ABSTRACT
Infecting mice with a mutant Moloney murine leukemia virus which contains the
bacterial suppressor tRNA
supF
in its LTR allows rapid cloning of proviral integration sites from genomic
tumour DNA. In a previous study E
[mu]
pim-1
/E
[mu]
L-myc
bitransgenic mice had been inoculated neonatally with MoMuLV
supF
virus. The retroviral infection led to acceleration of lymphomagenesis
indicating the proviral activation of further oncogenes cooperating with
myc
and
pim-1
in tumour development. Using a functional
supF
screen for analysis of genomic mouse tumour DNA libraries which had been
constructed in the phage vector EMBL3A, a common proviral integration site on
mouse chromosome 5 was cloned and found to be identical to the proviral
integration site
evi-5
which has recently been identified in an AKXD T-cell lymphoma and which is located 18 kb upstream of the
gfi-1
gene. Tumours bearing
evi-5
integrations showed an enhanced
gfi-1
expression level suggesting that
gfi-1
is the target gene for insertions at the
evi-5
locus. Together with three other previously described Moloney integration
clusters all responsible for enhanced
gfi-1
expression the number of tumours from infected double transgenic E
[mu]
L-
myc
/E
[mu]
pim-1
transgenic mice with retrovirally activated
gfi-1
added up to 53% underscoring the role of GFI-1 as an effective collaborator for MYC and PIM-1 in the process of lymphomagenesis.
Infection of mice with the non-acute transforming retrovirus Moloney murine leukemia virus (MoMuLV) has
become an established method to search for new genes implicated in the process
of lymphomagenesis (
1
,
2
; for review see
3
). Genomic DNA from the tumours that arise upon infection is analysed for
proviral integrations, and genes that are located in the vicinity of viral
sequences can be identified. Several mechanisms lead to the activation of genes
that are hit by proviral integrations: by promoter and enhancer insertions, the
expression of the host target gene becomes elevated by the retroviral
transcription control elements residing within the LTR sequences.
Alternatively, the proviral integration could either remove RNA destabilising
elements (
3
) or it could lead to a differently spliced gene products with new properties (
4
). Proviral integration sites can be isolated either by inverse PCR reaction or
by screening a genomic DNA library constructed from DNA of infected tissue or
cells with a virus specific probe. With this method, phage clones containing
genomic mouse sequence in their inserts that flank the proviral DNA can be
isolated (
2
).
Transgenic mice expressing activated oncogenes under cell type specific promoter
and enhancer elements have been proven to be valuable tools to investigate
oncogene cooperation
in vivo
(
5
-
7
). A well studied system have been E[mu]
c-myc
transgenic mice where the
c-myc
gene is linked to an immunoglobulin heavy chain enhancer (
8
,
9
). Here, the lymphocyte specific expression of the transgene leads to
development of pre-B or B-cell lymphoma (
8
,
10
). To search for oncogenes that cooperate with
c-myc
, a retroviral infection of newborn E[mu]
c-myc
transgenic mice had been performed and led to identification of the cytoplasmic
serine/threonine kinase PIM-1 and the zinc finger protein BMI-1 as strong collaborators of c-MYC in the development of B-cell lymphoma (
1
,
2
). Another retroviral integration site,
pal-1,
that was hit in a large proportion of tumours (
2
,
11
), was identified in this experiment. Subsequent studies showed that proviral
activation within the
pal-1
locus lead to the activation of a gene termed
gfi-1
(for growth factor independence 1) located several kb downstream of
pal-1
(
11
,
12
). The
gfi-1
gene had previously been identified in a search for genes that confer T-cells the independence of IL-2 (
13
). The efficient collaboration between
myc
and the
pim-1
gene was not only evident in MoMuLV infection experiments but could also be
shown by creating various E[mu]
myc/pim-1
double transgenic mice which show a dramatic acceleration in lymphomagenesis
compared to the single transgenic parent strains (
14
,
15
).
The analysis of tumours arising in E[mu]
myc/pim-1
bitransgenic mice also showed that all malignancies were still of clonal or
oligoclonal origin. This indicated that additional events were required for
full malignant transformation even in the presence of two activated
trans
-oncogenes. To search for these additional cooperating oncogenes that
synergize in the process of lymphomagenesis with both
myc
and
pim-1
, a MoMuLV infection of E[mu] L-
myc/pim-1
bitransgenic mice was carried out. The infection resulted in a profound
acceleration of tumour formation in these animals and the analysis of the
emerging tumours showed that in 37% of the cases the
pal-1/gfi-1
locus was affected by retroviral integration resulting in high level expression
of the
gfi-1
gene (
12
). This finding underscored the potential of the
gfi-1
gene to efficiently cooperate with both
myc
and
pim-1
genes.
In the infection experiment with E[mu]
pim-1
/E[mu]
L-myc
bitransgenic animals a replication competent mutant MoMuLV virus strain was
used which contains a 200 bp
supF
suppressor tRNA in its LTR sequence (
16
). In this paper we present the identification of proviral integration sites
from tumours that arose in infected bitransgenic mice by taking advantage of a
supF
selection procedure. To this end, we used the EMBL3A phage cloning vector to
construct genomic DNA libraries from DNA isolated from tumours of infected
animals. The EMBL3A vector contains two amber stop codons in genes essential
for its replication (
17
) so that plating the libraries on the
supF
-
bacteria strain MC1061 allowed exclusive isolation of phage clones with
retroviral
supF
and flanking mouse sequences in their inserts. Using this functional
supF
screen we could clone and analyse a common integration site which was
tentatively named
tmi-1
(for T-cell Moloney integration site 1) and which was affected by proviral
insertions in 22% of the tumours in the Moloney infected E[mu]
pim-1
/E[mu]
L-myc
bitransgenic mice. Detailed analysis revealed that
tmi-1
is identical to the recently discovered Moloney integration site
evi-5
on mouse chromosome 5 (
18
) and probably activates the expression of the
gfi-1
gene located ~18 kb downstream of
evi-5.
Transgenic E[mu]
pim-1
and E[mu]
L-myc
animals and the generation of doubly transgenic animals have already been
described (
14
,
25
). Mice originating from breedings between E[mu]
pim-1
and E[mu]
L-myc
parents were infected until 48 h after birth intraperitoneally with 50-100 [mu]l sterile MoMuLV
supF
containing supernatant from the producer cell line MoMuLV
sup
-1 (
16
). The supernatant of subconfluently growing virus producing cells was
concentrated 100-fold under N
2
pressure in a stirring cell (Amicon) to obtain a virus titre of 10
4
-10
5
p.f.u./ml. Virus containing supernatants were titrated by seeding 1 * 10
5
XC cells per well into a 24-well plate. The wells were inoculated with serial dilutions of virus
containing cell culture supernatant and the titre was determined from the
number of syncitia 24 h later (
26
). XC cells were maintained in DMEM with 10% FCS and antibiotics.
Genomic tumour DNA (100-500 [mu]g) was partially cleaved with
Sau
3A and enriched for fragments of 10-20 kb by a 10-40% sucrose gradient. The size-selected inserts were ligated to the
Bam
HI cleaved vector arms of the lambda replacement vector EMBL3A. For preparation
of the phage arm 100 [mu]g EMBL3A DNA were cleaved to completion with
Bam
HI and
Eco
RI. The original insert and the `stuffer' polylinker fragment were separated
from the cloning phage arms by a 10-40% sucrose gradient. The ligation reaction was packaged by using a
lambda
in vitro
packaging kit from Amersham. An aliquot of the packaging reaction was plated on
the
supF
+
bacterial strain LE392 to determine the titre of the library. Phage particles
(10
6
) were then plated with the
supF
-
bacterial strain MC1061 which only allowed propagation of phage clones
containing retroviral
supF
sequence in their insert. To molecularly clone the unrearranged genomic DNA at
the
tmi-1/evi-5
locus a genomic mouse library (Stratagene, liver, agouti, [lambda] FixII vector) was screened for overlapping clones.
For library construction and
supF
screening the following bacterial strains were used: LE 392
supE
44
supF
58
hsdR
514
galK
2
galT
22
metB
1
trpR
55
lacY
1; MC1061
hsdR mcrB araD
139 [Delta]
(araABC-leu)
7679 [Delta]
lacX
74
galU galK rpsL thi.
Based on rearrangements of the T-cells receptor [beta] locus, the immunoglobulin heavy and light chain genes and on the
presence of characteristic surface markers (B220, Thy 1.2, CD4, CD8) as
determined by FACS analysis (Becton-Dickinson FACSCAN) the tumours were classified as either of the T- or the B-cell lineage. The probes used to detect distinct
rearrangements of either the T-cell receptor gene locus or immunoglobulin genes in Southern blot analysis
with genomic tumour DNA have already been described (
27
).
Preparation of genomic DNA from mouse tail tips and tumour samples was performed
as previously described (
27
). Cleavage of DNA by restriction enzymes and DNA blotting procedures were
performed as described elsewhere (
28
). The
supF
probe was a 200 bp
Bam
HI-
Bam
HI fragment containing the complete sequence of the
supF
suppressor tRNA and had been kindly provided by Dr Stocking, Heinrich-Pette-Institut, Hamburg. The probes E2.1 and D22 that were used to detect
proviral insertions within the
tiam-1
locus were 1 kb
Eco
RI and
Sal
I genomic DNA fragments, respectively (
4
). The probe for GAPDH has already been described (
25
). To detect
gfi-1
integrations a 2.0 kb
gfi-1
mouse cDNA fragment was used which contained almost the complete coding sequence
with only the first five amino acids missing. The
evi-5
probe was a 1.4 kb mouse genomic fragment (
18
). As a
bla-1
probe a genomic 1.9 kb
Eco
RI-
Sal
I fragment and as a
pal-1
probe a genomic 1.3 kb
Bgl
II-
Eco
RI fragment from the respective locus were used (
2
). Preparation of RNA was as described (
29
) and Northern blotting was performed according to established procedures (
28
). For hybridisation of the
evi-5
probe with poly(A)
+
RNA from different mouse organs a Northern blot membrane from Clontech
(Multiple Tissue Northern Blot) was used.
Chromosomal localisation of the
tmi-1
locus and the mouse cell lines used for FISH analysis was performed as
described elsewhere (
19
).
To identify novel oncogenes cooperating with
myc
and
pim-1
in the process of lymphomagenesis we infected E[mu]
pim-1/
E[mu]
L-myc
double transgenic mice with a mutant replication competent Moloney murine
leukemia virus carrying the bacterial suppressor tRNA
supF
in the proviral LTR sequence (Fig.
1
;
12
,
16
). The infected bitransgenic animals succumbed to 100% with lymphoid
malignancies after an average latency period of 66 days. This was significantly
shorter compared to the onset of tumourigenesis in uninfected
pim-1/L-myc
transgenic control mice indicating the proviral activation of further oncogenes
cooperating in the process of lymphomagenesis (
12
,
14
). For cloning and analysis of the retroviral integration sites we made use of
the fact that the
supF
suppressor tRNA cloned into the LTR of the Moloney virus used in this infection
experiment helps to override amber stop codons in the
Aam32
and
Bam1
genes of the lambda phage vector EMBL3A (
17
) thereby allowing its replication and propagation. By using a
supF
probe for hybridisation of Southern blots with genomic DNA from mouse tumours
we confirmed that the integrated proviruses still contained the
supF
sequence and had not lost the suppressor tRNA due to recombination events (data
not shown). The Southern blots also showed that each tumour carried in average
four to five proviral integrations. We chose five tumours from MoMuLV
supF
infected E[mu]
pim-1/
E[mu]
L-myc
double transgenic mice with no virus insertions in genes known to be frequent
target sites for MoMuLV integrations (i.e.
myc
,
pim-1
,
pal-1
,
bla-1
and
bmi-1
) to isolate DNA for the construction of genomic DNA libraries with the EMBL3A
phage cloning vector. The titre of each library was evaluated by growth on the
supF
+
bacterial strain LE392 which allows replication of all phage particles. Plating
the libraries on the
supF
-
bacterial strain MC1061 led to replication of only those phage particles which
contained in their DNA inserts a MoMuLV
supF
LTR providing the essential suppressor tRNA for propagation. The numbers of
phage clones obtained with MC1061 bacteria (
supF
-
) are shown in Table
1
. The average size of the DNA inserts was in the range of between 12 and 17 kb
so that a reasonable portion of mouse DNA flanking the retroviral integration
site was isolated. All phage clones isolated by this screen contained
retroviral LTR with its
supF
sequence in their inserts as could be confirmed by Southern blots using a
supF
specific probe (data not shown).
To search for common integration sites which were affected by MoMuLV insertions
in several tumours, Southern blot hybridisation of tumour DNA with probes
obtained from the genomic mouse DNA sequences flanking the proviral insertion
sites in the
supF
phage clones was performed. For this purpose, a restriction map of several phage
clones was established. Figure
2
shows a partial restriction map of
supF
phage clone 25/33. The phage insert contained the complete proviral MoMuLV
supF
sequence an in addition 500 bp flanking sequences upstream and ~4 kb downstream of the 5'- and the 3'-LTR, respectively (Fig.
2
). The 2.2 kb
Pst
I-
Eco
RI fragment downstream of the 3'-MoMuLV
supF
LTR was designated probe I (Fig.
2
) and was used in Southern blot analysis with DNA from all tumours of the
infected
L-myc/pim-1
bitransgenic mice. Fourteen out of 63 tumours (22%) showed retroviral
integrations at this locus. The insertions were detected by Southern blot
hybridisation using three different restriction enzymes (Fig.
3
). The membranes were then stripped off the probe and rehybridised with the 200
bp
supF
fragment confirming that all signals representing alleles occupied by proviral
DNA also contained
supF
sequences (data not shown). We concluded that the genomic locus covered by the
insert of phage clone 25/33 contains a common proviral integration site which
was tentatively termed
tmi-1
(for T-cell Moloney integration site 1).
For further characterisation of the
tmi-1
locus a genomic mouse DNA lambda phage library (Stratagene, agouti, liver, [lambda]-FixII vector) was screened with the 2.2 kb
Pst
I-
Eco
RI fragment from phage clone 25/33 (probe I). Two overlapping clones could be
isolated and were used to establish a partial restriction map and to localise
the proviral integrations of several tumours (Fig.
4
). The positions of proviral
tmi-1
integrations were found to span a region of ~6-7 kb (Fig.
4
). Data from genomic Southern blots indicated in a
Hin
dIII digest the existence of two germline fragments of 7 and 2.3 kb (Fig.
4
). However, the restriction map derived from genomic phage clones isolated with
probe I indicated only one germline
Hin
dIII fragment (Fig.
4
). A similar situation is found for
Bam
HI and
Asp
718 digests suggesting that probe I contains sequences that recognise another
locus besides
tmi-1
. Indeed, FISH experiments to determine the chromosomal localisation of
tmi-1
confirmed this conclusion. In order to identify loci on mouse chromosomes by
FISH we had previously established two mouse lymphoma cell lines WMP-1 and WMP-2 by MoMuLV infection (
19
). These lines were obtained from a wild mouse strain (WMP) that carries
Robertsonian translocations which allow an easy morphological distinction
between different chromosomes (
19
). As a result,
tmi-1
was clearly localised to chromosome 5, however, some of the mitotic figures
revealed hybridisation to chromosome 14 (Zörnig and Möröy unpublished). Therefore, it is possible that the additional
2.3 kb DNA fragment that is recognized by probe I represents another genomic locus with sequence homology to
tmi-1
. Alternatively, this fragment could be the result of a restriction fragment
length polymorphism. This is not unlikely as the mouse strain that was used for
the MoMuLV experiment is different from the strain used for the construction of
the genomic library.
By using the 2.9 kb
evi-5
probe for a Northern blot hybridisation with total RNA from tumours bearing
proviral
tmi-1/evi-5
integrations no signal could be obtained (data not shown) raising the question
what target gene is affected by proviral insertion at the
tmi-1/evi-5
locus. Northern blot with poly(A)
+
RNA isolated from different mouse organs (Stratagene) the
evi-5
probe detected a 7.5 kb message in several organs, especially abundant in liver
where an additional 2.4 kb band appeared (Fig.
6
). These data suggest that although there is a transcribed sequence in close
vicinity to the
tmi-1/evi-5
integration, it is very unlikely that the expression of this
evi-5
associated gene is transcriptionally activated by proviral integrations in
lymphoid tumours.
For further investigation of a possible target gene activated by
tmi-1/evi-5
integrations we next analysed
gfi-1
expression in a northern blot hybridisation with total tumour RNA.
Interestingly, when the murine
gfi-1
cDNA was used as a probe all tumours harbouring a MoMuLV
supF
tmi-1/evi-5
insertion showed an increased
gfi-1
mRNA level compared with thymus and spleen from wild-type mice (Fig.
7
). Increase in
gfi-1
expression in these tumours is to the same extent as in tumours which exhibited
elevated
gfi-1
RNA amounts due to proviral insertions at two other integration loci immediately
5' and 3' of the
gfi-1
gene affecting its expression (
12
). These results suggest that
evi-5
is another common proviral integration site that activates
gfi-1
expression and thereby participates in the process of lymphomagenesis.
Table
2
lists second-site mutations at known common proviral integration sites in tumours with
tmi-1/evi-5
insertions from MoMuLV
supF
infected E[mu]
pim-1/
E[mu]
L-myc
double transgenic mice. Five of the tumours bearing a
tmi-1/evi-5
integration also show viral activation of the
tiam-1
gene (
4
). Five tumours show a further MoMuLV integration at the
pal-1
locus. Tumour 154 shows in addition to its
tmi-1/evi-5
integration a further direct activation of
gfi-1
by a MoMuLV
supF
insertion upstream of and close to the
gfi-1
gene. One tumour bearing a retroviral
tmi-1/evi-5
integration also exhibits an insertion in the
bla-1
locus which probably leads to activation of the zinc finger oncoprotein BMI-1 (
1
,
2
; A. Berns, personal communication).
In a previous study we reported retroviral activation of the
gfi-1
and the
tiam-1
gene in T-cell lymphoma in an infection experiment with E[mu]
pim-1
/E[mu]
L-myc
bitransgenic mice (
12
). For the infection of these animals a mutant Moloney virus strain was used
which contains the
bacterial suppressor tRNA
supF
in its LTR. We attempted to isolate proviral integration sites in a functional
supF
screening procedure: genomic DNA libraries were constructed with the EMBL3A
phage cloning vector which contains amber stop codons in two of the viral genes
essential for phage replication (
17
). When plated with the
supF
-
bacterial strain MC1061 only those phage clones could replicate that contained
the retroviral
supF
sequence in their insert. All 68 clones obtained from five different lambda
phage libraries which were constructed with the EMBL3A vector and genomic
tumour DNA contained retroviral
supF
sequences in their insert representing MoMuLV
supF
integration sites. This procedure allowed to identify a common proviral
integration site and demonstrated that MoMuLV retroviral insertion sites can be
isolated from tumour tissue quite efficiently by using the MoMuLV
supF
mutant and the appropriate selection procedure.
Table 2
.
Second site MoMuLV proviral insertions in tumours from infected double
transgenic mice
The integration site identified in the
supF
selection experiment was found to be identical to
evi-5
which was originally discovered as a novel common site of retroviral
integration in AKXD T-cell lymphoma and is located 18 kb upstream of
gfi-1
on mouse chromosome 5 (
18
). Although it was also reported that
evi-5
cosegregated with
gfi-1
it remained unclear what gene is activated and is therefore involved in the
process of tumourigenesis by the proviral insertion at
evi-5
(
18
). To address the question we used different genomic probes from the locus to
search for coding sequences. None of these probes revealed signals on Northern
blots with total RNA from tumours bearing
tmi-1/evi-5
integrations. It therefore seems very unlikely that proviral
tmi-1/evi-5
insertions lead to an elevated expression level of coding sequences nearby the
integration site.
When we analysed lymphoid tumours with
tmi-1/evi-5
MoMuLV
supF
integrations for
gfi-1
RNA amounts we found a significantly enhanced mRNA expression level which was
comparable to the
gfi-1
RNA amount found in lymphomas harbouring MoMuLV
gfi-1
insertions. Thus it seems very likely that proviral integrations in the
tmi-1/evi-5
locus lead to
gfi-1
overexpression whereby the retroviral LTR sequences would have to act over a
distance of 18 kb. This action over large distances is not unprecedented.
Proviral integrations at
Mlvi-1
and
Mlvi-4
map between 30 and 270 kb 3' of the
c-myc
gene and upregulates its expression (
20
). Another example is the transcriptional activation of the cyclin D1 gene (
21
). Here, retroviral integrations at the
Fis-1
locus activated expression of cyclin D1 over a distance of 50-300 kb (
21
). On the other hand, these findings indicate that besides the activation of the
gfi-1
gene another target could be activated by tmi-1/evi-5 integrations that is located several hundred kb apart.
Three other proviral insertion clusters have been described which also lead to
gfi-1
overexpression in Moloney virus induced murine lymphoma (
11
,
12
): virus integrations immediately upstream or downstream of the
gfi-1
coding sequence as well as provirus insertions at the
pal-1
locus enhance
gfi-1
transcription and thus are very likely to contribute to T- and B-cell lymphomagenesis. Taking all these
gfi-1
affecting integration sites together, the total percentage of tumours with
retrovirally activated
gfi-1
in the MoMuLV
supF
infected E[mu]
pim-1
/E[mu]
L-myc
bitransgenic mice adds up to 53% excluding double integrations (
12
). This high percentage confirms the important role of
gfi-1
as an effective collaborator of
myc
and
pim-1
in lymphomagenesis. Originally,
gfi-1
had been identified as a retroviral integration site in rat T-cell lines in a screen for IL-2 independence (
13
). Overexpression of the
gfi-1
gene together with other yet unidentified events leads to IL-2 independent growth of former strictly IL-2 dependent growing T-cells (
12
,
13
). Transfection experiments with CTLL cells have indicated that
gfi-1
expression is involved in an increase in proliferation rate (
12
) although it is very well possible that GFI-1 acts also by inhibiting apoptosis of IL-2 deprived T-cells. As the independence from growth factors like IL-2 is a prerequisite to tumour progression towards a more malignant state it would be a
reasonable model to assume that
myc
, a transcription factor important in the regulation of cell proliferation, and
pim-1
, a cytoplasmic serine/threonine kinase, would cooperate in the onset of tumourigenesis while engagement of
gfi-1
in a tripartite cooperation with
myc
and
pim-1
leads to tumour progression.
T- and B-cell lymphomagenesis is a multistep process in which activation of
several cooperating oncogenes and inactivation of certain tumour suppressor
genes contribute to malignant transformation of the lymphocytes. Therefore, it
is not surprising to find MoMuLV integrations at several common integration sites such as
tiam-1
and
pal-1
or
bla-1/bmi-1
in tumour DNA from the same infected mouse.
Tiam-1
has recently been identified in a search for genes that coordinate the invasive
behaviour of already transformed cells (
4
). Moreover, the TIAM-1 protein is thought to act as a GDP/GTP exchanger for the
rho
family of GTPases (
4
,
22
). Five tumours show a further MoMuLV integration at the
pal-1
locus. While transcriptional activation of a candidate gene within the
pal-1
locus has not been observed in tumours with
pal-1
integrations all these tumours express high levels of
gfi-1
mRNA confirming that retroviral insertions into the
pal-1
locus also activate the
gfi-1
gene (
11
,
12
). MoMuLV integrations at the
tmi-1/evi-5
and the
pal-1
locus in the same tumour might further upregulate
gfi-1
expression. BMI-1 belongs to the polycomb family (
2
) and seems to be important in regulating the proliferation of a number of
hematopoietic cells throughout pre- and postnatal life as well as for morphogenesis during embryonic
development (
23
). Deregulated overexpression of BMI-1 in lymphocytes of E[mu]
bmi-1
transgenic mice provokes a predisposition for lymphoma and strongly collaborates
with c-MYC in tumourigenesis (
24
).
We are indebted to A. Berns and the members of his lab for the evi-5 probe and for the phage clone [lambda]23AB. We thank A. Grzeschiczek for excellent technical assistance
and C. Stocking for providing the MoMuLV
supF
producing cell line. This work was supported by grants from the Deutsche
Forschungsgemeinschaft (DFG, SFB 215-D10) and the Fonds der Chemischen Industrie to T.M. and by a stipend from
the Graduiertenkolleg `Zell- und Tumorbiologie' to T.S.
+
Present address: Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer
Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
Tumour number
Cell type
Additional identified proviral
MoMLV supF integration sites
12
T
bla-1
21
T
pal-1
55
T
pal-1
84
preB
tiam-1
112
preB
tiam-1
113
T
tiam-1
129
preB
pal-1
146
Preb
pal-1
154
T
gfi-1
188
preB
tiam-1
199
T
tiam-1
209
T
pal-1
REFERENCES
Return