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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 3997-4010
REVIEW ARTICLE
Regulated Genomic Instability and Neoplasia in the Lymphoid Lineage
By
Gary J. Vanasse,
Patrick Concannon, and
Dennis M. Willerford
From the Departments of Medicine and Immunology, University of
Washington School of Medicine, Molecular Genetics Program, Virginia
Mason Research Center, and the Puget Sound Blood Center, Seattle, WA.
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INTRODUCTION |
ADAPTIVE IMMUNITY in mammals depends on
the generation of a vast repertoire of Ig and T-cell receptor (TCR)
specificities expressed on individual B and T lymphocytes. Antigen
receptor diversity is created by the assembly of variable region gene
segments by a process of somatic DNA rearrangement known as V(D)J
recombination. Additional genetic alterations in Ig genes occur during
immune responses through isotype class switch recombination and somatic hypermutation of variable regions in germinal center B cells. Thus,
lymphocytes breach genomic integrity at several developmental stages to
expand the germline genetic program. A central feature of neoplasms in
the lymphoid lineage is chromosomal translocations, which occur in the
majority of non-Hodgkin's lymphomas (NHL) as well as in many acute
leukemias and plasma cell neoplasms. Translocations in NHL
characteristically juxtapose a cellular proto-oncogene with one of the
antigen receptor loci, leading to deregulated oncogene expression. It
has been hypothesized that such oncogenic translocations arise as
byproducts of physiologic gene rearrangement processes, although
evidence to date has been mostly circumstantial. In recent years, there
have been important advances in several areas that make it appropriate
to re-examine the connection between genetic instability at the antigen
receptor loci and the pathogenesis of lymphoid malignancies. An
understanding of the major steps of V(D)J recombination at both the
genetic and biochemical levels has emerged, suggesting ways in which
the recombinase mechanism may protect broken DNA ends from exposure to
inappropriate repair mechanisms. It is now apparent that genetic
plasticity in lymphocytes, especially involving the Ig loci in B cells,
is much greater than previously appreciated. In particular, B-lineage
cells are now known to undergo V(D)J recombination and revise Ig locus
variable regions in response to antigenic stimulation. Germinal center B cells also undergo somatic hypermutation of Ig genes, a process that
is now understood to involve DNA strand breaks, as well as Ig isotype
switch recombination. Inasmuch as the most common types of nodal B-cell
NHL are postulated to arise in the germinal center, these recent
insights suggest that genomic instability that occurs in the context of
immune responses may contribute to the genetic alterations leading to
lymphoid neoplasia.
Given the vigor with which chromosomal DNA is broken and repaired in
lymphocytes during lineage development and antigen responses, it is
perhaps appropriate to ask what elements of antigen receptor diversification processes limit generalized genomic instability, ie,
what safety features prevent the occurrence of oncogenic events in most
individuals. Clues to such tumor-suppressor mechanisms may emerge from
the study of genes responsible for hereditary lymphoma susceptibility
disorders, including ataxia-telangiectasia (A-T), Nijmegen breakage
syndrome (NBS), and Bloom's syndrome. In the absence of genetic
predisposition, toxic exposures or immune system alterations (eg, such
as occurs in acquired immunodeficiency syndrome [AIDS]) may interfere
with mechanisms that assure genetic safety in the resolution of DNA
breaks. Further understanding of antigen receptor gene diversification
in lymphocytes and the regulation of these processes in immunity may
therefore yield testable hypotheses about the causes of lymphoma.
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PHYSIOLOGIC GENOMIC INSTABILITY IN LYMPHOCYTES |
Mechanism of V(D)J recombination.
Antigen receptor diversity is generated during development of B and T
cells by a process of somatic gene rearrangement, wherein variable
regions are assembled by apposition of germline variable (V), diversity
(D), and joining (J) segments (or V and J segments) to form a
contiguous exon (reviewed in Alt et al1). Sequence diversity is, in part, combinatorial, achieved by selection among multiple versions of each type of segment. V(D)J recombination may
involve segments located more than 2 Mb apart, yet almost always occurs
in cis. During B- and T-cell development, rearrangement of antigen
receptor genes occurs in a stepwise fashion to generate proteins that
interact with a membrane signaling complex. Successive versions of this
complex drive cellular expansion, mediate cellular differentiation, and
deliver survival and/or death signals that shape the composition of the
immune system (reviewed in Willerford et al2). Thus, V(D)J
rearrangement is not only a product of lymphoid differentiation, but is
also a critical mediator of the development process. Many of the genes
involved in V(D)J recombination have been identified, and some of the
steps are beginning to be understood at a biochemical level. The
physiology of this process has also been studied in mutant mice
generated by gene targeting strategies. A summary is provided in
Table 1.
V(D)J recombination is site-specific and is targeted by characteristic
recognition signal sequences (RSS) that flank the borders of
recombining variable region gene segments
(Fig 1). RSS are composed of conserved
heptamer and AT-rich nonamer sequences, separated by either a 12 or 23 nucleotide spacer. V(D)J recombination is initiated by DNA breaks
catalyzed by the lymphoid-specific recombinase components Rag-1 and
Rag-2, which occur precisely at the border between RSS and coding
segment.3-5 This cleavage reaction occurs efficiently in
vitro, beginning with nicking of 1 strand and followed by
transesterification of the opposing phosphodiester bond to produce a
blunt cut at the signal end and a sealed hairpin structure at the
coding end. V(D)J rearrangement occurs between RSS with dissimilar
spacer lengths (known as the 12/23 rule), a property that prevents
nonproductive V to V or J to J rearrangements.1 This
reflects an important, intrinsic property of Rag-mediated DNA scission:
creation of 2 DNA breaks is coupled6,7 and occurs within a
single synaptic complex that remains assembled after cleavage, where it
facilitates efficient rejoining.8 Mutational inactivation
of either Rag-1 or Rag-2 in mice prevents V(D)J recombination and
blocks T- and B-cell development at an early progenitor
stage.9,10 Mutations in the Rag genes account for a subset
of autosomally inherited severe combined immunodeficiency in
humans.11 Point mutations of Rag-1 or Rag-2 reducing the efficiency of V(D)J recombination have also been found in Omenn syndrome, an immunodeficiency characterized by activated, oligoclonal T
cells and hypereosinophilia.12
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| Fig 1.
Mechanisms for antigen receptor gene diversification in
lymphocytes. (A) V(D)J recombination is initiated by DNA cleavage at
RSS (triangles) bordering variable region gene segments. Rejoining of
ends is reciprocal and requires the DNA-PKcs and Ku proteins, as well
as XRCC4 and DNA ligase IV. (B) Class switch recombination is targeted
by an unknown mechanism involving switch regions located upstream of
constant region coding exons. Like V(D)J recombination, rejoining is
reciprocal and requires the DNA-PKcs and Ku proteins. (C) Somatic
hypermutation is targeted to Ig variable regions by a transcriptional
mechanism. DNA breaks occur in the course of the mutation process,
which leads to base substitutions as well as small insertions and
deletions.
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An important observation regarding the rejoining of DNA ends created by
Rag-mediated DNA scission is that all somatic cells possess the
required machinery, which overlaps with general cellular pathways
responsible for repairing double-strand DNA breaks.3,13,14 There is reciprocal rejoining of both coding and signal ends, so that,
depending on the respective orientation of recombining segments, the
intervening DNA is either excised as a circle15 or inverted
with re-establishment of chromosomal continuity. The distinct
structures of signal and coding ends created by Rag-mediated DNA
cleavage are processed by different pathways.16 Signal ends are usually religated without gain or loss of germline sequence. In
contrast, coding end processing is inherently inaccurate: first, opening of the sealed hairpin structure may occur asymmetrically, generating short complementary sequences at coding joints recognized as
P elements; second, open coding ends frequently lose several base pairs
to nuclease activity during processing; finally, expression of terminal
deoxynuclotidyl transferase (TdT) in developing lymphocytes adds bases
(N-nucleotides) in a template-independent fashion. These properties
produce extreme sequence diversity at the site of V(D)J joins, which
correspond to the antigen contact residues of TCR and Ig molecules and
act to expand the diversity of the immune repertoire.
A central component of the rejoining reaction is the DNA-dependent
protein kinase (DNA-PK) complex, which includes the DNA-PK catalytic
subunit (DNA-PKcs), along with the Ku70 and Ku86 nuclear antigens.17 DNA-PKcs is a member of the PI-3 kinase gene
family, which includes the ATM gene, responsible for the
lymphoma susceptibility and neuro-degenerative disorder A-T. DNA-PKcs
is mutated in the mouse Scid strain,18-20 which,
along with mice carrying insertional or targeted DNA-PKcs mutations,
exhibits defective V(D)J rearrangement and impaired lymphocyte
development.21-23 Defects in DNA-PKcs selectively impair
coding joint formation, whereas signal joints are relatively normal.24,25 The infrequent coding joints recovered from
DNA-PKcs-deficient cells often exhibit large deletions, which suggests
that alternative pathways for rejoining engender a lesser degree of
protection for DNA ends created during V(D)J recombination. The Ku70
and Ku86 nuclear proteins exhibit DNA end-binding activity and, in concert with DNA, activate the kinase function of the
DNA-PKcs.17 V(D)J recombination is impaired in cell lines
deficient in either Ku70 or Ku86.13,26,27 However, in
contrast to the defect with DNA-PKcs mutations, Ku70 and Ku86 are
required for rejoining both coding and signal ends, indicating that
these proteins have functional activities independent of the DNA-PK
complex. Mice lacking Ku86 or Ku 70 have severe defects in T- and
B-cell development, although leaky development of T cells is
observed in Ku70 / mice.28-31 In addition,
both types of mice exhibit growth retardation, indicating that the Ku
proteins function independently of the DNA-PKcs in general growth regulation.
The final step in the V(D)J reaction involves ligation of DNA ends. Two
essential components are the XRCC4 gene, which complements the defect
in V(D)J recombination and DNA repair in the radiation-sensitive XR-1
cell line,32 and DNA ligase IV.33 XRCC4 appears
to facilitate recruitment of the ligase to the rejoining
complex.34,35 Targeted null mutation of either XRCC4 or DNA
ligase IV produces a similar phenotype, with an arrest in lymphoid
development at an early progenitor stage. Interestingly, both of these
mutations lead to severe defects in brain development stemming from
death of postmitotic neurons, raising the possibility that some form of DNA rearrangement is required to form the nervous
system.36,37 Along these lines, it has recently been
determined that neural cadherin-like genes are organized into variable
and constant region clusters reminiscent of Ig and TCR
loci.38
The V(D)J recombination mechanism is shared by all 7 TCR and Ig loci.
However, the process is tightly controlled, so that rearrangement of
individual loci is restricted to the appropriate cell lineage and
developmental stage.2,39 Rag-mediated DNA cleavage requires
that target locus chromatin be in an open or accessible
conformation.3,40 Changes in chromatin structure accompany
transcription of antigen receptor loci in their germline configuration,
which invariably occurs before rearrangement and appears to play a key
role in regulating the initiation of the V(D)J reaction. This
conclusion is based on an elegant series of studies using gene
transfection, transgenic recombination substrates, and knockouts of
cis-regulatory elements that demonstrate that rearrangement requires
the enhancer and promoter elements that regulate germline
transcription.39,41,42 It is not known whether germline
transcription acts only in a permissive way to facilitate recombinase
access or plays a more direct role in targeting V(D)J recombination to
the appropriate locus.
Ig class switch recombination.
After immune stimulation, antigen-reactive B cells frequently undergo
isotype class switching, wherein exon clusters encoding the constant
regions for Ig , Ig , or Ig isotypes are juxtaposed with a
functional V(D)J-rearranged variable region to produce antibody
molecules with the same antigen specificity but with specialized
effector functions determined by the incoming constant region (reviewed
in Stavnezer43). These functions may include prolonged
plasma half-life, differential binding to distinct Fc receptor types on
effector cells, and exogenous secretion by mucosal epithelium. The
constant region clusters for the different IgH isotypes are located
3' of Cµ and C within the IgH locus. Class switching
involves a nonhomologous recombination event, resulting in the looping
out of intervening sequences.44 Recombination is targeted
by characteristic switch regions that span 2 to 10 kb and are located
5' of Cµ and each of the constant region clusters, excepting
C (Fig 1). Switch regions contain characteristic tandem arrays of
short repetitive sequences; however, there appears to be no sequence
specificity in switch region breakpoints.45,46 It is not
known how switch recombination is initiated, but the characteristic
targets are distinct from the RSS used in V(D)J recombination.
Correspondingly, class switching proceeds efficiently in the absence of
Rag-1 or Rag-2, whether driven by CD40 in cultured Rag-deficient pro-B
cells or in mature B cells from Rag-null mice carrying a rearranged IgH
allele.47,48
Class switching is regulated to a large extent by interaction with T
cells and includes signals from a variety of cytokines as well as by
CD40 ligand/CD40 interactions. These effects are mediated in part via
regulation of germline transcription, which originates at noncoding I
exons located 5' of the constant region clusters.43,49 Germline transcription is controlled by
upstream promoter regions as well as enhancer/LCR elements within the
IgH locus.50-52 The mechanistic role of transcription in
switch recombination has been investigated using gene targeting
techniques in mice.49,50,53-57 These studies suggest that
transcription alone is insufficient for activation of class switching,
unless the appropriate signals for transcript processing by RNA
splicing are included. Thus, germline transcription appears to play
more than a minimal function in regulating locus accessibility, and
either the RNA splicing machinery or the processed transcript itself
could play a role in the switch recombination process.
Switch recombination resembles V(D)J recombination in that DNA ends are
rejoined in a reciprocal fashion.44 This suggests that the
creation of 2 DNA breaks may be coupled, occurring within a synaptic
complex that sequesters DNA ends and facilitates rejoining. In support
of this notion, rejoining of DNA breaks created during class switch
recombination requires the DNA-PK complex. Switch recombination is
impaired in pro-B cells from Scid mice,47 as well
as in mice carrying rearranged Ig genes that lack Ku70 or Ku86.58,59 Thus, V(D)J and class switch recombination
represent 2 distinct ways in which physiologic DNA breaks are created
in the IgH locus in B cells, whereas resolution of these breaks shares a common pathway.
Somatic hypermutation.
The Ig repertoire created by V(D)J recombination during B-cell
development is further diversified during immune responses by the
induction of somatic hypermutation in the V regions of IgH and IgL
genes (reviewed in Neuberger and Milstein60 and Storb61). In germinal center B cells, mutations are
introduced into V region gene segments at a rate of approximately
1/1,000 bp per generation (Fig 1). Subsequent selection processes favor survival and expansion of cells with increased affinity for antigen. Somatic hypermutation of Ig genes is confined to a region within about
1.5 kb downstream of the promoter, and extensive studies using
transgenic mice indicate that transcription and somatic hypermutation
are intimately linked.62 Models for the mechanism of
somatic hypermutation have focused on DNA repair processes, and these
have been tested by examination of humans and mice with mutations
involving several of these pathways. A role for nucleotide excision
repair has been largely excluded by the finding that somatic
hypermutation is intact in humans with mutations in several genes
responsible for Xeroderma Pigmentosum or Cockayne syndrome, as well as
in mice with targeted mutation of XPC, XPA, or XPD.63 Studies in mice with mutations in DNA mismatch repair components PMS2
and MSH2 have yielded some conflicting results regarding a potential
role in somatic hypermutation (discussed in Wood64 and
Kelsoe65). Moreover, the recent finding that MSH2 is
required for effective humoral immune responses and maturation of
germinal centers66 complicates the interpretation of these
studies. Somatic hypermutation most commonly involves base
substitutions; however, recent studies have indicated that insertions
or deletions may also occur with significant
frequency.67,68 These imply that somatic hypermutation
involves DNA strand breaks, a hypothesis supported by recent
experiments in a constitutively hypermutating Burkitt's lymphoma cell
line.69 By artificially expressing TdT, these investigators
found that a substantial fraction of acquired mutations in IgH V
regions exhibited nontemplated base additions, indicating that the
mutation mechanism involved free DNA ends. These findings raise
important questions, including whether DNA breaks are an essential
element of the somatic hypermutation mechanism, whether such breaks
involve both DNA strands, and, if so, whether rejoining involves any of
the elements shared between V(D)J and class switch recombination.
Genetic plasticity of Ig genes in B cells.
Antigen receptor diversification in lymphocytes occurs not only during
development in the primary lymphoid organs, but also as part of a
complex response to exogenous stimuli delivered through the antigen
receptor itself. In immature IgM+ bone marrow B cells,
V(D)J recombination is reactivated in response to certain Ig receptor
signals, leading to replacement of functionally rearranged Ig V
regions. In this setting, receptor editing, as this process is known,
may help prevent the emergence of autoreactive Ig
specificities.70-73 Intensive diversification of Ig genes
is also induced by antigen receptor signals in peripheral B cells during the germinal center reaction (Fig
2). Germinal centers arise from a limited number of B cells activated
by antigen and migrating to primary follicles, where they interact with
follicular dendritic cells (FDC).74-76 After proliferative
expansion, discrete dark and light zones are identified. The dark zone
contains rapidly cycling centroblasts, whereas the light zone harbors
resting centrocytes derived from the centroblasts, FDC that sequester
antigen, and antigen-specific T cells and macrophages. Somatic
hypermutation occurs in proliferating centroblasts. After exiting the
cell cycle, progeny bearing mutated Ig genes migrate to the light zone,
where interaction with antigen and FDC induces the survival of cells bearing high-affinity antigen receptors. Ig class switching occurs within the centrocyte compartment and is facilitated by interaction with T cells.77 Centrocytes selected by antigen may
re-enter the dark zone and undergo further clonal expansion and somatic hypermutation or may exit the germinal center to differentiate into
memory B cells or plasma cells.
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| Fig 2.
The germinal center reaction. During immune responses,
antigen-responsive B cells proliferate within lymphoid follicles,
leading to formation of germinal centers. Rapidly cycling cells are
located in the dark zone, where somatic hypermutation of Ig V regions
occurs. Cells then exit the cell cycle and migrate to the light zone,
where cell fates are determined by interaction with antigen-bearing FDC
and antigen-specific T cells or undergo cell death. B cells with
high-affinity receptors undergo class switch recombination and may
either exit the germinal center to differentiate into memory cells and
plasma cells or re-enter the dark zone for additional rounds of
replication and hypermutation. Some B cells may also undergo V(D)J
recombination.
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It was recently discovered that, in addition to somatic hypermutation
and class switch recombination, germinal center B cells may also
undergo receptor revision through V(D)J recombination. Thus, Rag-1 and
Rag-2 are expressed in reactive germinal centers78,79 and
mediate successive IgL rearrangements.80,81 Rearrangements of the IgH locus were also detected on the normal allele in mice with
gene-targeted VDJ rearrangements of 1 allele.81 Recent studies cast doubt on the notion that Rag genes are re-expressed in
Rag-negative mature B cells, and important questions remain regarding
the origin of peripheral B-lineage cells undergoing V(D)J recombination
in response to antigen signals.82,82a Both somatic
hypermutation and receptor revision by V(D)J recombination have also
been identified in peripheral T cells; however, their role in
regulating the peripheral T-cell repertoire is not yet known.62,83
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CHROMOSOME TRANSLOCATIONS INVOLVING ANTIGEN RECEPTOR GENES |
Oncogene deregulation by control elements for antigen receptor gene
expression.
A genetic hallmark of NHL is the presence of chromosome translocations
involving the antigen receptor loci. Whereas oncogenic chromosome
translocations commonly associated with most myeloid and some lymphoid
leukemias result in the formation of a fusion gene and expression of a
novel chimeric protein, NHL translocations typically place a
structurally intact cellular proto-oncogene under the regulatory
influence of the highly expressed Ig or TCR genes, leading to effects
on cell growth, cell differentiation, or apoptosis.84-90
The molecular structure of a number of recurrent translocations has
been elucidated by breakpoint cloning and sequence analysis
(Table 2). Of these, the role of the
t(8;14) and t(14:18) translocations in the pathogenesis of lymphoma
have been explored most thoroughly.
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Table 2.
Examples of Recurrent Chromosome Translocations
Involving Antigen Receptor Loci in Lymphoid Malignancies
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Translocations involving c-myc are implicated in nearly all cases of
Burkitt's lymphoma. Although these most often involve the IgH locus in
t(8;14), variant translocations t(2;8)(p11;q24) and t(8;22)(q24;q11)
are also observed, juxtaposing c-myc with the Ig or Ig loci,
respectively.91-96 C-myc plays a broad role in
transcriptional regulation of cell growth, differentiation and
apoptosis (for reviews, see Henriksson and Luscher97 and Dang98). A role for t(8;14) in neoplasia was demonstrated
by transgenic mice expressing c-myc under the control of the IgH intronic enhancer (Eµ) that exhibit polyclonal hyperplasia of pre-B
cells and develop aggressive clonal B-lineage malignancies within
several months.99-101 The t(14;18)(q32;q21), which is found in more than 85% of human follicular B-cell lymphomas, places the
Bcl-2 gene in proximity to the IgH locus, usually directly upstream of
one of the JH segments.85,102-105 Resulting Bcl-2
overexpression prolongs survival of B cells through inhibition of
apoptosis.105-107 In Eµ-BCL2 transgenic mice, follicular
B-cell hyperplasia is observed, with some mice eventually developing
aggressive monoclonal B-cell lymphomas after a protracted latency
period.107-109 These experiments demonstrate that
deregulated expression of cellular proto-oncogenes by translocation
into the IgH locus is sufficient to initiate an oncogenic pathway.
Although there is a clear requirement for additional genetic changes
for the development of lymphoma, these findings indicate that even very
rare rearrangement events that involve antigen receptor loci have the
potential to cause malignant disease.
Evidence implicating physiologic rearrangement processes.
The correlation between genetic instability of antigen receptor genes
during lymphoid development and immune responses and lymphoid-specific
oncogenic chromosome translocations involving the same loci yields a
compelling hypothesis that NHL translocations arise from errors in
these physiologic processes. The vast majority of V(D)J rearrangements
occur on the same chromosome, even though the recombining sequences may
be located megabases away. In principle, the V(D)J recombination
mechanism is equally compatible with reciprocal rearrangements between
chromosomes. A variety of interallelic and interlocus V(D)J
rearrangements in lymphocytes have been described,110-113 and these studies suggest that, although uncommon, recombination between chromosomes occurs physiologically at low frequency. Evidence for oncogenic translocations mediated by V(D)J recombinase is based on
comparisons of translocation breakpoint sequences with normal antigen
receptor rearrangements and is thus largely circumstantial (reviewed in
Rabbitts,88 Tycko and Sklar,89 and Showe and Croce90). The t(14;18) translocations found in most cases
of follicular B-cell lymphomas bear perhaps the strongest resemblance to normal V(D)J joints. Chromosome 14 breakpoints frequently occur at
or near the RSS bordering DH or JH segments.85,102-105 In
addition, sequences resembling the RSS heptamer have been described in
proximity to breakpoints on 18q21.89,90,104 The t(7;9)
(q34:q32) of T-cell lymphoblastic lymphoma/leukemia similarly involves
breakpoints at RSS flanking D segments of the TCR gene on chromosome
7, whereas cleavage sites on chromosome 9 were flanked by consensus RSS
heptamer sequences and separated from AT-rich nonamer-like sequences by an 11- to 12-bp motif.114 Also reminiscent of V(D)J coding
joints is the frequent finding of nontemplated nucleotide additions
suggestive of TdT activity at translocation breakpoints.89
Despite the resemblance of some translocation breakpoints to V(D)J
coding joints, much of the available data on breakpoint sequences do not strictly conform to the attributes of V(D)J recombination. Putative
cryptic RSS are often located at some distance from the breakpoint or
bear poor resemblance to physiologic RSS, including the critical
heptamer sequence.89 In addition, precise determination of
the initial cleavage sites requires analysis of the reciprocal translocation breakpoint. This has only been performed in a few studies, several of which indicate that cleavage did not occur at RSS
borders.89,114-116 Taken together, the data available
suggest that most chromosome translocations involving antigen receptor loci do not represent the products of normal V(D)J reactions.
An alternative scenario proposed for the involvement of V(D)J
recombinase in chromosome translocation involves a hybrid mechanism, wherein physiologic DNA breaks within antigen receptor loci are rejoined with DNA breaks occurring elsewhere in the genome by other
mechanisms.89 Nonantigen receptor breaks could occur
randomly or could be targeted by particular features of the locus. For example, both the mbr and mcr breakpoint clusters accounting for most
translocations involving the bcl-2 locus contain
polypurine-polypyrimidine sequences similar to the Chi recombination
element present in Escherichia coli.117,118 Binding
of a 45-kD nuclear protein to the Chi-like sequences
appears to facilitate their cleavage by a nuclease present in early
B-cell extracts.118 Another potential rearrangement
mechanism is suggested by recent studies of the Rag-1 and Rag-2
proteins, which bear a resemblance to prokaryotic transposases.119,120 After endonucleolytic cleavage at RSS,
the Rag proteins remain complexed with signal ends, which may then engage in a nucleophilic attack on a DNA strand, leading to a transposition event. The second step appears to be sequence-independent and, therefore, could explain the absence of RSS on partner chromosomes involved in antigen receptor locus translocations. One or more of these
mechanisms may lead to abnormal rejoining events involving DNA ends
created during the V(D)J reaction.
Ig class switch recombination is implicated in t(8;14) in some cases of
sporadic Burkitt's lymphoma, wherein the chromosome 14 breakpoints
occur within the switch regions upstream of Cµ, C, or
C.116,121,122 Although such cases eliminate the Eµ
enhancer on the der14 chromosome, it is now clear that the IgH locus
contains other control elements. In particular, the 3' Ig
enhancer/locus control region located near C has potent long-range
cis effects in deregulating c-myc expression.52 Switch
recombination is also implicated in murine plasmacytomas occurring
spontaneously or induced by pristane or mineral oil in susceptible
strains.93,123-125 The majority of these tumors harbor
t(12;15), juxtaposing c-myc with the switch region on mouse
chromosome 12. Recently, a novel oncogenic mechanism based on switch
recombination has been identified in a multiple myeloma cell line. An
excised switch region product containing C and the 3' IgH
enhancer were found inserted into chromosome 11 in close proximity to
the cyclin D1 proto-oncogene, which is associated with cyclin D1
overexpression.126 This model widens the spectrum of
potentially oncogenic recombination events that may occur during IgH
switch recombination or, for that matter, during V(D)J recombination.
Somatic hypermutation of Ig variable regions has been found in most
types of nodal B-cell lymphoma, suggesting that the precursors of these
tumors have passed through a germinal center
reaction.67,127 Given the recognition that DNA breaks
accompany the hypermutation process, this diversification mechanism
could potentially contribute directly to lymphoma-associated chromosome
translocations.67-69 There is evidence that this may be the
case in some t(8;14) Burkitt's lymphomas. Such translocations
frequently involve a V(D)J rearranged IgH allele, and there are several
examples of breakpoints occurring within the V region, correlating with
the physiologic target of somatic hypermutation.67,127 In
addition to a possible role in some Ig locus translocations, the
somatic hypermutation process may introduce genetic alterations outside
of the Ig loci. When placed near the Ig loci by a translocation, the
c-myc and bcl-6 genes frequently develop point mutations characteristic
of somatic hypermutation, potentially contributing to the additional
genetic changes required for progression of the malignant
phenotype.128,129 Recently, it was shown that somatic
hypermutation also affects the germline bcl-6 gene in normal germinal
center B cells, indicating that this process is not completely
contained within the Ig loci under physiologic
circum- stances.130-132 Altogether, peripheral B cells
subjected to antigen stimulation may acquire DNA breaks by at least 3 distinct mechanisms. Each of these is potentially capable of being
rejoined via alternative DNA damage repair mechanisms to generate
chromosome translocations. Given the increased propensity for genetic
errors in this tissue compartment, cellular surveillance mechanisms
controlling DNA damage may be of particular importance in preventing
oncogenic events.
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GENETIC SUSCEPTIBILITY TO LYMPHOMA |
Aside from immunodeficiency syndromes that impair host defenses against
the Epstein-Barr virus, 3 inherited disorders in humans affect cellular
responses to DNA damage and are associated with a high risk of lymphoid
malignancies. As with tumor suppressor genes identified for other human
cancers, the genes responsible for A-T, NBS, and Bloom's syndrome
appear to function in pathways that monitor genomic integrity. These
genes may therefore offer important clues to how neoplastic outcomes
are prevented during antigen receptor diversification in lymphocytes.
Candidate tumor-suppressor genes can be studied using mice, in which
genetic manipulation may be combined with developmental and functional
models of the immune system. These approaches are beginning to yield
new models for the connection between genomic instability in
lymphocytes and chromosome translocations in lymphoid malignancies.
A-T.
A-T is an autosomal recessive disorder characterized by progressive
cerebellar ataxia, telangiectasias on sun-exposed surfaces, hypersensitivity to ionizing radiation, and cellular and humoral immunodeficiency (reviewed in Gatti133). The gene mutated
in A-T, ATM, encodes a member of the PI-3 kinase gene family
that includes the DNA-PKcs.134 Cancer affects approximately
38% of A-T patients, despite their reduced
lifespans.133,135,136 Although adults may develop a broad
array of solid tumors, 85% of malignancies in A-T patients are
lymphoid leukemias and lymphomas. In children, these include ALL and
lymphomas, predominantly of T-cell origin. Adults frequently develop
chronic T-cell leukemias (T-PLL/T-CLL) associated with recurrent
chromosome translocations involving the TCR /, , or loci
and the TCL-1 oncogene on chromosome 14q32. Interestingly, up
to half of the individuals without A-T who contract T-PLL are
heterozygous carriers of mutations in the ATM
gene.137-139 Loss of heterozygosity is observed for the
normal allele in leukemic cells, suggesting that ATM functions
as a classic tumor suppressor in this context.140 ATM
mutations, as well as reduced ATM expression, have also been
identified in a subset of B-CLL, suggesting that abnormalities in this
gene may be involved in a diverse spectrum of lymphoid
malignancies.141-143
Susceptibility to lymphoid malignancies in A-T is paralleled by a
general predisposition to chromosome translocations. Whereas fibroblasts exhibit increased but apparently random chromosomal rearrangement,144 a restricted set of translocations may be
seen in up to 10% of nonmalignant lymphocytes from A-T
patients.136 In some instances, these represent clonal
T-cell expansions bearing translocations similar to those seen in T-PLL
and are presumably premalignant. However, there is also a high
frequency of translocations that are apparently not associated with
oncogene activation and include V(D)J trans-rearrangements involving
TCR loci on chromosomes 7 and 14.111,113,136 Such
interlocus rearrangements can also be detected at a much lower
frequency in normal individuals. These findings suggest that ATM is
involved in limiting the number of interchromosomal rearrangements
mediated by the V(D)J recombinase, a property that might explain the
increased incidence of oncogenic translocations in A-T. It is striking
that the chromosomal abnormalities in A-T lymphocytes primarily involve
T cells, raising the possibility that the relative importance of
various tumor-suppressor mechanisms differs for antigen receptor
diversification in T versus B lymphocytes.
There is at present only a superficial understanding of how ATM
functions in cells. A-T patients and cell lines exhibit heightened sensitivity to agents inducing double-strand DNA breaks such as ionizing radiation or bleomycin. In addition, A-T cells exhibit abnormal cell-cycle regulation in response to DNA damage. ATM does not
appear to be directly involved in repairing DNA damage, inasmuch as
extracts from A-T cells do not exhibit any consistent abnormalities in
DNA repair proficiency.145 Similarly, a direct role for ATM
in V(D)J recombination has been discounted based on normal
rearrangement of plasmid recombination substrates in A-T
fibroblasts.146 ATM has protein serine-threonine kinase
activity, and a number of substrates have been identified in vitro and
in vivo, many of which tend to play roles in either cell cycle
checkpoint control or apoptotic pathways.147-150 In
particular, ATM phosphorylates p53 in response to DNA breaks, and
accumulation of p53 in response to DNA damage is delayed or absent in
A-T cells. The emerging picture of ATM function, based both on clinical
manifestations in A-T patients and biochemical characterization of ATM
and its interacting partners, identifies ATM as playing a signaling
role receiving information from molecules that detect DNA damage,
perhaps by direct binding to lesions, and relaying that information to
appropriate downstream effector molecules that trigger responses
ranging from cell cycle arrest to apoptosis. In this context, ATM could
respond to unresolved DNA breaks during V(D)J recombination, either
suppressing indiscriminate repair pathways or perhaps limiting the
survival of cells at increased risk for abnormal rejoining events.
NBS.
NBS was first described in 1981 and was initially thought to be a
clinical variant of A-T.151 NBS patients lack the
characteristic ataxia and telangiectasias seen in A-T and instead are
characterized by microcephaly, borderline mental retardation, and
significant growth retardation. However, the disorders share several
other features, including hypersensitivity to radiation, the
characteristic chromosomal rearrangements in lymphocytes involving
antigen receptor loci, and the predisposition toward malignancies,
particularly those of lymphoid origin (reviewed in Wegner et
al152). NBS is exceptionally rare, with fewer than 100 patients listed in the international NBS registry in Nijmegen. To date,
40% of these patients have developed a malignancy, of which 85% are
leukemias or lymphomas. In contrast to the situation in A-T, the most
common lymphoid tumors in NBS patients are NHL of B-cell origin.
The recent positional cloning of the NBS1 gene, which is mutated in the
majority of NBS patients, clearly demonstrates that A-T and NBS are
distinct disorders.153,154 The corresponding nibrin protein
has no significant structural similarity to any other known protein.
Biochemical studies show that nibrin forms part of a multiprotein
complex that includes the products of the Rad50 and Mre11 genes and is
distributed diffusely throughout the nucleus in
fibroblasts.155 Irradiation of subnuclear regions leads to
localization of these complexes to exposed regions where they form
nuclear foci, a process that is defective in NBS
cells.155,156 Mre11 has both a 3' to 5'
exonuclease activity and a single-stranded endonuclease activity and is
assumed to be directly involved in the repair of DNA
damage.157 Among the substrates it can cleave are hairpins
of the type formed after Rag-1/Rag-2-mediated
cleavage,158,159 suggesting a potential role for the
complex that includes nibrin in V(D)J recombination. Additional
circumstantial evidence is provided by studies in yeast, in which
repair of double-strand DNA breaks either by end ligation or
nonhomologous end-joining requires the complex of Xrs2 (nibrin
homologue)/Rad50/Mre11, as well as the Ku proteins.160
Given the partial overlap in the clinical and cellular phenotypes
between A-T and NBS, the relationship between the ATM and nibrin
proteins in the cellular response to DNA damage remains a compelling
question, the answer to which may be especially relevant to the
pathogenesis of lymphoid malignancies.
Bloom's syndrome.
Bloom's syndrome is an autosomal recessive disorder characterized by
growth retardation, erythemic telangiectasias in sun-exposed skin,
defects in humoral immunity, and an extraordinary incidence of cancer.
The disorder is associated with mutations in the BLM gene, which is
homologous to ReqQ DNA helicases in bacteria.161 A recent
report described 100 cancers arising in 71 of the 168 individuals from
the Bloom's syndrome registry.162 These included a broad
variety of tumors, occurring at a mean age of 25 years. The most common
cancer in Bloom's syndrome is NHL, accounting for 21% of tumors in
the registry, and the incidence of acute lymphoid leukemias is also
markedly increased. Cells from Bloom's syndrome patients exhibit a
mutator phenotype and are subject to chromosomal instability with a
variety of spontaneous abnormalities, particularly those involving
recombinational exchanges between homologous
chromosomes.163-165 These abnormalities differ from those seen in A-T, and the extent to which antigen receptor loci may be
affected is not known. The BLM gene does not appear to be directly involved in V(D)J recombination, which is normal in Bloom's syndrome fibroblasts using plasmid recombination assays.146
Moreover, somatic hypermutation of Ig genes occurs at normal levels in
lymphocytes from Bloom's syndrome patients.166 From the
available information, it is not clear whether the increased incidence
of lymphoid malignancies in Bloom's syndrome stems from abnormalities
in antigen receptor diversification processes or simply reflects a
general cellular defect in the maintenance of genomic integrity.
Susceptibility to lymphoma in gene-deficient mice.
Lymphomas arising spontaneously in genetically mutant strains of mice
represent potentially important models for understanding the
pathogenesis of lymphomas in humans. Unfortunately, the most common
lymphoid malignancies in mice are thymic lymphomas, which are difficult
to correlate with the major lymphoma subtypes afflicting man. Mice with
a null mutation in p53 have a high incidence of lymphomas, occurring in
up to two thirds of individuals.167,168 Although most are
thymic tumors, peripheral B-cell lymphomas have also been reported.
Mice with the Scid mutation affecting DNA-PKcs also have an
increased incidence of thymic lymphomas compared with the parental
strain, approaching 15% by 12 weeks of age in some
colonies.169 Several groups have recently shown that mice with combined Scid and p53-null mutations (Scid
p53/) develop disseminated B-lineage lymphomas, with
incidence approaching 100% and occurring at a younger age than
lymphoid tumors in either parental strain.170-172 Our group
has shown that aggressive Scid p53/ lymphomas
carry a recurrent pattern of chromosome translocations involving the
IgH locus near the telomere of chromosome 12.173 Chromosome
15 is the most common partner in these translocations. However, in
contrast to the t(12;15) in murine mineral oil plasmacytomas, the c-myc
oncogene does not lie on the derivative 12 chromosome, implying the
involvement of a distinct oncogene in Scid p53/ lymphomas. The tumors appear to arise at the pro-B-cell stage, coincident with physiologic IgH rearrangement, suggesting that the
translocations arise during attempted IgH locus rearrangement in
Scid pro-B cells. This conclusion is supported by the finding that a Rag-2-null mutation blocked the development of t(12;15) pro-B-cell lymphomas when introduced into the Scid
p53/ strain, demonstrating that initiation of V(D)J
recombination was a required element in the oncogenic pathway.
Pro-B-cell lymphomas bearing t(12;15) have also been observed in
Ku86/ p53/ mice (A. Nussenzweig, personal communication). Pro-B-cell lymphomas in Scid
p53/ mice appear to be pathogenetically distinct from the
thymic lymphomas typical of p53/ mice, which do not have
a predominant cytogenetic marker173,174 and which occur
with equivalent frequency in mice deficient in Rag-1 or
Rag-2.173-175
The predisposition to t(12;15) lymphomas conferred by combined
mutations affecting DNA-PK and p53 function suggests that physiologic suppression of oncogenic DNA rearrangements during V(D)J recombination has 2 important elements: efficient rejoining of DNA ends created by
Rag-mediated DNA scission and an intact cellular response to DNA damage
(Fig 3). p53 is stabilized by
posttranslational modification and accumulates rapidly in response to
double-strand DNA breaks, leading to a broad cellular response that
includes arrest of the cell cycle at the G1/S phase boundary and either
DNA repair or apoptosis.176-178 In lymphocytes, the
apoptotic response to p53 induction predominates.177,178
Lymphoid precursors in Scid mice accumulate Rag-mediated DNA
breaks within antigen receptor loci, which leads to activation of p53
and apoptosis.171,179,180 Impairment of the p53 DNA damage
response in Scid p53/ mice permits partial rescue
of early thymocyte development, presumably because T-cell precursors
survive long enough to undergo limited assembly of TCR genes by
otherwise inefficient, DNA-PK-independent
processes.170-172 By analogy, impairment of p53-dependent
apoptosis in Scid pro-B cells may permit illegitimate rejoining
of the cleaved IgH locus to chromosome 15 and other sites. p53 mutation
could also contribute to the development of lymphoma by facilitating
mutations elsewhere in the oncogenic pathway. In human lymphomas,
mutation of p53 is not a consistent finding, being primarily associated
with aggressive subtypes and with relapse, suggesting that it may occur
late in malignant evolution.181-183 Moreover, lymphoid
malignancies are not prominent among tumors in patients with
Li-Fraumeni syndrome, who carry germline p53
mutations.184-186 Given the complexity of the cellular
response to DNA damage, it may be that the functions of other genes
involved in these pathways are more relevant to containing
lymphoid-specific mechanisms of genomic instability.
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| Fig 3.
Model for susceptibility to chromosome
translocations mediated by V(D)J recombinase. Antigen receptor
diversification is initiated by DNA breaks, mediated in the case of
V(D)J recombination by the Rag-1 and Rag-2 proteins at RSS-coding
segment borders (depicted by triangles and squares, respectively). The
normal rejoining process resolves both sets of DNA ends efficiently, so
that the potential for aberrant recombination is minimal. Failure of
the normal rejoining process triggers cellular DNA damage sensors,
which effect cell death and thereby prevent oncogenic rearrangements.
Impairment in cellular DNA damage responses may allow alternative
DNA repair pathways to mediate rejoining of antigen receptor genes with
sites elsewhere in the genomesometimes including oncogene loci, which
give rise to lymphoma-associated chromosome translocations.
|
|
A number of other mouse strains with defects in V(D)J recombination
and/or DNA damage responses exhibit susceptibility to lymphomas. In
particular, mice lacking Atm have a high incidence of thymic
lymphomas. Cytogenetic studies indicate that these tumors carry complex
karyotypic abnormalities, including translocations involving chromosome
14, which harbors the mouse TCR/ complex.187,188 The
proximity of the TCR loci to the translocation breakpoints in
these tumors has not been reported. T-lymphoid malignancies are also
increased in mice with mutations in the V(D)J recombinase component
Ku70,31,189 combined Scid and PARP-null
mutations,190 as well as mice deficient in the DNA mismatch
repair gene MSH2.191-193 Cytogenetic studies, which have
provided seminal insights into the pathogenesis of lymphoid
malignancies in humans, have received limited application in
mouse lymphoid tumors. Although this undoubtedly reflects the
challenges inherent in the interpretation of mouse karyotypes,
techniques such as spectral karyotyping188 or panels of
chromosome-specific probes194 may facilitate a more
detailed characterization of lymphoid malignancies associated with
specific genetic defects in mice.
| |
SYNTHESIS AND CONCLUSIONS |
The capability of the immune system to recognize new pathogens and
mount adaptive responses depends on the creation of antigen receptor
gene diversity through the induction of targeted genomic instability.
In addition to ordered rearrangements during B- and T-cell ontogeny,
changes in the genetic makeup of lymphoid cells occur through reflexive
interactions with antigenic selection mechanisms, leading to rapid
evolution of the immune repertoire. Escape of these gene
diversification processes from their physiologic targets remains a
compelling explanation for many of the oncogenic genetic alterations
that are unique to malignancies of the lymphoid lineage. From the
standpoint of cancer biology, the genetic instability required for
adaptive immunity appears to violate fundamental cellular paradigms for
maintaining integrity of the genome. As the mechanisms of antigen
receptor gene diversification are elucidated, it is thus important to
ask how these processes are contained and targeted specifically to
their intended loci. Measures to ensure genetic safety could operate at
3 levels: by regulating specificity in the targeting of DNA breaks, by
monitoring the resultant DNA ends and facilitating appropriate
rejoining mechanisms, and by preventing indiscriminate DNA repair.
For V(D)J recombination, 2 features of the cleavage reaction may
contribute to specificity. Recognition of RSS by Rag-1 and Rag-2
defines the correct sites for DNA breaks. However, the sequence requirements observed during in vitro cleavage reactions are more degenerate than observed in vivo,5 suggesting that other
factors may assist in directing DNA scission to the appropriate target. Such factors may be tied to the regulation of V(D)J rearrangement by
transcription. Specificity is also enhanced by the preferential rearrangement of segments located on the same chromosome, even though
RSS pairs may be located megabases apart. In experiments using
extrachromosomal recombination substrates, a similar bias is seen for
intramolecular over intermolecular reactions, with a recent study
indicating that this bias occurs at the level of coding end
rejoining.195 If these results can be extrapolated to
chromosomal loci, they may shed light on an important mechanism for
suppressing chromosome translocation events. Cellular exposure to free
DNA ends during the V(D)J reaction is controlled in part by the
obligate coupling of 2 cleavage events. These occur within a single
synaptic complex that facilitates efficient rejoining.8 Similar principles may apply to class switch recombination, which, like
V(D)J recombination, involves reciprocal rejoining of ends and requires
the DNA-PK complex. Rejoining of antigen receptor DNA breaks by
DNA-PK-independent processes appears to entail a lesser degree of
protection for cut ends, and exclusion of indiscriminate DNA repair may
be an important factor in preventing aberrant rejoining events. For
example, Rag-mediated DNA cleavage is restricted to the G0/G1 phases of
the cell cycle,196 a feature that may preclude replication-dependent repair of unresolved DNA ends. Measures to ensure
precise targeting and rapid resolution of DNA breaks during antigen
receptor diversification may occasionally fail, necessitating cellular
responses that either interrupt the propagation of cells susceptible to
abnormal recombination events or otherwise prevent inappropriate
rejoining. Indeed, compared with other somatic cells, lymphocytes have
a low threshold for death after DNA damage, which may reflect an
adaptation to the oncogenic risks inherent in antigen receptor
diversification. In mice, the p53 pathway acts as a tumor suppressor
for lymphoid malignancies and is particularly important when rejoining
of V(D)J-mediated DNA breaks is impaireda point underscored by the
high incidence of lymphomas harboring recurrent chromosome
translocations observed in mice with combined mutations in DNA-PKcs and
p53. In humans, ATM could serve a related function by
activating p53 and other cellular responses to unresolved DNA breaks in
antigen receptor genes. Further studies of ATM and other
lymphoma susceptibility genes will provide additional insight into
these pathways.
The germinal center has emerged as a focal point for investigating the
pathogenesis of lymphoid malignancies, particularly B-cell NHL. In
physiologic terms, the germinal center is a veritable hotbed of genomic
instability, in which the iterative process of antigen receptor gene
mutation, selection, and proliferation leads to a rapid evolutionary
improvement in Ig affinity for antigen. This involves both gradualistic
changes, ie, accumulations of small mutations, as well as saltations
engendered by wholesale replacement of V regions. At the same time,
exchange of IgH constant regions through class switch recombination
broadens the effector functions of antibodies. The common subtypes of
nodal B-cell NHL resemble normal comp |
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INTRODUCTION
PHYSIOLOGIC GENOMIC INSTABILITY...