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Molecular Biology of Burkitt’s Lymphoma

From the Departments of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA.

Address reprint requests to Jon C. Aster, MD, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115.

ABSTRACT

The diagnostic category of Burkitt’s lymphoma encompasses a closely related group of aggressive B-cell tumors that includes sporadic, endemic, and human immunodeficiency virus–associated subtypes. All subtypes are characterized by chromosomal rearrangements involving the c-myc proto-oncogene that lead to its inappropriate expression. This review focuses on the roles of c-myc dysregulation and Epstein-Barr virus infection in Burkitt’s lymphoma. Although the normal function of c-Myc remains enigmatic, recent data indicate that it has a central role in several fundamental aspects of cellular biology, including proliferation, differentiation, metabolism, apoptosis, and telomere maintenance. We discuss new insights into the molecular mechanisms of these c-Myc activities and their potential relevance to the pathogenesis of Burkitt’s lymphoma and speculate on the role of Epstein-Barr virus.

BURKITT’S NON-HODGKIN’S lymphoma comprises a heterogeneous group of highly aggressive B-cell malignancies. Originally termed undifferentiated lymphoma in the Rappaport classification, these tumors were subsequently classified as small non–cleaved-cell lymphoma, Burkitt type, in the working formulation¹ and then recast again as Burkitt’s lymphoma (BL) in the Revised European-American Lymphoma classification.² The most recent World Health Organization classification maintains BL as a distinct category of peripheral B-cell lymphoma. Recognition and diagnosis of BL are of clinical importance, as this tumor responds best to specific chemotherapeutic regimens that differ from those used for other aggressive B-cell lymphomas, such as diffuse large-cell lymphoma.³

BL is invariably associated with chromosomal translocations that dysregulate the expression of c-myc,^4,5 a gene encoding a basic helix-loop-helix (bHLH) transcription factor that binds to DNA in a sequence-specific fashion. c-myc normally plays a central role in the transcriptional regulation of an emerging set of downstream genes that control diverse cellular processes, including cell cycle progression and programmed cell death (apoptosis). This review will focus on the molecular and biologic consequences of c-myc translocation and dysregulation in BL. We will also discuss the role of Epstein-Barr virus and other acquired genetic aberrations that collaborate with c-myc to cause BL.

CLINICOPATHOLOGIC FEATURES OF BL

BL principally occurs in three clinical settings (Table 1). Burkitt first described an endemic form found in equatorial Africa and subsequently in New Guinea that typically arises at extranodal sites in adolescents and young adults. Endemic BL subsequently proved to be a tumor of B cells latently infected with Epstein-Barr virus (EBV). A sporadic, morphologically identical form of BL occurring in the United States and other regions also usually arises at extranodal sites in adolescents or young adults.² Lastly, BL is common in HIV-infected individuals.⁶ Unlike endemic BL, only a subset of sporadic and human immunodeficiency virus (HIV)–associated BLs are EBV-associated.

View larger version (157K): [in this window] [in a new window]   Fig 1. Histopathology of BL. (A) Arrowheads denote some of the phagocytic macrophages responsible for the characteristic "starry sky" appearance (x200). The dark material within macrophages is the nuclear remnant of phagocytized tumor cells. (B) A monomorphous population of intermediate-sized BL cells and a macrophage containing nuclear debris are shown (x400). (C) Immunoperoxidase staining for Ki-67, a marker of growing cells, shows strong nuclear reactivity in virtually all BL cells (x400).

Insight into the cell of origin in BL has come from DNA sequence analysis of the productively rearranged IgM heavy-chain alleles of tumor cells. The survival of normal germinal center B cells depends on the expression of an Ig receptor capable of recognizing antigen with high affinity. The ability to make high-affinity antibodies requires somatic hypermutation, in which germinal center B cells acquire mutations at a high rate within the Ig genes in regions (termed complimentarity-determining regions) that encode the antigen-binding residues.²⁷ Because somatic hypermutation is apparently confined to germinal center B cells, it marks B cells as being of germinal center origin. Sequence analysis of the Ig variable heavy (V_H)- and light (V_L)-chain genes in endemic, sporadic, and HIV-associated BL has shown that they have undergone somatic hypermutation.^28-31 Additionally, the Ig genes of some endemic BLs show evidence of continuing somatic hypermutation, a phenomenon also seen in follicular lymphomas. These data are compatible with a germinal center B-cell origin for all forms of BL.^31,32

MECHANISMS OF c-myc DYSREGULATION IN BL

The sine qua non of BL is the inappropriately high activity of c-Myc, a 64-kd protein belonging to the family of bHLH transcription factors. c-Myc protein levels are upregulated in BL through several different mechanisms. Most importantly, the chromosomal translocations characteristic of BL always result in the juxtaposition of the DNA coding sequences for c-myc with sequences from Ig genes termed enhancers. Ig enhancer elements bind to B cell–specific factors capable of activating transcription from genes located up to 500,000 base pairs (500 kb) away. Because Ig enhancer elements are specifically active in mature B cells, their juxtaposition to c-myc in BL cells drives inappropriately high levels of c-myc mRNA and protein expression.^33,34 In addition, negative regulatory sequences residing within c-myc are often removed as a direct consequence of chromosomal translocation or are mutated through other mechanisms, further contributing to increased c-Myc activity.

Position of Breakpoints in Ig and c-myc
In 80% of cases of BL, the translocation partner for c-myc is the IgH locus, leading to the formation of a t(8:14)(q24:q32) (Fig 2A). In 15% of cases, the translocation partner is the locus at chromosome 2p11, whereas in the remaining 5% of cases, the locus at chromosome 22q11 is involved. The positions of the chromosomal breakpoints in c-myc and various Ig genes are widely dispersed, which makes it difficult to design sensitive polymerase chain reaction–based tests for c-myc/Ig fusion genes. As a result, the c-myc translocations involving chromosome 8q32 are only reliably identified by karyotyping of metaphase chromosomes or fluorescent in situ hybridization, which can, if necessary, be performed on interphase nuclei prepared from paraffin-embedded tissue (Fig 2B).

View larger version (32K): [in this window] [in a new window]   Fig 2. Detection of c-myc/Ig chromosomal translocations in BL. (A) Karyotypic appearance of c-myc translocations in BL. Idealized idiograms are shown. In the classic t(8:14), the c-myc gene is placed in apposition to the immunoglobulin heavy-chain locus (IgH) on the derivative chromosome 14 (14q+). In the variant translocations (t(2;8) and t(8;22)), the light chain loci are placed in proximity to the c-myc gene on the derivative chromosome 8 (8q+). The orientation of the translocated segment on each derivative chromosome is denoted by an asterisk. (B) Dual-color interphase fluorescence in situ hybridization.35 The diagrams (right) show the position of regions telomeric (T) and centromeric (C) of c-myc that are homologous to red and green fluorescent-labeled probes and the expected position of these probes on metaphase chromosomes. In interphase nuclei isolated from a paraffin-embedded BL tumor specimen (left), physical separation of red and green signals is observed that could be indicative of any of the three common c-myc translocations (t(2;8), t(8;14), or t(8;22)). Analysis of karyotypes prepared from metaphase cells of the same tumor confirmed the presence of the t(8;14) (not shown).

View larger version (28K): [in this window] [in a new window]   Fig 3. Positions of c-myc/Ig breakpoints in BL. (A) Germ-line configuration of c-myc. Positions of the c-myc coding sequence (blackened portions of exons), the P1 and P2 c-myc promoters, and subtype-specific BL breakpoints are depicted (arrowheads). All BLs carry one of three balanced chromosomal translocations, each involving c-myc on chromosome 8 band q24. The positions of the most common translocation breakpoints in endemic BL (eBL), sporadic BL (sBL), and AIDS-associated BL (aBL) are shown, as well as the positions of variant breakpoints that may be seen in any BL subtype. (B) Prototypical c-myc/IgH fusion gene in endemic BL. The positions of the IgH µ enhancer, µ switch region, and µ constant region coding sequences on the derivative chromosome 14 (14q+) are shown in relation to DNA sequences translocated from chromosome 8. The breaks in the IgH gene occur during attempted V(D)J recombination, leading to the juxtaposition of the IgH enhancer and c-myc. Transcription is driven from the normal P1 and P2 promoters. Mutations are frequently found in negatively regulatory regions of c-myc exons 1 and 2. (C) Prototypical c-myc/IgH fusion gene in sporadic BL. The breakpoints in the IgH gene occur during attempted Ig switching, leading to the juxtaposition of 3' IgH enhancer elements (such as E) and intron 1 sequences of c-myc. The translocation results in the loss of the 5' negative regulatory region of c-myc exon 1 and the P1 and P2 promoters. Transcription occurs from a cryptic promoter (P3). Mutations may be found in negative regulatory regions of exon 2. (D) and (E) Variant c-myc fusion genes involving the Ig and light-chain loci. In the variant translocations, the breaks in chromosome 8 occur in noncoding sequences lying 3' of c-myc, which are joined to sequences from either chromosome 2 (Ig locus) or 22 (Ig locus). Ig enhancer elements from either the (Ei and E3') or the locus (HuE) activate transcription from the normal P1 and P2 promoters. Mutations are frequently found in c-myc exon 1 and exon 2 negative regulatory regions.

Little is known about the mechanisms that cause the chromosomal breaks in c-myc. The breakpoints in c-myc occur at sites with no homology to V(D)J or switch recombinase recognition sequences, which suggests that they are unlikely to be dependent upon these activities. A recent observation of interest is that a very high proportion of mice with genetic defects that impair the repair of double-stranded DNA breaks develop pro–B-cell tumors, all of which have chromosomal translocations involving IgH and c-myc.⁴⁷ The relevance of this observation to the mechanism of sporadically occurring c-myc translocations in human BL remains to be ascertained.

Enhancer Elements Involved in c-myc Dysregulation in BL
The Ig enhancer elements that contribute to c-myc dysregulation normally specifically stimulate transcription in B cells, presumably because B cell–specific transcription factors associate with these DNA segments. Although the precise identity of the critical enhancer sequences in each type of c-myc translocation remains uncertain, experiments conducted with transgenic mice have shown that the juxtaposition of these sequences and c-myc creates an oncogene. A transgene consisting of the IgH Eµ enhancer fused to c-myc coding sequences (thus resembling the structure of the rearranged c-myc alleles seen in endemic BL, Fig 3B) is a potent inducer of B-cell tumors in mice, which suggests that Eµ elements may be sufficient to drive oncogenic levels of c-myc expression in tumors with the t(8;14). The Eµ enhancer is not juxtaposed in cis to c-myc in most sporadic BL translocations (Fig 3C), however, indicating that other IgH elements also contribute to c-myc dysregulation. Consistent with these observations, a human c-myc/IgH "minichromosome" deleted from the Eµ enhancer has unaltered capacity to produce B-cell lymphomas in a mouse model,⁴⁸ which supports a role for 3' IgH sequences, such as the E enhancer, in the upregulation of c-myc expression.⁴⁹

In (2;8) translocations involving the Ig locus (Fig 3D), it seems that three elements located 3' to C segments drive c-myc overexpression: the kappa intron enhancer (Ei), the 3' enhancer (E3'), and the matrix attachment region.³⁷ In (8;22) translocations involving the Ig locus (Fig 3E), the regulatory elements driving c-myc expression are less well defined but are encompassed by a 12-kb genomic fragment located 3' of C segments that include the human Ig enhancer.⁴¹

Role of c-myc Mutations and Deletions in Dysregulation
Expression of c-myc in BL seems to be further increased by the deletion and/or mutation of negative regulatory sequences within c-myc. The c-myc gene consists of three exons (Fig 3A). Exons 2 and 3 encompass the coding sequence for c-myc protein, and exon 1 encodes an unusually long 5' untranslated sequence that acts as a negative regulatory sequence by virtue of the presence of sequences that block transcriptional elongation.⁵⁰ In addition, intron 1 of c-myc contains a binding site for a nuclear protein known as c-myc intron binding factor that may function as a negative transcriptional regulator.⁵¹ In sporadic BL, the t(8:14) breakpoints tend to fall in the first intron, thus removing negative regulatory exon 1 and intron 1 sequences (Fig 3C). These breakpoints lie downstream of the normal P1 and P2 promoters, so that transcription is initiated from an alternative promoter within the first intron. In contrast, in endemic BL and cases of sporadic BL associated with variant translocations, chromosomal breakpoints 5' or 3' to c-myc preserve its normal genomic organization and transcriptional start sites (Fig 3B, 3D, and 3E). However, DNA sequence analyses in these tumors have shown that the c-myc regulatory regions within exon 1 and intron 1, including the binding site for the c-myc intron binding factor, are frequently mutated, which results in the abrogation of negative regulation.^52,53 Taken together, these data suggest that the removal or mutation of cis-acting negative regulatory elements cooperates with positive regulatory Ig enhancer elements to drive c-myc expression in BL.

Mutations within exon 2 of c-myc may also enhance function by causing amino acid substitutions that stabilize the c-Myc protein,^54,55 thereby increasing its concentration. One important residue in this regard, threonine 58,^56,57 is subject to a phosphorylation event that targets c-Myc to a complex of proteins with proteolytic activity known as the proteosome. Mutation of threonine 58 is common in BL and sharply reduces the proteosome-mediated degradation of c-Myc. More generally, BL cell lines show increased c-Myc half-life relative to control cells,⁵⁷ even those in which threonine 58 is not mutated. Hence, a variety of mechanisms may collaborate to enhance c-Myc stability in BL.

MOLECULAR MECHANISMS OF c-Myc FUNCTION AND REGULATION

c-myc plays an important role in many aspects of cellular homeostasis, and its activity is normally tightly regulated at posttranscriptional as well as transcriptional levels. Posttranscriptional controls are overcome in BL through overexpression of c-myc protein (c-Myc) and secondary genetic events that variously enhance transforming activities, antagonize the activity of negative regulatory factors, and/or downregulate c-Myc activities that tend to counteract cellular transformation (eg, induction of programmed cell death).

The intermolecular interactions that determine c-Myc function represent potential targets for therapy aimed at the specific genetic lesion in BL and are thus of great importance. Unfortunately, detailed understanding of c-Myc function has remained elusive, possibly because of the existence of multiple regulatory feedback loops that tend to obscure primary effects, and also due to c-Myc’s functional effects being strongly influenced by cellular context and its level of expression. Several recent reviews have covered this area in detail.^58-60 We focus here on those aspects of c-Myc’s molecular biology (summarized in Fig 4) that are well established or particularly pertinent to cellular transformation.

View larger version (18K): [in this window] [in a new window]   Fig 4. Protein-protein interactions regulating c-Myc activity. The function of c-Myc is modulated by the availability of its heterodimeric binding partner Max and the concentrations of competing Max/Max homodimers and Max/Mad heterodimers, all of which bind to a hexameric DNA sequence termed the E box. Myc/Max heterodimers bound to DNA recruit protein complexes (TRRAP) associated with histone acetylase activity that modify chromatin and activate transcription, whereas Max/Mad heterodimers recruit inhibitory complexes (Sin3/N-CoR) associated with histone deacetylase activity. The function of c-Myc is further regulated by other polypeptides that modulate its association with DNA and its stability (see text for details).

The current model of c-Myc regulation (summarized in Fig 4) predicts that the underproduction of inhibitory bHLH-LZIP proteins might have consequences similar to those of c-Myc upregulation. In support of this idea, homozygous mxi-1 knockout mice have an increased susceptibility to lymphoma.⁶⁸

View larger version (29K): [in this window] [in a new window]   Fig 5. Summary of cell biologic effects of c-Myc overexpression. Upregulation of c-myc disrupts many aspects of cell function, which leads to tumorigenesis.

Role of Other c-Myc–Binding Proteins
The binding of c-Myc/Max heterodimers to E-box elements is weak compared with other E-box–binding transcription factors, such as the upstream stimulatory factor,⁸¹ a characteristic that may facilitate additional regulation at the level of DNA binding. Consistent with the existence of proteins that modulate c-Myc heterodimerization and binding to DNA, the C-terminal domain of c-Myc interacts with a host of other proteins implicated in the regulation of transcription, such as YY-1, AP-2, BRCA-1, TFII-I, and Miz-1.^59,60 A number of other proteins bind to the N-terminal portions of c-Myc and may thereby alter its interaction with elements of the transcriptional machinery. In addition to TRRAP (see above), N-terminal domain–interacting proteins include p107, Bin1, MM-1, Pam, and Amy-1.^59,60 Many of these interactions seem to be mediated through the evolutionarily conserved and functionally crucial "Myc box" sequences (MB1 and MB2) within the N-terminal transcriptional activation domain.⁶⁰ The significance of modulation of c-Myc function by these proteins with respect to B-cell transformation remains to be investigated.

Transcriptional Repressor Functions of c-Myc
Experiments in cultured cells and in vitro assays have suggested that c-Myc is also a transcriptional repressor of some promoters through mechanisms that are not currently understood. Several potential targets of c-Myc–mediated transcriptional repression have been identified, including the genes for adhesion molecules leukocyte function–associated antigen (LFA) 1 and alpha3/beta1 integrin, the growth arrest genes gadd 45 and gas1, and the cyclin-dependent kinase (CDK) inhibitor p27.⁵⁸ Transcriptional repression often, but not always, correlates with the presence of a pyrimidine-rich sequence termed the initiator within promoter elements, and requires the c-Myc dimerization domain and the N-terminal Myc box 2.⁸²

CELL BIOLOGIC CONSEQUENCES OF c-myc DYSREGULATION

c-Myc influences multiple cellular processes that can contribute positively or negatively to cellular transformation. These pleiotropic effects are mediated through the binding of c-Myc/Max heterodimers to the promoter elements of a discrete set of downstream genes, thereby either inducing or repressing their expression. The recent development of cDNA chip technology has permitted screening of cultured human fibroblasts for genes that are regulated by c-Myc.⁸³ This study, which used an array consisting of 6,416 genes, detected 27 genes that were induced and nine genes that were repressed by c-Myc. Among these c-Myc targets were genes that encode proteins that regulate cell growth, division, death, metabolism, adhesion, and motility, all of which are potentially important in cellular transformation. These data complement the cell biologic effects of experimental c-Myc upregulation, which include increases in proliferation, apoptosis, and cellular metabolism. As has been discussed, BL cells also exhibit high rates of growth, apoptosis, and metabolism, which suggests that these effects of c-Myc are likely to be of pathogenetic relevance in human tumors.

Cell Cycle Progression
The role of c-Myc in cell cycle regulation is complex and incompletely understood, but its expression is strongly correlated with proliferation. It is normally expressed in all dividing cells, where it enhances cell cycle progression,^84-88 and is downregulated in cells undergoing cell cycle arrest and/or terminal differentiation.⁸⁹ The global nature of c-Myc’s effects on cell cycle progression has been demonstrated in c-myc knockout fibroblasts, which demonstrate a marked lengthening of both G1 and G2.⁸⁸ The most profound defect in cell cycle regulators in these cells is a 12-fold reduction in cyclin D1/CDK4 and cyclin D1/CDK6 activity, a pair of protein kinases that promote cell cycle progression. However, multiple other CDK activities are also diminished in c-myc knockout cells, and restoration of CDK4 and CDK6 activity does not correct the c-Myc defect, which indicates that c-Myc acts through multiple downstream effectors.⁸⁸ Other potential targets include the cell cycle inhibitor p27, which is upregulated in c-Myc–deficient cells⁸⁸ and downregulated by c-Myc overexpression,⁹⁰ a second cell cycle inhibitor, p21, which is downregulated by c-Myc, and cdc25A,⁹¹ a protein phosphatase upregulated by c-Myc that activates CDK2 and CDK4.

In addition to increased proliferation, several studies suggest that one important consequence of enforced c-Myc overexpression is the induction of genomic instability, which may contribute to subsequent transformation. Felsher and Bishop⁹² found that transient upregulation of c-Myc increased the transformation of Rat1 fibroblasts at least 50-fold. This protransforming effect correlated with the appearance of chromosomal abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. c-Myc expression also induced genomic changes in normal human fibroblasts, although these cells did not become tumorigenic. It is proposed that genomic instability stems from accelerated passage through the G1 phase of the cell cycle and perturbation of the G1/S phase checkpoint.

Whether this "promutational" effect of c-Myc applies to spontaneous human tumors, such as BL, is not yet clear. One consequence of such an effect that has therapeutic implications is that c-Myc might act as a "hit-and-run" oncogene by generating secondary transforming events, which then in turn make c-Myc dysregulation superfluous for maintenance of the transformed state.

Differentiation
Given that c-Myc promotes persistent cell cycling and that terminal differentiation requires exit from the cell cycle, it is not surprising that the overactivity of c-Myc inhibits differentiation in a number of cellular assays. A second potential mechanism by which inhibition could be maintained is through the repression of genes that direct differentiation, such as C/EBP.^93,94

Analysis of a mouse model for BL created with a transgene consisting of a c-myc coding sequence fused to the IgH Eµ enhancer first suggested that c-Myc partially inhibits the differentiation of primary B cells.⁹⁵ In the prelymphomatous state, these animals show increased numbers of pre-B cells, decreased numbers of mature B cells, and increased numbers of cycling cells in both early and late B-cell populations. More recently, complementary data have been obtained from a mouse model that permits c-myc expression to be turned off by treatment with doxycycline. In approximately 90% of established lymphoid and myeloid tumors, downregulation of c-Myc elicits cell cycle arrest, terminal differentiation, and tumor regression.⁹⁶ Tumors relapsed in a minor subset of doxycycline-treated animals despite c-Myc downregulation, thereby supporting the idea that c-Myc may sometimes act through a promutational mechanism.

Metabolism
c-Myc regulates diverse metabolic pathways. Of most obvious pertinence to BL, which is usually associated with very high levels of serum lactate dehydrogenase A (LDH-A), c-Myc activates LDH-A transcription.⁹⁷ High levels of LDH-A correlate with the Warburg effect (the tendency for tumor cells to produce increased lactic acid under aerobic conditions) and seem to help tumor cells to survive under hypoxic conditions. These effects of c-Myc and LDH-A on metabolism and survival have been clearly demonstrated in cell culture assays. Rat fibroblasts stably overexpressing either LDH-A or c-Myc produce increased amounts of lactate under aerobic conditions, whereas antisense LDH-A RNA antagonizes the growth of c-Myc–transformed fibroblasts under hypoxic conditions and reduces the growth of BL cell lines in soft agar.⁹⁷

c-Myc may also regulate genes involved in nucleotide synthesis,^98-104 protein synthesis,^105-107 and iron metabolism.¹⁰⁸ In the case of iron metabolism, c-Myc acts through an initiator element to negatively regulate the expression of H-ferritin (which acts to sequester Fe⁺²), and also upregulates IPR2, a factor that stabilizes the transferrin receptor and thereby increases cellular uptake of Fe⁺². The net effect of these alterations is to increase free intracellular Fe⁺². Of note, downregulation of the expression of the H-ferritin gene is required for cellular transformation by c-myc.¹⁰⁸

Apoptosis
In some cellular contexts, C-myc overexpression induces programmed cell death (apoptosis). This association may underlie the starry sky appearance that is characteristic of BL, which stems from a high rate of apoptotic cell death. Several lines of investigation suggest the existence of p53-dependent and p53-independent c-Myc–induced cell death pathways. The p53-dependent pathway is triggered by withdrawal of growth factors and is accompanied by the upregulation of the regulatory protein phosphatase cdc25A.^91,109 A second potential player in the p53-dependent pathway is ARF, a c-Myc–inducible gene encoding a protein that upregulates p53 expression. c-Myc overexpression in primary mouse embryo fibroblast cell lines selects strongly for spontaneous inactivation of the ARF-p53 pathway, consistent with its role in the death of cells with inappropriately high expression of c-Myc.¹¹⁰ The p53-independent pathway is induced upon glucose deprivation and correlates with increased levels of LDH-A.⁹⁷ The mechanism of this effect is unclear, but overexpression of LDH-A alone in certain cell types is sufficient to cause apoptosis upon withdrawal of glucose but not serum.

Murine models support a role for the p53-dependent death pathway in the antagonism of c-Myc–induced lymphomagenesis. Spontaneous inactivation of the ARF-p53 pathway is a frequent finding in tumors arising in Eµ-Myc transgenic mice,¹¹¹ and development of lymphoma is accelerated in Eµ–c-myc transgenic mice in an ARF-deficient genetic background.¹¹² Conversely, mice that are heterozygous for knockout of Bmi-1, a transcription factor that represses the expression of ARF, show decreased c-Myc–induced lymphomagenesis. As predicted, the decrease in Bmi-1 gene dosage increases both the c-Myc–dependent upregulation of ARF expression and apoptosis.¹¹³

Bcl2 and N-ras also protect cultured cells from c-Myc–induced apoptosis,^114,115 an activity that may partially explain their synergism with c-Myc in induction of aggressive murine lymphomas.^97,116-119 Bcl2 protects against both the p53-dependent and p53-independent c-Myc–induced cell death pathways. Constitutive activation of N-ras induces a form of cellular senescence in which cells permanently withdraw from the cell cycle, which is hypothesized to be a protective mechanism to eliminate cells with potentially oncogenic N-ras mutations. The prosenescent effect of N-ras is blocked by c-Myc, whereas the proapoptotic effect of c-Myc is antagonized by activated N-ras.¹²⁰

Immortalization
In addition to being able to prevent the induction of cellular senescence by activated oncogenes such as N-ras, c-Myc also seems to maintain the expression of telomerase, an enzyme with reverse transcriptase activity. Telomerase contributes to the immortalization of cells by permitting the indefinite maintenance of the ends of chromosomes (telomeres), which normally shorten as part of the aging process, eventually causing cellular senescence. Expression of the catalytic subunit of telomerase, telomerase reverse transcriptase, is directly induced by c-Myc at the level of transcription in normal human mammary epithelial cells and fibroblasts.^121,122 Enforced telomerase reverse transcriptase expression immortalizes rodent cells and makes cells more susceptible to transformation.^122,123

Cellular Adhesion
Increased expression of c-Myc leads to the downregulation of LFA-1 (integrin alphaLbeta2 integrin) in lymphoblastoid cell lines⁵⁹ and of extracellular matrix proteins such as collagen and fibronectin in fibroblasts.⁸³ It has been suggested that decreased expression of polypeptides that promote cellular adhesion might enable BL cells to escape immune surveillance.

FACTORS THAT COOPERATE WITH c-myc IN BL

Other Recurrent Genetic Aberrations in BL
A number of recurrent secondary aberrations are seen in BL, none of which seem to be specific. Given that c-Myc overexpression can induce p53-dependent and p53-independent apoptosis, it might be anticipated that secondary lesions in BL abrogating programmed cell death pathways would be found. Approximately one third of BLs have anomalies in chromosome 17p, the site of the p53 gene,¹²⁴ which correlates with the detection of p53 mutations in approximately one third of fresh BL tumor specimens.^125,126 It has been suggested that acquisition of p53 mutations and deletions is a late event occurring during BL progression.¹²⁷ Consistent with this possibility, inactivation of normal p53 by the E6 protein of human papilloma virus increases the tumorigenicity of BL cell lines, whereas restoration of normal p53 function in p53 mutant BL cell lines reduces tumorigenicity.¹²⁸ More recently, transcriptional silencing of p73, a functional homolog of the p53, through promoter hypermethylation was detected in 30% of BLs.¹²⁹ Another means of antagonizing cell death is to inhibit the death pathways themselves. A high fraction of BLs show hypermethylation and transcriptional silencing of the death-associated protein kinase gene,¹³⁰ which encodes a Ca²⁺/calmodulin–dependent serine/threonine protein kinase that enables the generation of death signals downstream of interferon gamma,¹³¹ tumor necrosis factor alpha, and Fas.¹³² The relevance of this secondary event is made more plausible by the observation that c-Myc induces the expression of thrombin receptor agonist peptide 1,⁸³ which binds the intracellular domain of the tumor necrosis factor receptor and may be involved in the generation of death signals from this receptor.

The BCL-6 gene on chromosome 3q27 is involved by translocations in approximately 20% to 30% of large B-cell lymphomas and is mutated in a wider spectrum of B-cell tumors of germinal center or post–germinal center origin, including 30% to 50% of BLs.^133,134 The type of mutations found in BCL-6 is characteristic of that seen in somatically hypermutated Ig gene segments, which suggests that they might be induced by the same mechanism. The pathogenetic significance of these acquired mutations is that they also seem to dysregulate BCL-6 expression. Other recurrent abnormalities include a host of chromosomal aberrations, including deletions and rearrangements of 1q and 6q and trisomy 7, 8, 12, and 18.¹³⁵ Chromosome 6q abnormalities are particularly frequent in BL,¹³⁶ which suggests that an important tumor suppressor gene is located in this region. The pathogenic significance of the various trisomies in BL, as in other forms of human neoplasia, remains unknown.

Role of EBV
Laboratory studies of BL lines from African patients led to the first identification of the novel herpes virus EBV,¹³⁷ which was subsequently detected in more than 90% of endemic BLs, approximately 20% of sporadic BLs, and approximately 40% of HIV-associated BLs.⁴⁰ Analyses of the highly polymorphic, tandemly repeated terminal repeat sequences of EBV in EBV⁺ BL have shown a monoclonal population of EBV genomes,¹³⁸ indicating that EBV infection precedes or occurs concomitantly with B-cell transformation. Despite the longstanding epidemiologic link of EBV infection to BL and its presence from the earliest stage of cellular transformation, the manner in which EBV contributes to the pathogenesis of BL remains uncertain. Since most humans are infected with EBV in childhood or adolescence and maintain a small pool of EBV⁺ B cells throughout life, it is clear that rare, presumably stochastic, events must supervene to produce BL.

One model for EBV’s contribution to the pathogenesis of BL views its role as a potentiator, with little effect on tumor maintenance. This model supposes that acute EBV infection leads to a polyclonal expansion of latently infected B cells.¹³⁹ These EBV⁺ cells initially demonstrate a "lymphoblastoid" pattern of EBV gene expression, wherein infected cells express six nuclear antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, and LP) and three membrane proteins (latent membrane proteins 1, 2A, and 2B). This pattern of viral gene expression is associated with B-cell proliferation and transformation in vitro and is also observed in immunoblastic EBV⁺ B-cell proliferations in immunosuppressed patients. Such acute EBV infection might predispose to BL indirectly by stimulating B-cell proliferation, thus increasing the likelihood of c-myc rearrangement. Persistent acute infection in patients who are immunosuppressed by HIV or chronic malaria may extend the period of B-cell proliferation and thus further elevate the risk of secondary events. It has also been suggested that EBV infection might elevate the incidence of chromosome breakage independent of its effect on proliferation.¹³⁹

The second model (which is not exclusive of the first) takes into account the observation that latently infected BL cells show a pattern of EBV gene expression distinct from that of EBV-infected B immunoblasts. In BL, expression of EBV genes is restricted to EBNA-1, RK-BARF0 (a recently described membrane-associated protein),^140,141 and EBV-encoded RNA (EBER)-1 and EBER-2, a pair of small EBV-specific RNA transcripts. This pattern of gene expression correlates with downregulation of adhesion molecules such as LFA-3 and intercellular adhesion molecule 1, HLA class I molecules, and immunogenic EBV proteins (eg, EBNA2 and latent membrane protein 1), which suggests that it may facilitate escape from immune surveillance. Although neither RK-BARF0 nor EBNA-1 is essential for B-cell transformation in vitro,^142,143 EBNA-1 can induce B-cell lymphomas in transgenic mice.¹⁴⁴ It has also been appreciated that loss of the EBV genome from the Akata cell line, which shows a BL-like pattern of latent gene expression, results in a decrease in oncogenic potential that can be restored through expression of EBER-1 and EBER-2.^145,146 Hence, the EBV genes that are expressed in BL may yet be proven to be involved directly in the maintenance of the transformed state.

An integrated model that takes all of these data into account supposes that the "lymphoblastoid" pattern of EBV gene expression initially drives a polyclonal B-cell proliferation. Upon the stochastic acquisition of a c-myc rearrangement, the nascent tumor cell clone then converts to the BL pattern of latent viral gene expression, possibly enabling its escape from immune surveillance. Of note, c-myc dysregulation abolishes EBNA2 dependence and causes a change in morphology from immunoblastic to BL-like in EBV-transformed B-cell lines in vitro, which suggests that this event may promote the change in the EBV gene expression pattern.¹⁴⁴ As noted earlier, c-Myc also downregulates the expression of adhesion molecules through transcriptional repression, an effect that may further contribute to avoidance of the host immune system.

FUTURE DIRECTIONS

As this review should make clear, a great deal has been learned about the role of c-Myc and a variety of EBV-encoded polypeptides in cellular transformation from the study of a variety of cell culture and animal models. An understanding of c-Myc function and binding partners has identified a number of potential therapeutic targets. Solutions for the structure of these protein complexes at high resolution afford an opportunity for the development of small molecules that specifically disrupt or modify the function of c-Myc. Recent successes using the tyrosine kinase inhibitor CGP57148B (STI571), which inhibits the Bcr/Abl fusion protein found in Philadelphia chromosome (t(9;22))–positive hematologic malignancies,^147,148 demonstrate the feasibility of therapy directed at tumor-specific oncoproteins.

Despite progress using in vitro and animal model systems, relatively few of c-Myc’s functions or interactions have been studied or validated in freshly excised BL tissue. Since the effects of c-Myc in particular are dependent on dose and context, it is an open question how relevant observations made in experimental assays are to the etiology of BL. To some extent, these difficulties are a reflection of the limitations of tissue-based research, which necessarily tends to be more correlative than hypothesis-driven.

These difficulties may soon be obviated by new high-throughput approaches to tumor classification that will permit a much more detailed description of gene and protein expression in primary BL cells. Recent examples of such approaches include the use of cDNA microarrays to identify gene expression signatures specific for a variety of B-cell lymphomas¹⁴⁹ and the use of high-resolution protein separation methods coupled to mass spectroscopy to identify tumor-specific polypeptides.¹⁵⁰ The validation of tumor markers identified through these types of screens will be assisted by the creation of tissue microarrays that permit the interrogation of hundreds of tumor samples simultaneously.¹⁵¹ It is anticipated that these systematic approaches will lead to new hypotheses concerning the molecular pathogenesis of BL and the identification of a number of novel biologic markers representing potential therapeutic targets.

ACKNOWLEDGMENTS

Supported by National Cancer Institute grant no. CA82308.

We thank Jeffrey Jorgensen, MD, and Jonathan Fletcher, MD, for images demonstrating the detection of the t(8;14) by fluorescent in situ hybridization.

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