Download - P 53 Tumour Biology
ARTICLE
Identification of novel oncogene RPL 10a using a Daudi cDNA library in p53 KO Mouse Embryonic Fibroblast Cells
Anil Babu. J. Gaurav. D. Dwivedi*
Department of Molecular Biology, Umeå University, Umeå-901 87, Sweden
*Correspondence: [email protected]
Summary
The p53 protein is involved in the regulation of cell cycle and apoptosis. Many cancers were investigated for the state of p53
and in over half of the cases, p53 action was found to be absent. Using the Western Blot study, we analyzed tumors from
Myc mice to comprehend the relation of p53 and ARF. Here we identified the novel RPL 10a in Knockout (KO) mouse
embryonic fibroblast (MEF) cells transformed by Viraport XR plasmid Daudi cell lines to determine the potential oncogenic
nature of RPL 10a Jointly, these conclusions create a hypothesis that RP-Mdm2 interaction serves as an important p53 stress-
signaling pathway which gets stimulated by abnormal ribosome biogenesis and proves vital for shielding against oncogenic c-
MYC-induced tumors.
Introduction
P53 is a tumor suppressor gene which is associated with
the regulation of various cellular activities which leads to
cellular stress like cell cycle arrest, senescence, and
apoptosis. Mutations in p53 gene and loss of its function
through deletion, mutation and epigenetic silencing are
frequently found in both familial and sporadic cancers.
More than 50 % of human cancers caused due to the
mutations in TP53 or inactivation of p53 pathway
(overproduction of p53 inhibitor MDM2 –murine double
minute2).Myc is a transcription factor which activates Arf
dependent p53 pathway (Evan and Vousden, 2001; Ko and
Prives, 1996).
Abnormal oncogenic factors are usually responsible in
activation of p53 through the p19ARF
pathway (Sherr and
Weber, 2000; Sherr, 2001). The intensity of p19ARF
protein
is increased by the tumorigenic signaling, as occurs with
overexpression of c-myc or mutational activation of ras
(Palmero et al., 1998; Zindy et al., 1998). The rising ARF
level is affecting the inhibition of Mdm2-mediated
ubiquitylation of p53 (Kamijo et al., 1998; Zhang et al.,
1998), where p53 undergoes degradation in 26S
proteasomes (Roth et., al.1998).
Hence, ARF increases the availability of p53 and results in
either p53-mediated cell cycle arrest or apoptosis. p19ARF
inhibits tumor development both through p53 dependent
and p53 independent pathway. The molecular mechanism
involved in p53 independent function by inhibiting the
processing the rRNA through interactions with
nucleophosmin (Kawagishi,Nakamura ,Maruyama
, Mizutani , Sugimoto , Takagi , Sugimoto).This pathway
apparently supports to protect the organism from cells
that recruit anomalous oncogenic factors and may be turn
into cancers (Sherr, 2001). Recent genetic studies are
supporting the evidence for p53 independent function of
Mdm2 (Freedman et., al 1999).
In this study we observed the different levels of Arf and p53 status in tumor samples which are harvested from lambda-myc mice, and searching for a novel oncogene in p53 KO mouse embryonic fibroblasts, so that we used p53 KO mouse embryonic fibroblast with retroviruses containing c DNA library from Daudi cell line for screening of novel oncogene. Ribosomal protein-Mdm2 interactions are focusing the new insights for the cancer therapy, through p53 independent Mdm2 (RING finger) functions (Marine,et., al 2010).
Significance
Ribosomal proteins are in the limelight of the modern cancer research. In our studies we found about the unique oncogenic capability of the human ribosomal protein L10a (RPL10a) in a p53-Knock out mouse embryonic fibroblast cells. The recent studies suggesting that similar interaction between RP-Mdm2 provokes the formation of Eµ Myc induced lymphomas. We put forward that the interaction between RPL10a and Mdm2 can imitate some effects of Myc over-expression, contributing in the tumor (lymphoma) progression in the loss of p53 function.
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Results
Western Blot analysis of p53 and ARF expressions
The λ Myc lymphoma samples contained different
levels of p53 , p19ARF
which is illustrated by the
Western Blot results. In case of sample 1 (λ 956) and
sample 2 (λ959) p53 expression is normal, while
sample 3 (λ 962) showed that p53 was mutated.
Figure 1. Western Blot Analysis of p53 and ARF Levels.
The above samples, sample 1 (λ 956),sample 2 (λ
959),sample3 ( λ 962), were analyzed by western blot
using anti-p53, anti-ARF, and anti-actin antibodies. The
analysis showed different variations of p53 and ARF
expression.Sample3 ( λ 962) indicated that overexpession
of ARF and mutated p53 also observed,while sample 1 and
sample 2 showed normal expression of p 53 ,however no
ARF.
Searching for novel oncogene by cDNA library
p 53 Knockout (KO) mouse embryonic fibroblast
(MEF) cells were infected with the Viraport XR
plasmid Daudi cDNA library, transformation was
determined by foci formation as mentioned in figure
2. PCR analysis of some foci results in the formation
of smear but some of the foci shows two or three
bands. Lane 1 represents DNA ladder (1Kb marker),
On Lane 2, 5 and 9 foci bands were not separated
well. Foci bands which were well separated and
strong were selected for sequencing.
Figure 2. Number of foci observed after infection of
p53KOMEFs.
Daudi cell line cDNA library were used to transform
p53KOMEFs. The cells were also aseptically cultured as a
negative control (uninfected). Followed by 19-days of cell
culture, number of foci was obtained as 8, and 12, foci for
retroviral control, cDNA library, and uninfected plates,
respectively.
Figure 3. PCR reaction and DNA extraction from agarose
gel.
The genomic DNA was extracted from 9 foci followed by
PCR analysis and loaded on an agarose gel .6 bands were
selected and sent for sequencing. The band 4 resembles to
the gene coding for RPL10a.
0
2
4
6
8
10
12
14
RV-cDNA cDNA Uninfected
Nu
mb
er
of
Foci
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Table 1. Sequencing Result of Band 3
Caaatgtgccttaatataggcgctggggcttgcccatggtgctcttgatatataaggcccggacattctgccagtttttcttgggcaatgacaccaagaagttgacagccaggtgaatgttatacacaagctcatcgtctgtcatcttcacgtgaccaacagctacagccagacataacaccttcttcatttggaacttgattgtggacttcacctcatccactttggccaccatgttttcgttgtgtgtgagcagggaagggaactttcctgccttatttaaacctgggccgaggattcgtggaatctgcttgatcagagactctgaggccaaaaacgcatcatatttcttggccagcttcttgaccagttttttattcttgttgagttttttcagcgcctcgatgtccatgtgggggatatccacggccttagcctcgtcacagtgctgctggtcccccaggacacacacagagaacttagggcggggagtggacttaagcctgacggtgcccgagaagcgcttgtccttctggggatcatagttcttcaagctgatctgcaactccaccgtctccaggaacttgcggcgcttgcgctggttcccgtgcaggacttcccgcaccgcctcgtacagggtgtcgcgagagactttgcctcgtgccgaattcgtcgacaattcgatccggcagtctagaggatggtccacccccggggtcggcagccttcacgtgggcggcgtgtatccaagctgcgatgccgtctactttgagggcggtgggggtggtcagcaggactgtgtaaggtcctttccagcgaggttctaggttcttagtctggtgtcggcggacccacactgt
Gel electrophoresis results of mini preparations
plasmid DNA.
Comparison between the Miniprepartions plamid
DNA digested with restiction enzymes(Eco RI) or un
digested using Gel electrophoresis analysis which
indicates that colony 3 Eco RI digested sample match
with the PCR product band 4 ,we isolated from the
PCR analysis (figure 3).The insert bands from Figure
4 match the PCR product band from figure 3.
Figure 4. Plasmid DNA digestion with EcoRI.
After incubation of plasmid samples with EcoRI, the
digested samples (D) and undigested plasmids (UD) were
loaded on an agarose gel. Two different DNA band sizes
were observed between UD and D plasmids extracted
from all colonies. The digested plasmid DNA samples 1, 3,
and 4 showed two bands representative for successful
enzymatic digestion.
After sequencing the selected 6 bands (Figure 3)
were identified as bands 1, 3 and 4(Figure 4)
representing novel oncogenes. The band 2 correlates
the genes from untransformed cells next to
transformed cells, and accidentally harvested. The
gene which is band 3 out of the three bands (Figure
4) selected for sequencing, the sequenced gene was
identified through BLAST search as Gene ID: 4736
(Ribosomal Protein L10a) and the details mentioned
in Table 1.
Discussion
Precise diagnostic and therapeutic stratagies could
be developed by understanding the biochemical,
cellular,and molecular mechanisms which are the
back bone of the different stages of cancer
development.
The western Blot analysis results show the p53 level,
sample one and two are almost similar, but it was
quite higher in third sample. In sample one and two
there is no Arf expression which is similar to loss of
Arf function observed in 24 % of overall Arf- MdM2-
p53 pathway deregulation (Eischen., et.al 1999 ), but
the third sample indicating the overexpression of
both p53 and Arf. In p53 KO cells, ARF directly binds
to c-Myc protein and inhibits c-Myc–induced
hyperproliferation and transformation with
associated inhibition of acknowledged c-Myc target
gene induction (Boone., et al 2010).The p53 is
stabilized by Arf by binding with MDM2, so it would
have also expressed at higher levels as p53, due to
deregulation of myc (Translocation of myc gene
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between chromosome 8 and chromosome 14-t8:14,
leads to Burkitt’s lymphoma.
The number of foci found in the plates is important,
which indicate the efficiency of transformation of
mouse embryonic cells to transformed cells. The p53
KO mouse fibroblast embryonic cells transformed
with the cDNA vector identified as the novel
oncogene ribosomal protein (RP L10a). Many of the
ribosomal proteins are majorly involved in the RP-
Mdm2 interaction to inhibit the ribosomal
biogenesis (Macius et al 2010). The development of
Eµ-Myc-generated B-cell lymphomas and intestinal
adenomas as a result of APC loss are delayed in mice
with decreased levels of Mdm2. Loss of RP-Mdm2
interaction provokes Eµ-Myc induced
lymphomagenesis. c-Myc upregulates ribosome
biogenesis through the activation of many nucleolar
proteins which involved in this process (Ruggero and
Pandolfi 2003). Eµ-Myc transgenic mice develop B
cell lymphomas (Adams et al., 1985) similar to that
which are observed in Bukitt’s lymphomas bearing a
translocated (t8;14) c-MYC allele(Alitalo et al.,
1987).Similar to the mammalian system, deficiency
of several Ribosomal proteins in zebrafish also
initiates p53 dependent cell growth arrest and
apoptosis. Overexpression of RP initiates tumor
formation (Chakraborty et., 2009). In case of p53
independent functions of Mdm2, likewise in Mdm2
(RING Domain basis) ubiquitylation contributes to
Ras-ERK- mediated degradation of the transcription
factor, FOXO3a to promote cell proliferation (Marine
et., al 2010). Another evidence for p53 independent
Mdm2 action is the ability of the Mdm2 to induce
monoubiquitylation of dihydrofolate reductase
(Maguire et., al 2008). The other proteins include Rb,
E2F1 and Samds are also supporting the Mdm2
oncogenic function of p53 independent and Mdm2
plays a key role in IGF1R mediated p53 independent
proapoptotic function. In fact identification of
MDM2-RP interactions provides the new thought
puts to develop anticancer drugs (Kawai et., al 2003).
Experimental Procedures
Analysis of Myc lymphomas
The λ Myc lymphoma samples were harvested from mice and kept frozen until added the lysis buffer to each pellet, dissolved it by pipetting up and down. Homogenize the samples with the help of 20 gauge needle and syringe. The samples were centrifuged at 1500 rpm for 5 minutes. The supernatant (Protein solution) transferred to fresh tube and replaced it on ice. BioRad protein assay solution was used for quantification of protein samples.
Western blot was performed using primary antibodies α-p53 (1:500/ Sigma), α-Arf (1:500/Santa Cruz), and anti-β-actin (1:20,000 /Sigma). After incubation the membrane was washed off and treated with developing liquid- SuperSignal West Dura (Pierce) followed by developed on X-ray film (Fujifilm Lifescience).
Harvesting of Foci, DNA extraction and its
amplification
p53 Knockout (KO) mouse embryonic fibroblast
(MEF) cells were plated at a density of 180,000
cells/6 cm on a petridish (Delta Si) and infected with
Viraport XR plasmid cDNA library supplemented with
8 µg/mL of polybrene (Sigma) and cultured in DMEM
(GIBCO) medium. The mouse embryonic fibroblast
(MEF) cell culture plate and mouse embryonic
fibroblast (MEF) cells containing viral vector were
used as control to compare the cell morphology.
Media was drained off and then PBS added to the
cells. Foci harvested under the inverted microscope
by using the pipette tip and transferred to eppendrof
tube.
Identification of novel oncogene RPL 10a
Direct PCR lysis reagent (Viagen Biotech) used to
isolate genomic DNA from foci. Lysates were
analyzed using PCR under conditions specified in the
Viraport Manual. After size separation on an agarose
gel, specified bands were then isolated for further
analysis. QIAquick Gel Extraction kit was used to
remove agarose from DNA. The eluted DNA (30 µl)
from gel extraction procedure was sent for DNA
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sequencing (GATC Biotech), the remaining samples
pooled (120 µl) together in one eppendrof tube and
used for cloning (ligation, transformation). Ligation
reaction was carried out with the pGEMT easy
cloning kit (Fermentas).
The mini preparation plasmid DNA was digested with
restriction enzyme Fast Digest EcoR1 (Fermentas)
and separated by agarose gel electrophoresis.
Acknowledgments
We thank to Dr. Jonas Nilsson, Mr. Linus Plym Forshell, Mrs. TachaZi Plym Forshell and Mr. Pramod.K, for their guidance and the knowledge they imparted to us. We also thank to our laboratory mates for their cooperation and support.
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