Vol.2, No.9, 1065-1071 (2010) Health
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
P53 pseudogene: potential role in heat shock induced
apoptosis in a rat histiocytoma
Amere Subbarao Sreedhar
Centre for Cellular and Molecular Biology, Hyderabad, India; assr@ccmb.res.in
Received 1 June 2010; revised 2 July 2010; accepted 5 July 2010.
The p53 tumor suppressor gene is either non-
functional or highly and frequently mutated in
majority of cancers. In our study towards un-
derstanding cellular adaptations to stress using
a rat histiocytic tumor model, we have identified
mis-sense mutation in p53 that led to premature
termination of translation at the carboxyl-termi-
nus. Further, the cDNA isolated from heat stre-
ssed cells producing two amplicons with cDNA
specific primers (N-terminus) suggested oc-
currence of possible pseudogene(s). A com-
parative analysis between different tumor cell
lines of rat origin and rat genomic DNA using
p53 gene specific primers resulted in the ampli-
fication of a processed pseudogene and its
positive interaction with wild type p53 probe on
Southern blot analysis. The genomic DNA se-
quence analysis, and sequence comparison
with cDNA discovered that the processed pseu-
dogene lacks DNA binding domain and nuclear
localization signal, however, contains the ribo-
somal entry and stop signals. Rat genome
BLAST analysis of the pesudogene suggested
chromosome-18 localization which was in addi-
tion to 14, 13, 10, 9 localization of the cDNA. In
the interest of unraveling hidden dimensions of
p53 tumor suppressor gene, our study explores
the probability of p53 functional pseudogenes in
rat histiocytoma.
Keywords: Pseudogene; P53; Tumor; Rat
The tumor suppressor p53 is a multifunctional protein
that is involved in a variety of biological processes such
as growth arrest, apoptosis, differentiation and senes-
cence [1,2]. Aberrant expression of this gene results in
either a gain of transforming potential or a loss in tumor
suppressor activity [3,4]. The p53 gene mutation, dele-
tion, insertion or protein sequestration etc are often
found in many cancers [5,6] and these mutations affect
the p53 binding to DNA [7]. Analysis of the degeneracy
of p53 DNA-binding site suggests that there may be as
many as 200-400 p53 target sequences or perhaps more
[8]. Despite the high frequency with which p53 is mutated
during tumor development, a substantial proportion of
tumors still express the wild type p53 [9]. This could be
the reason in spite of exhaustive information on p53 modi-
fications the corresponding role of p53 modification in
experimental animal tumor models is poorly understood.
We are investigating the role of p53 in heat stress-in-
duced rat histiocytic tumors models. In the process of
elucidating heat stress induced cell death pathways and
evaluating the functional significance of p53 in heat
shock induced cell death in tumor cells, we have identi-
fied mutated form of p53 with two functional alleles by
reverse transcriptase polymerase chain reaction, and the
deletion and addition of nucleotides had resulted in
C-terminal deletion of 50 amino acids. We demonstrated
that Fas/CD95 induced apoptosis requires p53, and hy-
pothesized that C-terminal deletion and loss of oligo-
merization domain and nuclear localization signal proba-
bly are responsible for p53-transcritpion independent
apoptosis as suggested [10,11]. In the present study we
show that there are two processed pseudogenes for p53
in this tumor model and one of them also has ribosomal
entry site. A comparative genome analysis further re-
vealed that the processed pseudogene is predominantly
present in all the rat and mouse species but absent in
2.1. Animal Handling
All animal maintenance and handling was accomplished
as per the institutional ethical committee approval at
Centre for Cellular and Molecular Biology, Hyderabad,
A. S. Sreedhar / HEALTH 2 (2010) 1065-1071
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2.2. Tumor Growth and Cell Culture
AK-5 tumor cell line is established from i.p injections of
cell-free ascites fluid of a chemically induced and estab-
lished rat liver tumor, Zajdela ascetic hepatoma (ZAH).
These cells possess typical characteristics of macro-
phages. Single clone of AK-5 tumor, called BC8, was
adapted to grow in culture for several generations in
Dulbecco’s Modified Eagle’s Medium (DMEM) with
10% heat inactivated fetal calf serum (FCS) in the pres-
ence of penicillin (100 U/ml) and streptomycin (50
g/ml) is used in the present study. Rat fibroblasts (F111)
was procured from ATCC and maintained similar to BC8
as mentioned above. BC-8 cells (8 × 106 cells) were used
for injection either for s.c. or i.p. of six-week-old naïve
male Wistar rats and tumor growth was monitored. The
i.p tumor development approximated by the mean total
cell mass calculated from the percentage of packed cells
and the total ascites weight.
2.3. Genomic DNA Isolation
For normal rat live genomic DNA, six month old male
Wistar Rat was scarified as per institutional animal eth-
ics recommendations and genomic DNA was isolated
from the liver by phenol: chloroform method and used in
the present experiment.
2.4. RNA Isolation and cDNA Library
The control and heat stressed tumor cells are subjected
to single step total RNA isolation using Trisol reagent,
the integrity of RNA was examined by 1% agarose gel,
and 5 µg total RNA was used for cDNA preparation by
reverse transcriptase system containing the MMLV re-
verse transcriptase enzyme and oligo d(T) primer and the
cDNA prepared was used for further experiments.
2.5. Primers Used for the Polymerase Chain
PCR primers were designed for the cDNA clone span-
ning the coding sequence of rat wild type p53 (Acc. No.
X13058). Four sets of primers for the amplification of
full length as well as partial cDNA amplification were
made and used in the present study (Figure 1(a)). Primer
set II, P1: Forward-5’ atggatccatggaggattcacagtcg 3’, P2:
Reverse-5’ atgaattcgcacagggcatggtcttc 3’; Primer Set III,
P3: Forward-5’ atggatcctctgccagctggcgaagacat 3’, P4:
Reverse-5’ atgaattcggacaggcacaaacacga 3’; Primer Set
IV, P5: Forward-5’atggatcctgaggttcgtgtttgtgc 3’, P6:
Reverse-5’ atgaattctgtcagtctgagtcaggc 3’; and the Primer
set I is the combination of primers P1 and P6. Care has
been taken while designing the PCR primes to have 50%
GC content. The PCR conditions are as follows, 94˚C
1 min followed by 94˚C—1 min, 55˚C—1 min, 72˚C—1
min × 30 cycles unless otherwise indicated.
2.6. Southern Blot Analysis
The full length wild type p53 cDNA (1.2 kb) was radio-
labeled using 32P-dATP by random primer labeling.
Hundred nanograms of the template DNA was incubated
II   IIIIVVTA PRD
1100 101300 301393
P1 P3P5
P2P4 P6
P1 P3P5
P2P4 P6
ami noacids
Figure 1. Structural organization of p53. (a) Genome organization depicting the exons of p53 and indicating
the primers P1-P6 matching regions; (b) Schematic representation of coding region (cDNA) indicating prim-
ers P1-P6; (c) Functional domains showing the conserved regions of p53, appropriate amino acid lengths
(1-393) are mentioned. DBD: DNA binding region; NLS: nuclear localization signal.
A. S. Sreedhar / HEALTH 2 (2010) 1065-1071
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(37˚C, 15 min) with dNTPs exempting dATP and in the
presence of 10 µci of 32P-dATP, random primer, Klenow
enzyme (5 U) and reaction buffer. After the reaction, la-
beled template was purified through sephadex G-50 col-
umn, probe containing 1 × 108 µci per microgram DNA
was used for hybridization. PCR amplicons first run on
1% agarose gels were vacuum transferred to N+ nylon
membrane (Amersham), UV cross-linked and hybridized
with radiolabeled probe for overnight. Blots were washed
under stringent conditions (sodium phosphate buffer +
SDS) and exposed to X-ray film, and photographed.
2.7. Cloning and Sequence Characterization
The PCR amplicons are purified using PCR Wizard puri-
fication system (Promega, USA) either cloned in TOPO
cloning vector and or taken to automated DNA sequence
analysis (Model 3730, M/s Applied Biosystems, USA).
The obtained DNA sequences were subjected to blast
analysis (Entrez at http://www.ncbi. nlm.nih.gov) and the
deduced amino acid sequences were analyzed at h t tp://
www.expasy.ch, and http://www. isrec.isb-sib.ch.
3.1. Heat Stress Induces p53 Transcription
In continuation of our interest to know the functional
significance of p53 in rat histiocytic tumor models, we
compared control cells with heat stress and found that
heat stress enhanced p53 transcription (Figure 2(a)).
Interestingly when heat stressed samples were sub-
jected for partial PCR analysis we found that primer set
II gave two prominent amplicons, while primer sets III
and IV giving single amplicon (Figure 2(b)). The PCR
amplicons obtained by all the primer sets were excised
from agarose gel, purified using PCR product purifica-
tion kit (PCR Wizard, Qiagen) and re-amplified using
same set of primers. All the amplicons showed signifi-
cant re-amplification suggesting that these amplicons
are p53 gene specific (Figure 2(c)). However to con-
firm and avoid ambiguity with p53 sequence specificity,
all the products hybridized with wild type radio labeled
p53. Except the lower band of the amplicon with
primer set II, all other amplicons showed signficant
binding to the radiolabeled probe (Figure 2(d)). The
amplicons were cloned in TA cloning vector (Promega)
and subjected to automated DNA sequencing. The se-
quences obtained were aligned with wild type p53
cDNA sequence and found to be homologous (data not
shown). While full length did not show any duplication,
only primer set II showing such amplicon suggested
presence of possible pseudogenes.
3.2. BC8 Genome Contains a Processed
In addition to the two alleles reported [10] the additional
amplicons obtained may be related to processed p53
alleles originating from the genomic DNA. Therefore the
genomic DNA from the tumor cells was isolated and
subjected to genomic PCR using p53 cDNA specific
primer sets I, II, III, and IV. While primer sets I, II, and
IV were giving a single amplicon, primer set III did not
yield any amplification (Figure 3(a)). Genomic southern
however identified only the full length amplicon ampli-
fied using the primer set I (Figure 3(b)). These results
therefore suggested a processed pseudogene of p53 in
these tumor cells.
rathistiocytom a(BC8) ratlivercells
rathistiocytom a(BC8)
RNA Southernanalysis
Figure 2. Reverse transcription and polymerase chain reaction. (a) The total RNA from control and heat shocked
BC8 tumor cells was isolated and subjected to RT-PCR analysis with primer set I. The RNA loading control was
also shown with intact 28S and 18S RNA; (b) The cDNA of heat shocked BC8 cells was used as a template to am-
plify p53 with primer sets II, III, and IV. Note only the primer set II showing two amplicons; (c) Re-amplification of
first round PCR products after gel elution with appropriate primer sets mentioned; (d) Southern blot analysis of
re-amplified PCR products.
A. S. Sreedhar / HEALTH 2 (2010) 1065-1071
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rathistiocytoma(BC8) ratascites(AK5)ratfibroblasts(F111) ratliverce lls
(a) (b )
(c) (d)
Southern analysis
Figure 3. Genomic PCR and Southern analysis. (a) BC8 genomic PCR analysis with four primer sets, I, II, III, and IV; (b)
Genomic Southern analysis of PCR products obtained from Figure 3(a); (c) Genomic PCR analysis of rat fibroblasts, rat
histiocytoma (AK5), and rat liver cell; (d) Genomic Southern analysis of PCR products obtained from Figure 3(c).
Processed pseudogenes arise through a mechanism
whereby a spliced mRNA is reverse transcribed and sub-
sequently inserted into the genome [12]. If pseudo-
genes are formed in this way during evolution, these
pseudogenes should present in the rat genome and
should coexist with all the cell types. To examine this,
transformed rat fibroblast cells (F111), parental ascites
rat histiocytoma (AK5) were compared with the normal
rat genomic DNA.
3.3. Blast and In-Silico Translational Analysis
We went ahead of cloning p53 pseudogene and se-
quence alaysis of cloned product using automated
DNA sequencing. From the sequence analysis we
found that there is indeed a processed pseudogene
having a potential to provide two gene products with
different reading frames. A comparative sequence alig-
nment of processed pseudogene with full length RT-
PCR product of rat histiocytoma additionally showed
high sequence homology. Analysis of pseudogene re-
vealed loss of DNA binding region (nt 700-860) and
nuclear localization signal (nt 1030-1080) of cDNA
(Figure 4). Whole rat genome Blast analysis with
cDNA sequence identified its chromosome localization
on chromosomes 14, 13, 10, 9 and 2, and the pseu-
dogene sequence Blast identified its additional local-
ization at chromosome 18 (Table 1).
The p53 gene is frequently lost or rearranged in a large
variety of cancers, and most of the alterations in p53 are
found in the core domain that interfere with p53 DNA-
binding activity [5]. Although p53 has been a wonder
molecule and the guardian of genome, mutation of p53
affects its native functions including the antiapoptotic
function. Several p53 mutant cells are reported to have
lost apoptotic functions but not the cell cycle inhibition
[13,14]. While our earlier study suggesting that loss of
C-terminal 50 amino acids could have played a role in
p53-transcription independent apoptosis via Fas/CD95
translocation from golgi to plasma membrane [11,15], a
report from Zhu et al. [16] indicated that the N-terminal
43-63 amino acid are more than sufficient to activate
p53 transcription dependent apoptosis. Further, induction
of pro-apoptotic factor Bax, a known transcriptional cli-
ent for p53 [17], and subsequent activation of intrinsic
apoptotic death pathway through mitochondrial dysfunc-
tion [18] directed us to look for possible processed genes
in the tumor genome.
By definition, pseudogenes lack a function. However,
the classification of pseudogenes generally relies on
computational analysis of genomic sequences using
complex algorithms [19]. It has been established that
quite a few pseudogenes can go through the process of
A. S. Sreedhar / HEALTH 2 (2010) 1065-1071
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transcription, either if their own promoter is still intact or
in some cases using the promoter of a nearby gene; this
expression of pseudogenes also appears to be tis-
sue-specific [20]. Pseudogenes are often referred to in
the scientific literature as nonfunctional DNA. Failure to
observe pseudogenes coding for a product under ex-
perimental conditions is no proof that they never do so
inside an organism. Homologous recombination between
the intact functional p53 gene and the p53 pseudogene is
thought to have occurred in such a perturbed intracellu-
lar environment with genomic instability, thus inactivat-
ing the intact allele of the functional p53, therefore the
persistence of pseudogenes is in itself additional evi-
dence for their activity. Natural selection would remove
p53 cDNAvs. Pseudogene
Figure 4. Blast analysis of p53 cDNA and pseudogenes showing the loss of DNA binding domain (DBD) and nuclear localization
signal (NLS) in the pseudogene.
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Table 1. Genome blast analysis showing the chromosome localization of cDNA and pseudogenes.
S. No. Accession number chromosome E value % identity
cDNA NW 047430.2
NW 001084694.1 14 0.0 84
NW 047390.2
NW 001084680.1 13 0.0 78
NW 047334.2
NW 0010884656.1 10 1e-140 100
NW 047813.2
NW 001084694.1 9 0.0 84
NW 047627.2
NW 001084680.1 2 0.0 87
pseudogene NW 047518.2
NW 001084740.1 18 0.0 99
NW 047430.2
NW 001083694.1 14 0.0 84
NW 047390.2
NW 001084680.1 13 3e-23 76
NW 047334.2
NW 001084656.1 10 1e-42 81
NW 47813.2
NW 001084880.1 9 5e-86 95
this type of DNA if it were useless, since DNA manu-
factured by the cell is energetically costly. As the func-
tion of more pseudogenes is being uncovered by testable
and repeatable science, it is evident that these genetic
elements, which are copiously spread in the genomes of
different organisms, have been created with purpose.
In addition, and in contrast to previously believed in-
formation that pseudogenes are non functional copies of
genes [21,22], growing evidence suggests that at least
some pseudogenes are functional. It has been demon-
strated that pseudogenes notably arise from seemingly
absent or disabled promoters, premature stop codons,
splicing errors, frameshift-causing deletions and inser-
tions, etc., and do not necessarily abolish gene expres-
sion [23,24]. McCarrey et al. [25] have suggested that
pseudogenes can be functional in terms of the regulation
of the expression of its paralogous genes, otherwise an-
tisense to pseudogenes should not interfere with cellular
functions. In support of this earlier we have used N-
terminal siRNA to p53 and could inhibit its functions
[10]. With respect to the evolution of regulatory func-
tions of pseudogenes we must now conclude that tran-
scribed pseudogenes are not necessarily without function.
Indeed, they would appear to be especially suited to
roles involving the antisense regulation of the active
genes to which they are related [24]. In summary we
report a processed pseudogene and additional transla-
tional products for p53 in a rat histiocytoma that differ
from the parental tumor and from the rat genome may
have function roles upon stress and tumorigenesis.
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