P53
Loss of heterozygosity at the short arm of chromosome 17 has been associated with tumors of the lung,colon and breast. This region of chromosome 17 includes the p53 gene. The P53 gene was originally discoveredbecause the protein product complexes with the SV40 large T antigen. It was first thought that P53 was adominant oncogene since cDNA clones isolated from tumor lines were able to cooperate with the RAS oncogenein transformation assays. This proved to be a misleading since the cDNA clones used in all these studies weremutated forms of wild-type p53 and cDNAs from normal tissue were later shown to be incapable of RAS co-transformation. The mutant p53 proteins were shown to be altered in stability and conformation as well asbinding to hsp70.Subsequent analysis of several murine leukemia cell lines showed that the P53 locus was lost by eitherinsertions or deletions on both alleles. This suggested that wild-type p53 may be a tumor suppressor not adominant proto-oncogene. Direct confirmation came when it was shown that wild-type p53 could suppresstransformation in oncogene cooperation assays with mutant p53 and ras.It has now been demonstrated that mutation at the P53 locus occurs in cancers of the colon, breast, liverand lung. Indeed, p53 involvement in neoplasia is more frequent than any other known tumor suppressor ordominant proto-oncogene.The protein encoded by P53 is a nuclear localized phosphoprotein. A domain near the N-terminus of the p53protein is highly acidic like similar domains found in various transcription factors. When this domain is fused tothe DNA-binding domain of the yeast GAL4 protein, the resulting chimera is able to activate transcription fromgenes containing GAL4 response elements. This suggests that p53 may be involved in transcriptional regulation.p53 has been shown to bind DNA,
in vitro
, that contains at least 2 copies of the motif 5'-PuPuC(A/T)(A/T)GPyPyPy-3'. This sequence motif suggests that p53 may bind to DNA as a tetramer. Binding as a tetramericcomplex explains the fact that mutant p53 proteins act in a dominant manner. They are present in complexeswith wild type p53 and alter the function of the normal tetramer.Like pRB, p53 forms a complex with SV40 large T antigen, as well as the E1B transforming protein ofadenovirus and E6 protein of human papilloma viruses. Complexing with these tumor antigens increases thestability of the p53 protein. This increased stability of p53 is characteristic of mutant forms found in tumor lines.The complexes of T antigens and p53 renders p53 incapable of binding to DNA and inducing transcription. Acellular protein, originally identified in a spontaneous transformed mouse cell line and termed MDM2, has beenshown to bind to p53. Complexing of p53 and MDM2 results in loss of p53 mediated trans-activation of geneexpression. Significantly, amplification of the MDM2 gene is observed in a significant fraction of most commonhuman sarcomas.Phosphorylation also regulates the activity of p53. The level of p53 is low after mitosis but increases duringG
1
. During S phase the protein becomes phosphorylated by the M-phase cyclin-CDK complex of the cell cycleand also by casein kinase II (CKII). Sequences at the N-terminus of the p53 protein function as a transcriptionactivator indicating the role of p53 in the transcription of genes involved in suppression of cell growth. One majorcell-cycle regulating gene that is a target for p53 is the CDK inhibitory protein (CIP), p21
CIP
. Activation of p53results in increased expression of p21
CIP
with a resultant arrest in the G
1
and G
2
phase of the cell cycle.Additionally, p53 protein has been shown to block the binding of DNA polymerase-
α
to SV40 large T,blocking replication of SV40 DNA. It is suggested that p53 may also regulate the initiation of DNA synthesis. Dueto the involvemenof p53 in both transcription and DNA replication, the various mutants of p53 may affect theseproperties in different ways. This may account for why some mutants lose tumor suppressor activity while othersbehave as dominant oncogenes.back to the top
Retinoblastoma (RB)
In the familial form of this disease individuals inherit a mutant, loss of function allele from an affected parent.A subsequent later somatic mutational event inactivates the normal allele resulting in retinoblastomadevelopment. This leads to an apparently dominant mode of inheritance. The requirement for an additionalsomatic mutational event at the unaffected allele means that penetration of the defect is not always complete.In sporadic forms of tumors involving the RB locus 2 somatic mutational events must occur, the second ofwhich must occur in the descendants of the cell receiving the first mutation. This combination of mutationalevents is extremely rare.The locus of the RB gene, identified cytogenetically, is chromosome 13q14.1. A 4.7 kb RB transcript hasbeen identified (by chromosomal walking and subsequent Northern blotting with genomic DNA probes) andsubsequently cloned. The RB gene encompasses 27 exons that span 180 kb of chromosome 13. Two of theintrons in this gene are extremely large, 35 kb and 70 kb. The RB RNA encodes a p110 kDa protein (pRB) of 928amino acids. pRB is a nuclear localized phosphoprotein. pRB is not detectable in any retinoblastoma cells.However, surprisingly detectable levels of pRB can be found in most proliferating cells even though there is arestricted number of tissues affected by mutations in the RB gene (i.e. retina, bone and connective tissue).Many different types of mutations occur to result in loss of RB function. The largest percentage (30%) ofretinoblastomas contain large scale deletions. Splicing errors, point mutations and small deletions in thepromoter region have also been observed in some retinoblastomas.The germ line mutations at RB occur predominantly during spermatogenesis as opposed to oogenesis.However, the somatic mutations occur with equal frequency at the paternal or maternal locus. In contrast,somatic mutations at RB in sporadic osteosarcomas occur preferentially at the paternal locus. This may be the
Page 3 of 6Tumor Suppressors
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