H- and K-Ras
transformation appears to be restricted to the Siat1 gene, at
least amongst its immediate family members, as none of the
other sialyltransferases analyzed showed notable changes in
their transcript levels In several human cancers where
activating Ras mutations are common, it may be 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 three pathways: the
Raf-MEK-ERK signalling cascade which promotes proliferation
through the activation of transcription factors; the PI3
kinase pathway where lipid kinases generate second
mes-sengers which have diverse effects on cellular physiology
and the RalGEF signalling cascade which involves 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 effectors and thus signal through one pathway
only H-RasV12S35 binds only to Raf and is unable to
activate the two other signalling cascades whereas H-RasV12C40 binds exclusively to PI3 kinases and the H-RasV12G37 mutant specifically activates the RalGEF pathway When the three constructs coding the mutant Ras proteins were transfected into 3T3 fibroblasts only the H-RasV12G37 mutant was able to induce the increased expression of ST6Gal I similar to the wild type oncogenic H-RasV12 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 the clones with high levels
of H-RasV12SC40 showed increased amounts of 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 always accom-panied by a concomitant increase in ST6Gal I enzyme activity
The RalGEF pathway is the least well documented of the three major signalling pathways and most of the physiolo-gical consequences of RalGEF activation are still outstand-ing issues However its importance 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 in 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 expression of the Neu5Aca2,6Galb4GlcNAc epitope synthesized by ST6Gal I were found 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 of tissue specific splice forms of its mRNA derived from selective transcription of multiple promoters
In order to localize the promoter region targeted by Ras we wanted to identify the 5¢UT isoform, which is induced by the RalGEF signal In all cell lines studied that express activated K-RasS12or H-RasV12, the ST6Gal I transcripts found represent the isoform transcribed from the P3
Fig 7 Mapping of P3 within the complete
Siat1 genomic structure Schematic
represen-tation of the mouse ST6Gal I gene Siat1 This
gene spans over 130 kb on chromosome 16.
The transcription start sites at the four major
promotors are indicated by arrows The open
reading frame is encoded by exons II through
VI Exon I is an invariant 5¢UT exon found in
all Siat1 mRNA The two locations 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 small
arrows: P1, liver; P2a-c, B cells, P4, lactating
mammary gland.
Trang 10housekeeping promoter Although enhanced steady state
transcription is the most obvious explanation for the
accumulation of ST6Gal I mRNA in the presence of
K-RasS12or H-RasV12it 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 changes of a
particular class of ST6Gal I 5¢UT transcripts (including P3)
are primarily the result of transcriptional activity at 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 by exons Q, O and 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 sequence
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 than 820 bp apart) and
are coexpressed, there are two possible explanations for the
presence of these alternative 5¢UT leader sequences 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 usually found much
closer together than those for exons Q and R, normally less
than a hundred and usually within 30 nucleotides of each
other [48] On the other hand, Q and R may represent
transcription initiation sites of two related and overlapping
housekeeping promoter regions within the Siat1 gene
However, neither Q nor R appears to be favoured by the
activation through the RalGEF pathway although some
differences could be observed between cell lines
A preliminary analysis of the sequences directly upstream
of the two transcription start sites identified several putative
consensus sequences for transcription factors typical of
housekeeping promoters As both of these regions are
equally responsive to Ras, shared consensus sequences for
transcription factors could be a key to understand their
regulation It is therefore interesting to note that two
sequences recognized by Ras-responsive element binding
protein-1 are present within a few hundred nucleotides of
each transcription start site
An additional observation suggests that the transcription
is initiated at a true housekeeping promoter Although the
amount of ST6Gal I mRNA produced in Ras transformed
cells is close to the levels found in liver [49] the specific
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 in 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¢-RACE
experiments described in this study
The key effect of Ras on growth is to overcome contact
inhibition between cells [35] The 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 and that the Ras 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 also 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 metastatic potential of tumours
Acknowledgements
MD is the recipient of a postdoctoral fellowship from Le STUDIUM (Orle´ans, France) This work was supported by grants from the Ligue Nationale contre le Cancer (comite´s de´partementaux du Loiret et du Loir et Cher), the Centre National de 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 the Orle´ans chapter of the Lions Club FD acknowledges grants from MURST and the Universita` di Bologna We are grateful to Dr J Lau (Roswell Park Cancer Institute, Buffalo, NY, USA), Prof E He´bert (CBM, Orle´ans, France), Dr B Miller (Michigan Cancer Foundation, Detroit, MI, USA), Dr A Scibetta (Cancer Research UK, Guy’s Hospital, London, UK), Dr S Tsuji (The Glycoscience Institute, Tokyo, Japan) and Prof
B M Willumsen (University of Copenhagen, Denmark) for their generous gifts of plasmids or cell lines and to Dr C LeNarvor and Dr
C Auge´ (Universite´ Paris-Sud, Orsay, France) for kindly providing Galb1,4GlcNAcb-OCH 2 Ph.
References
1 Campbell, S.L., Khosravi-Far, R., Rossman, K.L., Der Clark, G.J., Gilbert, F & Glick, M.C (1984) Change in glycosylation of membrane glycoproteins after transfection of NIH 3T3 with human tumor DNA Cancer Res 44, 3730–3735.
2 Bos, J.L (1995) p21ras: an oncoprotein functioning in growth factor-induced signal transduction Eur J Cancer 31A, 1051– 1054.
3 McMahon, M & Woods, D (2001) Regulation of the p53 path-way by Ras, the plot thickens Biochim Biophys Acta 1471, M63– 71.
4 Santer, U.V., Gilbert, F & Glick, M.C (1984) Change in glyco-sylation of membrane glycoproteins after transfection of NIH 3T3 with human tumor DNA Cancer Res 44, 3730–3735.
5 Santer, U.V., DeSantis, R., Hard, K.J., van Kuik, J.A., Vlie-genthart, J.F., Won, B & Glick, M.C (1989) N-Linked oligo-saccharide changes with oncogenic transformation require sialylation of multiantennae Eur J Biochem 181, 249–260.
6 Le Marer, N., Laudet, V., Svensson, E.C., Cazlaris, H., Van Hille, B., Lagrou, C., Stehelin, D., Montreuil, J., Verbert, A & Delannoy, P (1992) The c-Ha-ras oncogene induces increased expression of b-galactoside a-2,6-sialyltransferase in rat fibroblast (FR3T3) cells Glycobiology 2, 49–56.
7 Vandamme, V., Cazlaris, H., Le Marer, N., Laudet, V., Lagrou, C., Verbert, A & Delannoy, P (1992) Comparison of sialyl- and a1,3-galactosyltransferase activity in NIH3T3 cells transformed with ras oncogene: increased b-galactoside a2,6-sialyltransferase Biochimie 74, 89–99.
8 Delannoy, P., Pelczar, H., Vandamme, V & Verbert, A (1993) Sialyltransferase activity in FR3T3 cells transformed with ras oncogene: decreased CMP-Neu5Ac Galb1–3galnac:a2,3-sialyl-transferase Glycoconj J 10, 91–98.
9 Le Marer, N & Stehelin, D (1995) High a2,6-sialylation of N-acetyllactosamine sequences in ras-transformed rat fibroblasts correlates with high invasive potential Glycobiology 5, 219–226.