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Previous Article | Next Article 
Molecular and Cellular Biology, December 2003, p. 9245-9250, Vol. 23, No. 24
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.24.9245-9250.2003
Copyright © 2003, American
Society for
Microbiology. All Rights Reserved.
Maureen J. O'Sullivan,2 Jennifer Doig,1 Ann-Marie Ritchie,1 David J. Harrison,2 David W. Melton,1 Mark J. Arends,3 Martin L. Hooper,1 and Charles E. Patek1*
Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, Western General Hospital, Edinburgh EH4 2XU,1 Division of Pathology, University of Edinburgh Medical School, Edinburgh EH8 9AG,2 Department of Pathology, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 2QQ, United Kingdom3
Received 30 June 2003/ Returned for modification 22 August 2003/ Accepted 18 September 2003
 |
ABSTRACT |
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 Top Abstract IntroductionMaterials and
METHODSResultsDiscussionReferences |
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4A/+ mice produced viable
K-rastm4A/tm4A offspring with the
expected Mendelian ratios of inheritance, and these mice expressed the
K-ras 4B splice variant only.
K-rastm4A/tm4A mice were fertile
and showed no histopathological abnormalities on inbred (129/Ola) or
crossbred (129/Ola x C57BL/6) genetic backgrounds. The results
demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal
mouse development, at least in the presence of functional K-Ras
4B. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and
METHODSResultsDiscussionReferences |
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21 kDa) GTPases which cycle between inactive
(GDP-bound) and active (GTP-bound) conformations at the plasma
membrane, by interaction with a variety of guanine nucleotide exchange
factors and GTPase activating proteins in response to stimulation by a
diverse array of cell surface receptors, including the epidermal growth
factor receptor and cytokine receptors such as interleukin-2
(14). Following
activation, Ras proteins bind and activate a plethora of downstream
effector proteins, including Raf kinases and phosphatidylinositol
3-kinases, and by this means control many cellular functions, including
proliferation, differentiation, migration, and apoptosis
(4,
11,
28). Activating point mutations leading to constitutive activation of the Ras proteins are associated with some 30% of all human malignancies (1), and these mutations render Ras insensitive to the regulatory action of GTPase activating proteins, leading to excessive and inappropriate signaling, resulting in the promotion of cellular transformation. However, while mutationally active ras genes are generally believed to act as dominant oncogenes, recent reports indicate that wild-type K- and N-Ras proteins have tumor suppressor activity (7, 32). Thus, K- and N-ras appear to exert a dual function in that they promote cancer development as gain-of-function oncogenes when mutated and inhibit cancer by tumor suppressor activity when wild type (proto-oncogene).
The high degree of homology between Ras
proteins suggests that functional redundancy may exist among these
proteins, yet mounting evidence exists for unique roles for the
ras gene family members. For example, most human cancers with
ras activating mutations are associated exclusively with a
particular ras gene, suggesting tissue-specific activity:
K-ras mutations are prevalent in lung (
30%),
colon (40%), and pancreatic (90%)
cancers, while H-ras and N-ras activating mutations
are associated with bladder cancer and myeloid leukemia, respectively
(1). Furthermore, while
N-ras and H-ras are dispensable for development both
individually and in combination
(10,
26), mice harboring a
homozygous K-ras null mutation
(K-ras-/-) are not viable and die
between day 12.5 post coitum and term (depending on the genetic
background) due to cardiac, liver, neurological, and hematopoietic
defects (18,
19). Thus, of the
classical ras gene family members (K-, N-, and
H-ras), only K-ras is necessary for embryonic
development. However, the findings that (i)
N-ras-/- mice are healthy but
N-ras-/-,
K-ras+/- mice die in utero
(18), (ii)
K-ras-/-
N-ras-/- mice exhibit a more severe
phenotype than K-ras-/- mice
(18), and (iii) fewer
than expected H-ras-/-
N-ras-/- mice survive embryogenesis
(10) underline the
essential role for K-Ras in development and imply partial functional
overlap between different Ras proteins.
Understanding the role(s) of K-Ras in development and tissue function is an important route to gain insight as to how K-ras activating mutations promote neoplastic change. However, this is complicated by the fact that the K-ras gene encodes two protein isoforms, K-Ras 4A and K-Ras 4B, of 189 and 188 residues, respectively, by alternative splicing of the fourth coding exons 4A and 4B, and K-ras activating mutations that usually affect codons 12, 13, and 61 jointly affect both isoforms (5, 9, 21). The isoforms differ significantly at their C termini after residue 165, including the hypervariable domains and CAAX motifs. These regions are involved in membrane association, which is essential for Ras function (16) via a series of posttranslational modifications, which include isoprenylation, endoproteolysis, and methylation (25). However, these modifications differ between the isoforms due to the sequence differences at the hypervariable domain: K-Ras 4A, like N- and H-Ras, is palmitoylated at cysteine residues upstream of the CAAX motif, which are replaced with a polylysine domain in K-Ras 4B.
The difference in C-terminal modifications of Ras proteins leads to alternative trafficking pathways to the plasma membrane (3) and ultimately localization to different plasma membrane microdomains (23, 24). Indeed, reports that H-, N-, and K-Ras differentially affect Raf-1, phosphatidylinositol 3-kinase, and Rac (28, 29, 31) suggest that they elicit divergent biological responses by interacting with different subsets of downstream effectors. Thus, the posttranslational differences between K-Ras 4A and K-Ras 4B could affect their membrane localization and, therefore, interaction with different membrane targets. The specific association between K-Ras 4B and the guanine nucleotide exchange factor Smg GDS (27) and reports that the oncogenic mutant (G12V) K-Ras 4A and 4B differ in their ability to activate Raf-1, induce transformed foci, enable anchorage-independent growth, and promote cell migration in vitro (28) and that K-ras 4A and 4B are expressed differentially during mouse development and in adult tissues (22, 30) further suggest that they have distinct biological actions. Thus, the cooperative effects of both isoforms could account, at least in part, for the high frequency of K-ras activating mutations in human cancers.
To
examine the role of the individual K-ras splice variants in
development, gene targeting was used to delete exon 4A and generate
K-ras 4A-deficient mice. It was found that
K-rastm4A/tm4A mice are healthy
and fertile. The result demonstrates that expression of K-ras
4A is dispensable for normal development, at least in the presence of
functional K-Ras 4B.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and
METHODSResultsDiscussionReferences |
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4A) was used to delete
exon 4A of K-ras to generate a mutant allele designated
K-rastm4A. The 2.8-kb 5' and the
1.3-kb 3' arms of homology were isolated by HindIII
and XbaI-EcoRI digests, respectively, of an 11.5-kb
EcoR1 fragment (from BALB/c mice) stretching from upstream of
exon 3 to upstream of exon 4B
(13). The neomycin
resistance cassette (containing a phosphoglycerate kinase I promoter)
and the herpes simplex virus thymidine kinase gene cassette were
employed for positive and negative selection, respectively (Fig.
1A).
|
4A/+ embryonic stem
cells.
The targeting vector
was linearized with EcoRI, and 150 µg of DNA was
electroporated (800 V and 3 µF, Gene Pulser, Bio-Rad) into
strain 129/Ola-derived HM-1 male mouse embryonic stem cells, which
harbor an inactivating deletion in the X-linked Hprt gene
(20). Homologous
recombination in colonies resistant to G418 (300 µg/ml;
Invitrogen) and ganciclovir (2 µM; Sigma) was identified
initially by PCR and confirmed by Southern analysis. Embryonic stem
cell clones were screened with the primer set neo22
(5'-CGATAGAAGGCGATGCGCTGCGAAT-3')
and Px4BA
(5'-ATAACTGTACACCTTGTCCTTGACT-3'),
positioned in the neomycin cassette and exon 4B,
respectively (Fig. 1A),
which amplify a 2.1-kb product. The PCR conditions were 1.0 µM
each primer (Sigma), 1x PCR buffer (Invitrogen), 1.5 mM
MgCl2 (Invitrogen), 200 µM each deoxynucleoside
triphosphate (Amersham), 1 unit of Taq DNA polymerase
(Invitrogen), and 100 ng of genomic DNA per reaction.
Following denaturing for 4 min at 94°C, DNA was amplified for
45 cycles (94°C for 1 min, 58°C for 1 min, and
72°C for 2.5 min). Homologous recombination was confirmed by Southern blotting. Genomic DNA (20 µg) was digested with HindIII or PvuII and, following electrophoresis, was transferred to a nylon membrane (GeneScreen) in denaturing buffer (0.5 M NaOH, 1.5 M NaCl). The membrane was neutralized and prehybridized at 65°C for 2 h (10% dextran sulfate, 1%sodium dodecyl sulfate, 6x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 200 µg of herring sperm DNA per ml). The 3' internal and 5' external probes (Fig. 1A) were labeled with [32P]dCTP by random priming (Stratagene) and hybridized to the membrane at 65°C overnight. Membranes were washed at room temperature with 2x SSC (twice for 5 min), at 65°C with 2x SSC-1% sodium dodecyl sulfate (twice for 30 min), then at room temperature with 0.1x SSC (twice for 30 min). Membranes were exposed to film overnight at -70°C.
Production of
K-Ras 4A-deficient mice.
Embryonic stem cells harboring the
heterozygous deletion of exon 4A
(K-rastm4A/+) were injected into
day 3.5 post coitum C57BL/6 blastocysts, which were implanted into the
uteri of day 2.5 post coitum CD1 pseudopregnant recipients. Male
chimeras were mated with 129/Ola or C57BL/6 females to generate inbred
and crossbred lines, and germ line transmission of embryonic stem cell
markers was identified by coat color (light yellow and agouti,
respectively).
Since long-range PCR proved unreliable for
analyzing tail biopsy DNA, mice were genotyped with two different PCRs.
In reaction 1, the primer set neo52
(5'-GATGCCTGCTTGCCGAATATCATGG-3')
and neo22
(5'-CGATAGAAGGCGATGCGCTGCGAAT-3'),
positioned in the neomycin cassette (Fig.
1A), amplify a 206-bp
product in K-rastm4A/+ and
K-rastm4A/tm4A mice. In reaction
2, the primer set Px4AS
(5'-CATTGGTGAGAGAGATCCGACAGTAC-3')
and Px4AA
(5'-TCACACAGCCAGGAGTCTTTTCTTC-3'),
positioned in exon 4A (Fig.
1A), amplify a 72-bp
product in wild-type and
K-rastm4A/+ mice. In both
reactions, DNA was denatured for 4 min at 94°C and amplified
for 30 cycles at 94°C for 30 s and 58°C for
30 s. The genotypes were subsequently confirmed by Southern
blotting with internal and external probes as described above.
In
the inbred colony, first-generation
K-rastm4A/+ males (which do not
harbor the Hprt null mutation) were mated with wild-type
129/Ola mice to generate an inbred stock that was wild type for
Hprt. In the crossbred colony, the Hprt mutation was
allowed to segregate. All animal work was carried out under Home Office
license, with ethically approved methods as set out by the UK
Coordinating Committee on Cancer Research in their guidelines on the
Welfare of Animals in Experimental
Neoplasia.
RNA analysis. K-ras 4A and 4B expression was determined in tissues from adult crossbred (129/Ola x C57BL/6) mice by reverse transcription-PCR (RT-PCR) with primers positioned within exons 1 and 4B, which amplify both isoforms in the same reaction (22). RNA was extracted with Trizol reagent (Invitrogen), and first-strand cDNA synthesis was performed with the SuperScript preamplification system, with 1 to 5 µg of RNA (Invitrogen). For each sample, controls without reverse transcriptase were included and were negative in all cases.
Histology. Mice were sacrificed by cervical dislocation, and tissues were fixed overnight in 10% neutral buffered formalin except for the large and small intestines, which were fixed in Methacarn (methanol, chloroform, and glacial acetic acid, 4:2:1 by volume) overnight. Tissues were wax embedded, and 5-µm sections were cut and stained with hematoxylin and eosin.
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RESULTS |
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TopAbstractIntroductionMaterials and
METHODSResultsDiscussionReferences |
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4A was designed to replace exon 4A of K-ras
with a neomycin resistance cassette (Fig.
1A). Of 324 embryonic stem
clones screened by PCR, 17 were positive for homologous recombination,
amplifying the correct 2.1-kb PCR product (Fig.
1B). The genotype of seven
of these clones was confirmed by Southern analysis with a 5'
external probe that identified a 5-kb wild-type and a 4-kb targeted
band and a 3' internal probe that identified a 2-kb wild-type
and a 6-kb targeted band (Fig.
1C). Of nine chimeras
(generated from three different embryonic stem cell clones), five
transmitted embryonic stem cell-derived coat color markers through the
germ line.
Since the effects of mutations, including the
K-ras null mutation
(18), can vary greatly
with the genetic background, the consequence of deleting K-ras
4A expression was examined in inbred (129/Ola) and crossbred (129/Ola
x C57BL/6) strains of mice. The embryonic stem cell-derived
offspring were genotyped at weaning by PCR (with the primer sets
neo22/neo52 and Px4AS/Px4AA, which amplify the neomycin cassette and
exon 4A, respectively; Fig.
2A) and by Southern blotting (with the 5' external and 3'
internal probes; Fig. 2B),
and it was found that K-rastm4A/+
and wild-type mice were present in the expected Mendelian ratio (54 and
61, respectively; 
21 = 0.426,
P =
0.514).
|
4A/+ mice were genotyped by
PCR (Fig. 2A) and Southern
blotting (Fig. 2B).
Wild-type, K-rastm4A/+, and
K-rastm4A/tm4A offspring were
present in the expected Mendelian ratios on both crossbred (48, 101,
and 58, respectively; 22 =
1.087, P = 0.581) and inbred (11, 20, and 10,
respectively; 22 = 0.073,
P = 0.964) genetic backgrounds, indicating that all
K-rastm4A/tm4A mice develop
normally. In addition, further breeding studies found that male and
female K-rastm4A/tm4A offspring
were present in the expected Mendelian ratios in both crossbred (113
and 113, respectively) and inbred (20 and 25, respectively;
21 = 0.556, P =
0.456) stocks. Male and female
K-rastm4A/tm4A mice were fertile,
and females successfully weaned their young.
Detailed
histopathological analysis was undertaken of 24 crossbred mice at 3
months, including 12 wild-type and 12
K-rastm4A/tm4A mice, with six
males and six females in each cohort. No abnormalities were detected at
necropsy, and examination of liver, kidney, large and small intestine,
stomach, pancreas, spleen, thymus, heart, lung, brain, ovary, uterus,
seminal vesicles, and testis found no difference between
tissues from wild-type and
K-rastm4A/tm4A mice. Inbred
K-rastm4A/tm4A mice
were also fertile and outwardly healthy, and histopathological analysis
of a single animal at 3 months found no abnormalities. The oldest
K-rastm4A/tm4A mice in the
crossbred colony are currently 8 months old, and these too are
outwardly healthy.
K-ras
4A-deficient mice express the K-ras 4B splice variant
only.
To confirm that the
modification of the K-ras gene, by the introduction of the
targeting vector, resulted in the deletion of the K-ras 4A
splice variant, RT-PCR was used to examine K-ras expression in
the large intestine of wild-type and
K-rastm4A/tm4A mice. Only the
K-ras 4B splice variant was expressed by
K-rastm4A/tm4A mice (Fig.
3).
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|
DISCUSSION |
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TopAbstractIntroductionMaterials and
METHODSResultsDiscussionReferences |
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4A/tm4A mice on both
inbred and crossbred genetic backgrounds developed normally, and the
adult mice are fertile and healthy. The finding that K-Ras 4A is dispensable for development suggests that the embryonic lethality of K-ras-/- mice (which do not express either isoform) may result solely from failure of expression of the K-Ras 4B isoform. However, the very fact that K-ras 4A is expressed during development, albeit in a spatially and temporally regulated manner (22), raises the possibility that the lethal K-ras-/- phenotype could result from loss of synergistic function of the two protein isoforms or else from loss of a critical function(s) that can be performed by either isoform, such that one or the other is necessary but in the absence of both development cannot occur. While the latter possibility is less likely, given that only the K-Ras 4B isoform is expressed ubiquitously throughout development (22), questions concerning whether expression of the K-Ras 4B isoform is essential for normal development and whether K-Ras 4A influences its action wait to be addressed by comparing the phenotypes of K-ras-/- and K-ras 4B-deficient mice on the same inbred genetic background. It is nevertheless important to stress that whatever the result of these future studies, our conclusion that K-Ras 4A is dispensable for normal mouse development in the presence of functional K-Ras 4B remains valid.
The C-terminal modifications of Ras proteins are essential
for protein function
(16), and functional
differences between Ras proteins may reflect, at least in part, these
different modifications. Therefore, the findings by Hancock et al. that
N-, H-, and K-Ras 4A undergo similar C-terminal modifications which
involve palmitoylation of cysteine residues upstream of the common CAAX
motif (14) are
significant because these proteins are dispensable for normal
development. Furthermore, the viability of
K-rastm4A/tm4A mice is unlikely
to reflect a compensatory upregulation of these closely related Ras
proteins, since the simultaneous deletion of both K-ras splice
variants does not result in the upregulation of H-ras or
N-ras expression in either tissues or fibroblast cultures from
K-ras-/- embryos
(18). Likewise,
H-ras-/-
N-ras-/- mutant mice show no change
in K-ras expression
(10).
Importantly,
since N-ras-/-,
H-ras-/-, and
K-rastm4A/tm4A mice are fertile,
it is now possible, by crossing these mice, to formally test whether
the viability of K-rastm4A/tm4A
mice is indeed independent of N- and H-ras expression. Also,
since K-Ras 4B, unlike K-Ras 4A, associates with the plasma membrane by
virtue of a polybasic domain and, thereby, may localize to different
membrane microdomains, it could selectively activate Ras effectors
required for embryonic development. Indeed, Ras proteins, including the
K-Ras isoforms, differentially activate particular downstream effectors
(28,
29,
31). Since
K-rastm4A/tm4A mice are viable,
their analysis can also address whether both K-Ras isoforms
differentially affect Ras effector signal transduction pathways in
vivo. This information is crucial in view of current research interest
in anti-Ras agents, some of which target posttranslational
modifications that affect one isoform and not the other, in cancer
therapy
(2).
Previous
studies based on RT-PCR and Northern blotting reported that
K-ras 4A is expressed in adult liver, stomach, small
intestine, colon, and kidney but disagree concerning its expression in
the lung (22,
30). Furthermore, these
studies reported that K-ras 4A is invariably the minor isoform
expressed. The present findings that K-ras 4A is expressed in
a wide range of adult tissues, including lung and in some cases at
levels equal to or greater than that of K-ras 4B (Fig.
4) imply it has an
important role in tissue homeostasis in general, even though it is
dispensable for normal development. In view of recent reports that
N-ras-/- and
H-ras-/- mice exhibit phenotypes
but only after challenge with exogenous agents
(6,
15), a similar approach
with K-rastm4A/tm4A mice,
combined with examination of the long-term consequence of K-Ras
4A-deficiency, may yield insight concerning the role(s) of K-Ras 4A in
tissue function.
The finding that K-Ras 4A is dispensable for
development also presents an opportunity to examine the role of the
K-Ras isoforms in tumorigenesis in vivo. While expression of a
mutationally activated K-ras 4B transgene can promote
development of lung and intestinal tumors
(12,
17), evidence suggests
that mutant K-Ras 4A could also be implicated in tumorigenesis: (i)
expression of a K-ras 4A transgene encoding an activating
mutation can transform cells in vitro and with greater efficiency than
K-ras 4B (28);
(ii) mutationally activated K-ras 4A is expressed by the SW480
human colorectal carcinoma cell line
(5); (iii) the level of
K-ras 4A expression in lung correlates with lung tumor
susceptibility among inbred mouse strains
(30); and (iv) here we
found that K-ras 4A is expressed in the colon, lung. and
pancreas, where tumors with K-ras activating mutations
generally arise in human malignancy
(1). Since mutant K-Ras
isoforms differ in their ability to promote cell transformation and
cell migration (28), it
is conceivable that synergistic interactions between activated K-Ras 4A
and K-Ras 4B could play a crucial role in driving tumor development. In
view of recent evidence that wild-type K-Ras exhibits tumor suppressor
activity (32),
K-rastm4A/tm4A mice can be used
to address not only the role of K-Ras 4A in neoplastic progression in
vivo, but also whether both isoforms exhibit tumor suppressor
activity.
Examination of the role of wild-type K-Ras in adult
tissues, where tumors with oncogenic K-ras mutations normally
arise, is one approach to gain valuable insight concerning how these
mutations promote tumorigenesis. While this has been hindered by the
embryonic lethality of the K-ras null mutation
(18,
19), we envisage that
future studies with
K-rastm4A/tm4A mice will
contribute significantly to understanding why K-ras activating
mutations are so prevalent in human malignancies and why they are
linked with particular types of epithelial cell-derived
tumors.
|
ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present
address: Department of General Medicine, Sir Charles Gairdner Hospital,
Nedlands, Perth 6009, Western Australia,
Australia.
|
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