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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: gadu0002@student.umu.se

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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