Molecular processes of chromosome 9p21 deletions causing inactivation of the p16 tumor suppressor gene in human cancer: Deduction from structural analysis of breakpoints for deletions

doi:10.1016/j.dnarep.2006.05.021

DNA Repair

Volume 5, Issues 9–10, 8 September 2006, Pages 1273-1281

https://doi.org/10.1016/j.dnarep.2006.05.021 Get rights and content

Abstract

Chromosome interstitial deletion (i.e., deletion of a chromosome segment in a chromosome arm) is a critical genetic event for the inactivation of tumor suppressor genes and activation of oncogenes leading to the carcinogenic conversion of human cells. The deletion at chromosome 9p21 removing the p16 tumor suppressor gene is a genetic alteration frequently observed in a variety of human cancers. Thus, structural analyses of breakpoints for p16 deletions in several kinds of human cancers have been performed to elucidate the molecular process of chromosome interstitial deletion consisting of formation of DNA double strand breaks (DSBs) and subsequent joining of DNA ends in human cells. The results indicated that DSBs triggering deletions in lymphoid leukemia are formed at a few defined sites by illegitimate action of the RAG protein complex, while DSBs in solid tumors are formed at unspecific sites by factors unidentified yet. In both types of tumors, the intra-nuclear architecture of chromatin was considered to affect the susceptibility of genomic segments of the p16 locus to DSBs. Broken DNA ends were joined by non-homologous end joining (NHEJ) repair in both types of tumors, however, microhomologies of DNA ends were preferentially utilized in the joining in solid tumors but not in lymphoid leukemia. The configuration of broken DNA ends as well as NHEJ activity in cells was thought to underlie the features of joining. Further structural analysis of other hot spots of chromosomal DNA breaks as well as the evaluation of the activity and specificity of NHEJ in human cells will elucidate the mechanisms of chromosome interstitial deletions in human cells.

Introduction

Chromosome interstitial deletion (i.e., deletion of a chromosome segment in a chromosome arm) is a major genetic event causing inactivation of tumor suppressor genes in human cancer cells [1], [2]. Such deletions also cause activation of a few oncogenes [3], [4]. Elucidation of the mechanism(s) for chromosome interstitial deletions is, therefore, indispensable in understanding the molecular mechanisms of human carcinogenesis. Interstitial deletions are supposed to be caused by DNA double strand breaks (DSBs) and subsequent illegitimate joining of DNA ends by homologous or non-homologous end joining repair. Structural analysis of breakpoints for chromosome interstitial deletions has been thought to be a powerful way to deduce molecular processes of its occurrence, since the breakpoints retain “traces” of DSBs and their subsequent repair. The analysis gives us information on:

(1)
Locations (clustering) of breakpoints on a genomic segment, which enables us to deduce genomic/chromosomal features making DNA susceptible to DSBs.
(2)
Structures of breakpoint junctions, which enables us to deduce DSB repair pathways used in joining broken DNA ends.

Section snippets

Genomic sequences susceptible to DSBs triggering deletion

The p16 (also called CDKN2A) tumor suppressor gene, and two related genes, p14ARF and p15 (also called CDKN2B) are involved in regulation of the cell cycle and/or apoptosis [5], and the genes are located in a 40-kb region in 9p21 (Fig. 1A). Homozygous deletion (i.e., deletion of both alleles) at chromosome 9p21 removing the p16 gene is frequently occurs in a variety of cancers, therefore, the p16 locus was considered to be a paradigm for loci that are subjected to interstitial deletions during

Genomic/chromosomal feature(s) responsible for the susceptibity to DSBs

Only a few defined RSS-like sequences were defined as breakpoint sites in lymphoid leukemia in spite that the 9p21 segment contained thousands of such sequences. Thus, the susceptibility of a genomic fragment to DSBs by the RAG complex was unlikely to be determined by the homologies with the consensus RSS. In addition, a 10-kb region encompassing the p16 locus contained the largest number of breakpoints for deletions in solid tumors, although a variety of sites in the region were subjected to

DSB repair activities to join DNA ends giving rise to chromosome interstitial deletions

Next, structures of breakpoint junctions were studied to deduce DSB repair pathways used in the joining of broken DNA ends. In Table 1, structures of breakpoint junctions for interstitial 9p21 deletions in lymphoid leukemia and solid tumors are summarized [7], [8], [9], [23], [24], [25]. First of all, it was noted that no significant homology has been found between the distal and proximal breakpoints in any of the cases. Thus, DSBs triggering 9p21 deletions were exclusively joined by

Prospective

The fundamental molecular processes for chromosome interstitial deletions in human cancer have been deduced by the analyses of breakpoints for deletions in human cancers. However, at present the following issues remain unknown.

(1)
Environmental and endogenous factors causing DSBs triggering deletions in the development of solid tumors.
(2)
Molecular basis for the architecture of chromatin making DNA susceptible to DSBs.
(3)
Molecular basis for the predominance of microhomology mediated-NHEJ in the repair of

Acknowledgement

This research was supported by Grants-in-Aid from the Ministry of Health, Labor and Welfare of Japan for the Third Term Comprehensive Control Research for Cancer, for Research on Human Genome and Tissue Engineering and for Cancer Research (16S-1).

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Several mechanisms are involved in the inactivation of p16INK4a and p14ARF, including chromosomal deletion, somatic mutation, and methylation of CpG islands extending from the promoter region to the first exon.39 The deletion at chromosome 9p21 in humans, which removes the CDKN2A tumor suppressor gene, is a genetic alteration frequently observed in several human cancers,40 prompting speculation that biochemical pathways regulated by these proteins must be disabled for normal cells to be transformed into tumor cells.39 Consistent with these findings, we previously showed that AID expression in biliary cells induces somatic mutations in the promoter region of the CDKN2A gene.10
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Inhalation of radon is closely associated with an increased risk of lung cancers. While the involvement of Ink4a in lung tumor development has been widely described, the tumor suppressor gene has not been studied in radon-induced lung tumors. In this study, loss of heterozygosity (LOH) analysis of the Cdkn2a locus, common to the Ink4a and Arf genes, was performed on 33 radon-induced rat lung tumors and showed a DNA loss in 50% of cases. The analysis of p16^Ink4a protein expression by immunohistochemistry revealed that 50% of the tumors were negative for this protein. Looking for the origin of this lack of expression, we observed a low frequency of homozygous deletion (6%), a lack of mutation, an absence of correlation between promoter methylation and Ink4a mRNA expression and no correlation between LOH and protein expression. However, a tendency for an inverse correlation between p16^Ink4a and pRb protein expression was observed. The expressions of p19Arf, Mmd2 and Mdm4 were not deregulated and only 14% of the tumors were mutated for Tp53. These results indicated that Ink4a/Cdk4/Rb1 pathway deregulation, more than Arf/Mdm2/Tp53 pathway, has a major role in the development of these tumors through p16^Ink4a deregulation. However, all known mechanisms of inactivation of the pathway do not play a recurrent role in these tumors and the actual origin of the lack of p16^Ink4a protein expression remains to be established.
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Molecular processes of chromosome 9p21 deletions causing inactivation of the p16 tumor suppressor gene in human cancer: Deduction from structural analysis of breakpoints for deletions

Abstract

Introduction

Section snippets

Genomic sequences susceptible to DSBs triggering deletion

Genomic/chromosomal feature(s) responsible for the susceptibity to DSBs

DSB repair activities to join DNA ends giving rise to chromosome interstitial deletions

Prospective

Acknowledgement

J. Biol. Chem.

Blood

Cell

Genomics

DNA Repair (Amst)

J. Biol. Chem.

Short, direct repeats at the breakpoints of deletions of the retinoblastoma gene

Proc. Natl. Acad. Sci. U.S.A.

How many tumor suppressor genes are involved in human lung carcinogenesis?

Carcinogenesis

Site-specific deletions involving the tal-1 and sil genes are restricted to cells of the T cell receptor alpha/beta lineage: T cell receptor delta gene deletion mechanism affects multiple genes

J. Exp. Med.

Activation of the beta-catenin gene by interstitial deletions involving exon 3 in primary colorectal carcinomas without adenomatous polyposis coli mutations

Cancer Res.

The Pezcoller lecture: cancer cell cycles revisited

Cancer Res.

Review of alterations of the cyclin-dependent kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human leukemia-lymphoma cells

Leukemia

Molecular processes of chromosome 9p21 deletions in human cancers

Oncogene

The RAG proteins and V(D)J recombination: complexes, ends, and transposition

Annu. Rev. Immunol.

Probing the chromosome 9p21 region susceptible to DNA double-strand breaks in human cells in vivo by restriction enzyme transfer

Oncogene

Modulation of DNA damage and DNA repair in chromatin

Prog. Nucleic. Acid. Res. Mol. Biol.

Apoptotic triggers initiate translocations within the MLL gene involving the nonhomologous end joining repair system

Cancer Res.

Chromosome translocations: dangerous liaisons revisited

Nat. Rev. Cancer