Title: Critically review the roles of the isoforms of the p53 tumour suppressor
To achieve the tumour suppression action, p53 specifically binds to consensus sequences of DNA as a tetramer complex and give a specific action (Funk et al, 1992). Furthermore, p53 also involved in cell cycle arrest through cyclin dependent kinase inhibitor WAF located on chromosome 6p21.2 (El-Deiry et al, 1993). Wild type p53 function can be regulated in the presence of p53 isoforms because in the study of co-transfection of p53 wild type with its isoforms p53β and ∆133p53, co-transfection with the former induced p53 mediated apoptosis whereas co-transfection with latter against p53 mediated apoptosis. Furthermore, p53 isoforms have their specific functions and activities which were different and independent to its wild type (Bourdon, 2007).
2. Literature Review with discussion
2.1 HISTORY BACKGROUND OF p53 and its isoforms
p53 gene is one of the most important genes in human health and diseases and even it can be called ‘Guardian of Genome'(Lane, 1992). Last 30 years ago, p53 was discovered as a protein which is co precipitated with simian virus 40 T antigen (Lane and Crawford, 1979; De Leo et al 1979). After nearly 2 decades of p53 discovery, its siblings p73 (Kaghad et al 1997) and p63 (Yang et al, 1998) were discovered and both of which are homology to p53. Although their sequences are similar, their biological roles are not totally similar which was shown in transgenetic knockout mice studies. Moreover, p53 family genes are conserved among organisms from drosophila to man.
In whole body irradiation mouse, p53 protein accumulation can be detected by monoclonal antibody in splenocytes, thymocytes and osteocytes but not in hepatocytes which shows p53 accumulation is tissue dependent manner in response to irradiation (Midgley et al, 1995).
The ratio of p53 isoforms is crucial in adjustment of cell proliferation and tumour formation because the p53 isoforms can mediate p53 functions of transcription and apoptosis (Bourdon, 2007).
2.2 STRUCTURES OF p53 ISOFORMS
Human p53 gene is composed of 393 amino acids (Vogelstein and Kinzler, 1994) with 11exons and located at chromosome 17q13 (McBride et al, 1986). During translation from mRNA of p53, different in initiation start codon and two internal ribosomal ribosome entry sites (IRES) give two major forms of p53 which are full length and truncated amino terminal isoforms (Ray et al, 2006). Moreover, p53 transcription can be started from two different locations one in upstream of exon 1 and another from intron 4's internal promoter. Variations in promoter give an amino terminally truncated form of p53 translation started at codon 133(∆133p53). Furthermore, alternative splicing at intron 9 create other two isoforms of p53 named p53β and p53γ. Oligomerization domain cannot be seen in these two of p53 isoforms. Thus, p53 gene has 9 different isoforms which are named similar nomenclature in p63 and p73 (Murray-Zmijewski et al, 2006). They are
p53 and its isoforms localisation
In p53 isoforms, some stay in nucleus but some in cytoplasm. Such as ∆133p53 and β isoforms usually homing in the nucleus but ∆133p53γ presents only in the cytoplasm. In contrast, ∆133p53β was found not only in nucleus but also in cytoplasm. In addition, some isoforms can exist in both nucleus and cytoplasm as shuttling form like p53γ. Therefore, the C terminal amino acids may control the cellular localisation of the p53 isoforms (Bourdon et al, 2005).
Amino truncated form of p53 (∆Np53) impairs in transcriptional activation and does not form complex with MDM2. Moreover, ∆Np53 also possesses negative feedback effect on p53 biological functions (Courtois et al, 2002). ∆p53 also effects on some cell cycle protein p21 and 14-3-3σ but not on the MDM2, bax and PIG3 (Rohaly et al, 2005). Contrary to this finding, Chan and Poon 2007 pointed out that ∆p53 did not involve in intrinsic transcriptional activity and possess dominant negative activity to full length p53. They mentioned that this may be the reason of ∆p53 fail to import into nucleus because of losing nuclear localization signal. Although p53β has more affinity to p21 and Bax compare with Mdm2, p53 preferentially bind to Mdm2 and p21 than Bax. p53β facilitates p53 transcriptional activity together with p53 by means of complex through Bax promoter but not p21 promoter.
p53β enhanced the p53 transcriptional activity by binding to varieties of promoter (Promoter dependent manner) while ∆133p53 gives dominant negative affect on p53 (Bourdon et al, 2005).
Both ∆N p63/p73 can block the functions of p53 as well as Tap63/p73 by direct protein interaction (Benard et al, 2003).
2.2.3 Recognition by antibodies
Due to lack of specific antibodies to p53 isoforms, though some experiments show p53 isoforms are endogenously expressed they could not be detected. However, there is specific anti p53β antibody which recognized to p53β and ∆133p53β isoforms. Hence, it can be said that p53γ, ∆133p53γ and ∆133p53 are expressed at protein level and due to their subcellular localisation give their specific activities. Nonetheless, now some new antibodies for p53γ and ∆133p53 are on research fields.
∆p53 mRNA was not detected in most studies by using PCR technique although they use appropriate primers.
Table1. p53 isoforms and their specific antibodies
Although, there are antibodies for p53 and its isoforms but most antibodies does not recognized specifically. Among mouse monoclonal antibodies, DO-1 and DO-7 only detect p53, p53β and p53γ. 1801 antibody recognizes p53 and most of its isoforms; nonetheless, it does not recognize ∆133p53 isoforms. Although DO-12 antibody can bind to most p53, the binding is weak and sometimes long time exposure with antibodies may require. 421 antibodies also weakly bind to p53 and usually it can only detect wild type p53. KJC 8 rabbit polyclonal antibody is a p53β isoforms specific one. CM-1 antibodies can be called almost perfect one because it can recognizes p53 and most its isoforms.
2.3BIOLOGICAL ACTIVITES OF p53 AND ITS ISOFORMS
2.3.1Involvement of p53 and its isoforms in differentiation and development
Strikingly, dual gene structure of p53 gene is conserved among organisms from human through Drosophila to Zebrafish but splicing variants is different. p53 isoforms have their own specific biophysical activities like p63 and p73.
The p53 gene also play critical role in neural tube closure because in p53-/- female died during embryogenesis between birth and weaning due to interruption in physiological closure of neural tube. As a result, a variety of neural tube defect such as exencephaly, ancephaly, a range of craniofacial malformation (Armstrong et al, 1995).
Although p53 involves in stress response, cell cycle arrest, apoptosis and senescence, until recently there is no direct genetic evidence that how p53 involved in organogenesis. However, in Zebrafish, there is one isoforms named ∆113p53 which is homologue to homosapien ∆133p53 which involved in organ formation. In this organism, ∆113p53 is controlled by def (Digestive organ expansion factor) gene and loss of function mutation in def gene led to hypoplasia of digestive organ (Chen et al, 2005).
p73, a family member of p53, although it is homolog in sequence and similar in function with p53, it has 7 isoforms as a result of alternative splicing in C terminal, from p73 alpha to eta ( Liu et al, 2004).
In p73 isoforms, TAp73 involved in cell cycle arrest and apoptosis through binding of p53 RE but ∆N isofroms give dominant negative function on p53 family members by interaction with p53 RE (Benard et al: 2003, Melino et al: 2003). However, its action also can achieve by interacting with other response element different from p53 RE.
2.3.2 Transgenic animal studies
Knock in animal studies
In knock in mutant mice (p53+/m) which express only carboxyl terminal result from deletion of first 6 exons , these mammals are more resistance to cancer than wild type (p53+/+). However, the phenotype of early onset of ageing was found in these littermates (Tyner et al, 2002). On the other hand, ‘Super p53 mice' who carried supernumerary of p53 transgenic allele in addition to their endogenous two alleles show better response to DNA damage and more protective ability to cancer when compare with normal mice. Moreover, the characteristic of premature ageing was not seen in these mice. (Garcia-Cao et al, 2002). Thus, some questions appear on this, will this approach enhance the future tumour therapy?
Knock out animal studies
2.4THE p53 AND ITS ISOFORMS IN CANCER
p53 gene is critical in tumourigenesis which is show in double homozygous knockout mice (p53-/-). In these mice, in spite of developmental process is normal, these animals die 6 months of age from variety of neoplasms (Donehower et al, 1992).
Due to tissue specific expression ∆p53 cannot be detected in 21 human tissues (brain, heart, lung, liver, colon, bone marrow, thymus, spleen, testis, prostate, uterus, skeletal muscle, stomach, kidney, placenta, fetal brain, fetal liver, salivary gland, adrenal gland, thyroid, breast) and certain forms of tumours such as 106 breast tumours, 80 AML, 40 head-neck tumours. (Murray-Zmijewski et al, 2006). In breast cancer, although p53 mutation can be seen in only 25% of cases, the most expressed form is ∆133p53.
In squamous cell carcinoma of head and neck (SCCHN), p53β is a major isoform which was analysed in tumour samples. Other p53 isoforms can also be detected in certain tumour and normal epithelium samples but ∆p53 isoforms are interestingly undetectable (Boldrup et al, 2007).
In human neuroblastoma cell line SK-N-AS p53β which is one of the C terminal truncated p53 isoforms was cultivated; hence, the truncated isoforms may critical in pathogenesis of Neuroblastoma (Goldschneider et al, 2006).
Oral lichen planus (OLP), a premalignant chronic inflammatory lesion of unknown aetiology, p53 protein is highly expressed in this disease. Moreover, some p53 isoforms such as p53β and ∆133p53 were detected in many samples but p53γ, ∆133p53β and ∆133p53γ expressed in a few samples (Ebrahimi et al, 2008).
Therefore, the abnormal expression of p53 isoforms may strike on the normal p53 functions; consequently, more and more genetic damages are susceptible and enhance the tumour progression.
Mutation in p53 gene does not always inactivate p53 transcriptional activity because in 60% of p53 mutation the affect on p53 transcription is silent and only 15% of mutation can interrupt the p53 transcription.
2.4.2. Their roles in Apoptosis
2.4.3 p53 isoforms and Chemo sensitivities
2.5 Will they play as therapeutic role not only in cancers but also in other diseases?
Although most studies are now trying to explore p53 and its isoforms by using immunostaining or RNA microarray, until recently specific gene or protein markers are unavailable in the market. However, it is undeniable in the immediate future one can create the new methods to detect p53 and its isoforms.