Molecular Biology, Vol. 39, No. 3, 2005, pp. 313–320. Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 355–363. Original Russian Text Copyright © 2005 by Umanskaya, Bystritskiy, Razin.
REVIEWS UDC 577.21
Chromosome Rearrangement Breakpoint Clustering: The Role of Clonal Selection O. N. Umanskaya, A. A. Bystritskiy, and S. V. Razin Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334 Russia; e-mail:
[email protected] Received November 25, 2004
Abstract—Chromosome rearrangements may result in fusion genes that encode chimeric proteins. The breakpoints of many such rearrangements cluster in definite genomic regions. In addition, many breakpoint clusters contain specific genomic elements, such as topoisomerase II consensus sites, nuclear matrix attachment sites, and various nucleotide sequences capable of assuming noncanonical secondary structure. Studies on breakpoint location are reviewed in terms of the available data on chromatin structure. In addition, the relationship between the location of breakpoints and the domain organization of the respective proteins, which has not been dealt with in published studies, is analyzed. The possible mechanisms of chromosome rearrangements are discussed. Key words: chromosome rearrangements, double-strand breaks, chimeric genes, nuclear architecture, protein domains
INTRODUCTION It is known that many tumor cells carry chromosome rearrangements [1]. In many cases, a specific chromosome rearrangement is correlated with the tumor type (Ewing’s tumor, Wilms tumor, various leukemias, etc.), which serves as an important diagnostic sign at early stages of the disease [2]. However, it has not been proved as yet that chromosome rearrangements are the direct cause of tumors (see [3] for review). Rearrangements often result in fusion genes whose expression yields chimeric proteins. From the practical viewpoint, the existence of these genes allows the rapid, simple RT–PCR method to be used for diagnosing tumors. The analysis of the structure of chimeric proteins makes it possible to put forward some hypotheses concerning the possible role of these proteins in tumor growth. GROUPS OF CHIMERIC PROTEINS At present, more than 200 genes are known to be involved in the formation of chimeric proteins [1]. In some cases, the expression of fusion genes leading to the formation of chimeric proteins is strongly correlated with the development of tumors and leukemias. Some leukemias are accompanied by chromosome rearrangements involving the MLL gene encoding a transcription factor. Cells of Ewing’s tumor are characterized by chromosome rearrangements damaging the EWS gene, which also encodes a transcription factor. These rearrangements give rise to chimeric transcription factors capable of interacting with a different set of promoters. Other examples are reported in some recent reviews [4, 5]. Sev-
eral chimeric proteins have been found in cells of some tumors. For example, Ewing’s tumor cells express proteins encoded by chimeric genes resulting from the fusion of the EWS gene with genes FLI1, FEV, CHOP, ERG, and ETV1. Cells of some other sarcomas contain the chimeric protein that results from the fusion of the EWS and ETV4 genes. Figure 1 shows examples of chimeric gene families. Note that most genes that form these families encode transcription factors or protein kinases [6]. For example, the RET gene encodes receptor tyrosine kinase, which is often structurally altered in thyroid tumors (see [7] or review). Complex interactions may occur between individual members of gene families. Chimeric genes resulting from the fusion of the same gene with different partners may be characteristic of essentially different tumor cells. For example, the myelodysplastic syndrome is characterized by a rearrangement between the ETV6 and PDGFRβ genes; and chronic myeloid leukemia, by a rearrangement between the PDGFRβ and D10S170 genes. A translocation between the gene of RET tyrosine kinase and the D10S170 are found in many cases of thyroid papillary carcinoma [7]. Thus, a relationship between entirely different types of tumors, leukemias and carcinomas, can be observed at the level of the genes involved in the rearrangements that result in chimeric transcription units (Fig. 2) (see [8] for review). BREAKPOINT CLUSTER REGIONS Some of the numerous genes involved in translocations interact with a wide range of partners, whereas
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Fig. 1. Examples of the “chimeric families” ETV6 (on the left) and RET (on the right) and their translocation partners. Rectangles show individual genes (designated by abbreviations in the rectangles). Genes encoding transcription factors are shown in gray. The lines between the rectangles indicate the existence of translocations between the given genes.
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Fig. 2. Relationships between families of various tumors. Designations are the same as in Fig. 1. The myelodysplastic syndrome and chronic myeloid leukemia are characterized by the rearrangements between the ETV6 and PDGFRβ genes and between the ETV6 and D10S170 genes, respectively. The translocation between the D10S170 gene and the gene encoding RET tyrosine kinase is often observed in thyroid papillary carcinomas.
others interact with only one or a few partners. This is shown in Figs. 1 and 2. The genes located in the centers of the “stars” are involved in the largest number of translocations (see [5] for review). The gene encoding transcription factor MLL (also called HRX and ALL1; see [4] for review) is one of the best known genes whose rearrangements are associated with malignant transformation. This gene is 86 kb in length, consists of 37 exons, and is involved in dozens of different translocations. Cloning and sequencing numerous joint sites of chimeric genes demonstrated that most breakpoints in the MLL gene are clustered within a 8.3-kb fragment containing exons 7–13 (Fig. 3). This fragment is termed the breakpoint cluster region (bcr), i.e., the DNA region where most breakpoints are located. It contains cleavage sites for topoisomerase II and nuclear matrix attachment sites [9]. These clusters characterized by the presence of specific structural elements, including nucleotide sequences in which chromatin loops are attached to the nuclear matrix or nuclear scaffold (nuclear matrix) attachment regions (S/MAR), sites of DNA cleavage
by topoisomerase II, and sites of hypersensitivity to DNase I, are not unique for the MLL gene. Breakpoint cluster regions have been found in the genes encoding transcription factors AF4 and AF9 (see [10] for review). These genes are the most common translocation partners of MLL. In the AF4 locus, breakpoints are clustered in a region located between exons 3 and 7 and occupying about one-third of the gene length [11]. The length of bcr1 in the 5'-end region of the AF9 locus is 6% of the entire gene length; bcr1 contains part of intron 4, exon 5, and one half of intron 5. Rearrangements in this region are observed in acute myeloid leukemia. In the 3'-terminal region of the AF9 gene, cluster bcr2 is located between exons 7 and 9; like the bcr of the MLL gene, it contains topoisomerase II cleavage sites. Both bcr1 and bcr2 of AF9 are flanked by S/MAR elements [12]. The gene of acute myeloid leukemia (AML1) is another example of a gene involved in leukemia [13]. Several genes are known to form chimeras with AML1. The ETO gene involved in the t(8;21) translocation is the most frequent partner of AML1. Like MLL, AML1 also codes for a transcription factor, with breakpoints also clusMOLECULAR BIOLOGY
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CHROMOSOME REARRANGEMENT BREAKPOINT CLUSTERING MLL 10 kb
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Fig. 3. Breakpoint clusters of the genes encoding MLL and RET. The upper row shows the scheme of genes. Exons are shown in black; each bcr is shown as a gray rectangle. Lower, cDNA is shown. Gray rectangles show protein domains: AT-hook (AT), zinc fingers (CxxC), the PHD-finger domain, the transactivating domain (TA), the SET domain (SET), the cadherin domain (CDH), the transmembrane domain (TM), and the catalytic domain of tyrosine kinase (YT). The lower row shows the RET cDNA map, with the junction of exons 11 and 12, between which a bcr is located, shown in black.
tering in AML1 and ETO [14]. Intron 5 of AML1 contains three neighboring fragments 2.8, 5.3, and 2.3 kb in length, where breakpoints cluster. Taking into account the intervals between them, these clusters take up 7% of the entire gene length. Three breakpoint clusters (2.6, 2.5, and 2.6 kb) occupying 10% of the gene length have been mapped to intron 1 of ETO. Four of the aforementioned six clusters, namely, bcr2 and bcr3 of AML1 and bcr1 and bcr2 of ETO, contain topoisomerase II cleavage sites. In solid tumors, EWS, the gene of Ewing’s tumor, is often involved in the formation of chimeric proteins (see [5] for review); this gene is also involved in many translocations. As in the cases of MLL and AML1, most breakpoints in the EWS gene cluster in a relatively short region between exons 7 and 9 [15–16]. Most other breakpoints are located between exons 9 and 12. The length of this region is 4.7 kb, i.e., even shorter than the bcr of the MLL gene (8.3 kb). Thus, we may consider breakpoints to cluster in the EWS gene. Note that topoisomerase II cleavage sites have also been found in this bcr. Thus, breakpoint clustering is a common characteristic of various rearrangements. Moreover, all these rearrangements are characterized not only by clustering, but also by some related properties, e.g., the presence of certain characteristic sequences in the clusters. This seems unlikely to be a mere coincidence. So, what is the mechanism of this similarity?
longer (about 40 kb) [18]. In each case, the reading frame is preserved, because the coding gene region is not affected. The transformation is most likely to result from the oncogene expression deregulation, which appears because the coding gene region is placed under the control of the immunoglobulin promoter or enhancer [19]. Since the immunoglobulin gene cluster usually serves as a partner of BCL6 in translocations, it has been hypothesized that these translocations result from errors in V(D)J recombination (see [20] and references therein). Apparently, the same mechanism underlies the translocations of many other genes of immunoglobulins and T-cell receptors.
RECOMBINATION MECHANISM The c-myc and IgH genes were the first ones that were demonstrated to be involved in translocations [17]. Later, rearrangements involving immunoglobulin and T-cell receptor loci were described. The BCL6 gene is one of the genes involved in these translocations. Breakpoints cluster in two regions of this gene: the major breakpoint region (MBR) 4 kb in length located in the untranslated exon 1, and the alternative breakpoint region (ABR), which is considerably
The clustering of breakpoints and the structural specificity of these clusters suggest other possible mechanisms. Some of the clusters described contain topoisomerase II recognition sites and S/MAR elements, i.e., the regions where chromatin loops are attached to the nuclear matrix (see [23] for review). The nuclear matrix is known to possess recombinogenic activity [24]. Topoisomerase II is one of the main components of the nuclear matrix [25]. This enzyme catalyzes illegitimate recombination in vitro,
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However, V(D)J recombination is not the only possible mechanism of chromosome rearrangements. Although there is evidence that V(D)J recombinase is a factor in some translocations that do not involve its natural targets (antibody and T-cell receptor genes), most data disprove this hypothesis [21]. It is conceivable that V(D)J recombination only occurs in cells of lymphoid origin. What occurs in other cells then? There is evidence that the circular protein translin, whose main biological role is thus far unknown, plays an important role in some cases. However, translin is known to be transferred to DNA breaks remote from one another. The consensus translin binding site is often located near illegitimate recombination breakpoints [22]. Nevertheless, these data are insufficient to demonstrate that translin actually plays an important role in all chromosome rearrangements in tumor cells.
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especially if its ligating activity is selectively suppressed by certain inhibitors [26, 27]. These inhibitors, e.g., etoposide (VP-16) are used in cancer chemotherapy. However, treatment with these drugs often causes secondary leukemias [28]. As with primary leukemias, the secondary leukemias induced by cancer chemotherapy with topoisomerase II inhibitors are often accompanied by MLL or, less often, AML rearrangements, with the positions of breakpoint clusters remaining the same as in primary leukemias [28]. These data agree with the model according to which the rearrangements are induced by DNA breaks caused by topoisomerase II. The double-strand breaks caused by the enzyme may be repaired incorrectly, thus resulting in various chromosome rearrangements. This model does not consider the specific mechanism of religation. The fragments are joined via ligation of double-strand breaks by one of the standard mechanism for the repair of these lesions [29]. Nonetheless, nonhomologous recombination plays a critical role in the formation of these chromosome rearrangements. However, it cannot be excluded that homologous recombination is also involved in the formation of chromosome rearrangements, although extremely few experimental data confirm this suggestion. Several translocations between homologous repetitive sequences located in different genes have been described [30]. PROTEIN STRUCTURE AND APPARENT BREAKPOINT CLUSTERING Regarding chromosome rearrangements typical of tumor cells in general and leukemia cells in particular, note that tumor cells result from the selection of cell clones possessing some specific characteristics, including the capacity for infinite proliferation. The expression of chimeric genes may help a cell to acquire the properties that are necessary for malignant transformation. Therefore, it is interesting to determine the nature of the chimeric genes resulting from certain translocations and their protein products. The most important questions in this regard are the following. Are reading frames preserved in fused transcripts (or deregulated genes)? How is the structure of the chimeric protein related to the structure of the “fused” proteins and is it related to the functions of these “precursors?” There is the answer to the former question. Almost all chimeric transcripts characteristic of chromosome aberrations retain the reading frame for both components of the combined protein [6]. Parallel analysis of translocation partner genes, the chimeric genes resulting from these translocations, and the proteins encoded by them provides a new insight into breakpoint clusters.
The bcr of the MLL gene has been studied in most detail. This cluster is located between exons 7 and 13. The structures of the wild-type protein MLL and its chimeric derivatives were reviewed recently [4]. In brief, breakpoints are almost always located after the CxxC domain in the N-terminal region of the protein. The chimeric derivatives of MLL are believed to cause transformation; however, the mechanism remains unknown. Apparently, the main stage is MLL binding with the target sequence. In addition to the CxxC domain, which is necessary for sequence-specific binding with DNA, MLL has three AT-hook domains in its Nterminal region. Translocations eliminate the parts of the gene encoding PHD, transactivator domains, and SET domains necessary for wild-type MLL functioning but not required for DNA recognition. A 47-kb bcr accounts for more than one-third of the AF4 (MLLT2) gene length. At first glance, such a long fragment can hardly be called a cluster of breakpoints. However, most of these 47 kb (namely, 36 kb) is occupied by an intron, and the “projection” of the bcr on the AF4 protein mRNA is only 170 bases out of a total of 9390 bases of the mRNA length. The structure and functions of the AF4 protein have been poorly studied; however, its comparison with similar proteins found recently [31] showed the presence of a conserved region that seems to be the strong transactivator domain (STD). Note that the AF4 bcr is located before the part of the gene encoding this domain; therefore, the STD is preserved in chimeric proteins containing AF4. The fusion of MLL and AF4 yields a chimeric protein that contains both the DNA binding site of the MLL protein and the STD of AF4. In the EWS gene, the bcr is located between exons 7 and 9. In addition to frequent rearrangements, this fragment often undergoes alternative splicing. The mRNA of one of the forms of this protein, EWSb, lacks exons 8 and 9. Exons 1–7 encode the transcription activation domain; and exons 11–17 encode the RNA-binding domain. In the cases of rearrangements, the N-terminal region of EWS binds with the C-terminal regions of various transcription factors, mainly from the Ets family. Therefore, the bcr of the EWS gene is located so that the activation domain of the EWS protein remains uninvolved in rearrangements and is included in chimeric proteins (see [5] for review). The receptor tyrosine kinase RET has a receptor domain in the N-terminal region and a cytoplasmic catalytic domain in the C-terminal region. Apparently, the chimeric protein ELE1–RET plays an important role in carcinogenesis [7]. All exons beginning from exon 12 encode the kinase domain. When chimeric genes are formed, the reading frames of both partners are preserved, and the catalytic domain of the fused protein is the same as in the original kinase. The function of the N-terminal region of the ELE1 protein is MOLECULAR BIOLOGY
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believed to consist in the activation of the androgen receptor. This region contains a supercoil (coiled-coil) domain [32] through which the fused protein ELE1– RET may interact with molecules ELE1 or ELE1–RET. These examples show a common property of all these translocations: the boundary between the components of chimeric proteins is always such that the functional domains of the original proteins remain intact. In other words, the localization of all breakpoints in the genes is definitely related to the structure of the corresponding proteins. This relationship may be pronounced to different degrees; however, it is found in all genes involved in the chromosome rearrangements characteristic of tumors. Functionally reasonable location of chromosome breakpoints in tumor cells with some or other translocations is unlikely to result from clustering primary chromosome breaks. Note that the positions of primary breaks have not been analyzed at all. For many years, translocations found in tumor cells, which are undoubtedly a result of selection, have been the objects of study. In the framework of the generally accepted carcinogenesis theory, a neo-Darwinian mechanism of tumor growth is assumed [33]. First, an aberrant cell is formed by some mechanism or another; then, it undergoes clonal selection. Some mutations may give the cells selective advantages; others may be lethal. Some other mutations may be indifferent for cells and, hence, overlooked by a researcher. Apparently, most translocations are indifferent at the protein level. Taking into account the number of genes in the human genome, we may assume that the probability of the formation of a functionally active chimeric protein as a result of an accidental chromosome rearrangement is extremely low. If we also take into account the number of different cell types and tissue-specific differentiation, proliferation, and apoptosis pathways, the probability that this protein will give the cell a selective advantage will be even lower. Therefore, it is conceivable that the breakpoint clusters described in the literature contain only those breakpoints that are responsible for the chromosome rearrangements “useful” for the host cells, i.e., those which allow the tumor to develop to a detectable state. Thus, the rearrangement clusters found in leukemias and other tumors may actually result from the selection of cell clones that are the subject of subsequent study. In this case, we may explain clustering as follows. There is no mechanism for clustering primary breakpoints of chromosome rearrangements per se. The observed clusters only reflect the fact that only some cells with chromosome rearrangements survive; these are the cells in which breakpoints are located in definite, small regions of a limited number of genes. A similar theory was put forward earlier [34]. Evidence for the effect of protein structure is too strong to be ignored. MOLECULAR BIOLOGY
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THE ROLE OF NUCLEAR ARCHITECTURE IN CHROMOSOME REARRANGEMENTS As we noted above, the genomic fragments where chromosome breakpoints cluster contain critical sites. The best known of them are S/MAR elements and topoisomerase II cleavage sites. These characteristics of breakpoint clusters are especially interesting in terms of the model assuming that the sites of primary chromosome breakage cluster in bcr. However, as noted above, the clusters of chromosome breakpoints that have been studied in most detail to date may have resulted from the selection of cell clones. Questions arises as to whether clusters of primary breakpoints actually exist and, if so, what the role of DNA spatial organization in the formation of these clusters is. To answer the first question, consider the genes whose rearrangements cannot give the cells any selective advantage. Obviously, the rearrangements of most of these genes have no practical importance and, hence, have not been studied. However, there are exceptions. These are the genes whose rearrangements cause hereditary diseases. In terms of the problem considered here, the human dystrophin gene is of special interest. Rearrangements of this gene are being intensely studied. It has been found that breakpoints within the dystrophin gene are distributed nonrandomly. There are two recombination hotspots. In the given case, there are grounds to believe that the sites of primary breaks cluster in these hotspots, because the product of the dystrophin gene is unimportant for cell viability and only plays a role at the level of muscle tissue. Loop domains of the dystrophin gene have been mapped in our laboratory [35]. The positions of recombination hotspots are strongly correlated with the positions of the long zones of the attachment of DNA loops to the nuclear matrix. We demonstrated earlier that the bcr of AML1 and bcr of ETO contained S/MAR elements and were attached to the nuclear matrix even in the cell lines in which these genes were not rearranged. Together with the aforementioned data that most breakpoint clusters contain S/MAR elements and topoisomerase II cleavage sites, these results indicate that the spatial organization of DNA in the nucleus largely contributes to the localization of chromosome rearrangements. This problem requires further study. The positions of S/MAR elements have only been determined in two genes, MLL and AML1. In each of the genes, this position coincides with the bcr. There is also evidence that S/MAR elements are located in gene AF9 (except for the long introns 2 and 3 [12]). However, in this case, the mapping of S/MAR elements was only performed in bcr-containing zones [12]; therefore, it is unknown whether these zones differ from the rest of the gene. The MLL [36], AML1 [14], and ETO [14] genes were also studied with respect to topoisomerase II cleavage in vivo. These
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experiments dealt with bcr-containing zones rather than whole genes. Obviously, in terms of the problem considered here, it would be especially interesting to compare breakpoint clusters with other regions of the genes. Such a study has not been carried out thus far. Note that the spatial organization of DNA in the nucleus has an effect on the frequency of translocations between different chromosomes. Note that chromosomes (chromosome territories) are nonrandomly distributed in the interphase nucleus [37–38]. Chromosomes rich in genes (e.g., human chromosomes 9 and 19) are located near the center of the nucleus, whereas chromosomes poor in genes (chromosomes 18 and 22) are located at its periphery [37]. The location of several chromosomes at the same “nuclear orbit” substantially increases the probability of recombination between them. For example, chromosomes 9 and 22, which contain almost equal numbers of genes, are located at the same “orbit,” and translocations between them are comparatively frequent. In some cases, specific chromosomes are not located at the same orbit, but are associated with each other throughout the cell cycle (or at least some of its phases) [39]. A detailed description of the spatial organization of chromosomes in the interphase nucleus and its role in chromosome rearrangements is beyond the scope of this review. Additional information may be found elsewhere [39–41]. CONCLUSIONS Taking into account the above data, a preliminary conclusion may be as follows: breakpoint clusters found in tumor cells are the result of the fact that the study was performed precisely with the clones of tumor cells that were preserved in the course of selection from a population of clones with numerous, dispersed primary translocations. The existence of breakpoint clusters is clearly confirmed by a well-known anthropic principle: the universe is such as we observe it merely because we could not have appeared in any other universe [42]. Similarly, the positions of rearrangement breakpoints are such as we observe them because all other rearrangements are either not expressed phenotypically or lethal for cells. However, this hypothesis does not exclude that primary breakpoints may have already clustered around or within these “functionally important” clusters in some (or even many) cases, as is apparently the case with the MLL and AML1 genes. Apparently, nuclear architecture determines, in some way or another, the positions of translocations. The existence of primary breakpoint clusters does not contradict the idea of the subsequent selection for “functionally useful” variants. Nor is it surprising that some clusters of primary breakpoints are located between the fragments of the gene encoding various functional domains of proteins. According to a generally accepted model, functional protein
domains are historical units of protein evolution. Ancestral domains (more precisely, the DNA fragments that encode them) were reshuffled in the course of evolution, which has led to the formation of currently existing proteins. Probably, the genes that encode modern proteins contain some traces of these ancient events. The boundaries between domains at the gene level may still be more prone to recombination than the bodies of domains themselves [43]. Many questions raised in this review remain unanswered. To resolve the uncertainty, it is necessary to study the distribution of primary breakpoints that have not undergone subsequent selection; however, this task is apparently too difficult. Several approaches may be suggested for studying “invisible” translocations, whereas studying lethal translocations is a real challenge for a researcher. Nevertheless, these studies could extend our knowledge of the structure and functions of chromatin, transcription factors, and other regulatory proteins. This will help us to understand the evolution of proteins and genomes, as well as the evolution of organisms, including humans. ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research (project no. 02-04-48 369) and the Program “Molecular and Cell Biology” of the Presidium of the Russian Academy of Sciences. REFERENCES 1. Mitelman F., Johansson B., Mertens F. 2003. Mitelman Database of Chromosome Aberrations in Cancer. [web page] http://cgap.nci.nih.gov/Chromosomes/Mitelman. 2. Yoshino N., Kojima T., Asami S., Motohashi S., Yoshida Y., Chin M., Shichino H., Yoshida Y., Nemoto N., Kaneko M., Mugishima H., Suzuki T. 2003. Diagnostic significance and clinical applications of chimeric genes in Ewing’s sarcoma. Biol. Pharm. Bull. 26, 585–588. 3. Mitelman F. 2000. Recurrent chromosome aberrations in cancer. Mutat. Res. 462, 247–253. 4. Ayton P.M., Cleary M.L. 2001. Molecular mechanisms of leukemogenesis mediated by MLL fusion proteins. Oncogene. 20, 5695–5707. 5. Kim J., Pelletier J. 1999. Molecular genetics of chromosome translocations involving EWS and related family members. Physiol. Genomics. 1, 127–138. 6. Rabbitts T.H. 1999. Perspective: Chromosomal translocations can affect genes controlling gene expression and differentiation–why are these functions targeted? J. Pathol. 187, 39–42. 7. Nikiforov Y.E. 2002. RET/PTC rearrangement in thyroid tumors. Endocrin. Pathol. 13, 3–16. 8. Bohlander S.K. 2000. Fusion genes in leukemia: An emerging network. Cytogenet. Cell Genet. 91, 52–56. 9. Strissel P.L., Strick R., Rowley J.D., Zeleznik-Le N.J. 1998. An in vivo topoisomerase II cleavage site and a MOLECULAR BIOLOGY
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MOLECULAR BIOLOGY
Vol. 39
No. 3
2005
