Int. J. Cancer: 51,892-897 (1992) 0 1992 Wiley-Liss, Inc.
PLOIcatdon of the lnrernationa Unton Against Cancer
HOX GENE EXPRESSION IN NORMAL AND NEOPLASTIC HUMAN KIDNEY C. C I L L O ~P.~BARBA’, ~, G. FRESCHI~, G. BUCCIARELLI~, M.C. MAGLI’and E. BONCINELLI~ ‘InternationalInstitute of Genetics and Biophysics, Naples; and 2Cattedradi Patologia Chirurgica e Propedeutica Clinica, Universita di Firenze, Florence, Italy.
As a consequence of transformation, cancer cells generally lose some of their differentiative properties. Thus, alterations interfering with the genetic mechanisms required to maintain embryonic determination could lead to tumorigenesis. Homeobox genes are a network of genes encoding nuclear proteins containing DNA-binding homeodomains that are highly conserved throughout evolution. They are expressed in a stage-relatedfashion in the developingembryo and, in adult life, in normal tissues. In mice and humans, homeobox genes of the HOX family are organized in 4 clusters on different chrornosomes which have presumably evolved by duplication of a primordial gene cluster. Strikingly, the order of genes within each cluster is also highly conserved throughout evolution, suggesting that the physical organization of HOX genes might be essential for their expression. Recent reports indicate that homeobox mutant mice display morphologicalabnormalities or show neoplasticalterations, and that growth factors can turn on homeobox genes. We have studied the expression of the Antennapedia-like HOX genes in normal human kidney and in renal carcinomas. The great majority of the HOX genes analyzed are expressed in a peculiar manner in normal kidney: blocks of genes, even entire HOX loci, are coordinately regulated. Alterations in HOX gene expression in renal carcinoma can be observed in 2 genes of the HOX-2 locus, HOX-2A and HOX-2E, which are actively expressed in normal kidney and silent in cancer biopsies. The HOX-3H gene is not expressed in normal kidney whereas the HOX-3H transcripts are present in renal carcinomas. Homeobox genes within the 4 HOX loci can be aligned on the basis of the maximal sequence homology of their homeodomains: this alignment defines I 3 paralogous gene groups. In renal carcinomas, genes of group I 0 (HOX- ID, 2F, 3E, 48) display a marked difference in their transcript classes when compared to those of normal kidney. Our findings suggest an association between altered HOX gene expression and kidney cancer. o 1992 Wiley-Liss,Inc. Cancer is a complex, multi-step process involving the aberrant expression of genes that regulate cell growth. The biochemical function of proto-oncogenes allows the pathway of cell growth to be divided into several important steps (Weinberg, 1989). One of these entails the trans-activation of target genes involved in cell proliferation (Varmus, 1987). The products of several nuclear oncogenes (i.e. fos, jun, erb A)(Distel et al., 1987; Bohmann et al., 1987; Green and Chambon, 1986) as well as the tumor-suppressor gene Rb (Chellappan et al., 1991), are DNA-binding proteins with transactivating activity. Thus, the role played by proteins that can exert effects on gene expression is becoming increasingly important to an understanding of the molecular basis of neoplasia. Homeobox-containing genes regulate a number of developmental processes. Genes of this family, although different from one another, contain a common sequence of 183 nucleotides which encodes a 61 amino-acid domain called the homeodomain (HD) (Gehring and Hiromi, 1986). The homeodomain is a DNA-binding domain capable of recognizing specific sequences by virtue of a helix-turn-helix structural motif. On the basis of structural similarities and direct evidence that Drosophila homeodomain proteins are capable of binding DNA sequences and modulating transcriptional activity, it is generally accepted that homeodomain proteins are transcriptional regulators (Hoey et al., 1988; Han et al., 1989). The homeobox was originally identified in the homeotic genes
responsible for the determination of segment identity in Drosophila development (Levine et al., 1984). Subsequently, homeobox-containing genes have been found in a number of evolutionarily distant organisms including nematodes and vertebrates (Akam, 1989; Scott et al., 1989). Most homeobox genes analyzed so far belong to class I, for which Antennapedia (Antp) is the prototype (Gehring and Hiromi, 1986). Mammalian class-I homeobox (HOX) genes are organized in 4 clusters on chromosomes 2, 7, 12 and 17 (Acampora et al., 1989; Kappen et al., 1989). HOX genes are expressed during mammalian embryogenesis with a complex pattern of positional and temporal specificity (Simeone et al., 1986). Every HOX gene is expressed in the embryonic central nervous system (CNS) (Mavilioet al., 1986). In adult life, some of the HOX genes are expressed in normal tissues (Acampora et al., 1989). The hypothesis of an association between genes that control transcription and the oncogenic process has recently been strengthened by a number of independent observations. First, constitutive expression of the HOX 2.4 gene can be oncogenic in mice (Perkins et af., 1990). Second, thepbx homeobox gene in the t(1; 19) translocation of pre-B acute leukemias is aberrantly expressed (Kamps et al., 1990). Third, altered expression of the tcl-3 (HOX11) gene in the t(10; 14) translocation has been reported in some T-cell leukemias (Hatano et al., 1991). Fourth, the coordinate regulation of HOX genes may play an important role in human hemopoietic differentiation (Magli et al., 1991). Given this association between HOX genes, developmental processes and oncogenesis, we wished to determine whether the physical organization of HOX genes reflects a regulatory network involved in kidney organogenesis and/or in its neoplastic transformation. In this study we have analyzed the expression of a panel of 38 HOX genes in normal adult human kidney and in renal-cell carcinomas. Some of the HOX genes tested show marked differences in expression in cancer specimens when compared to normal kidney, suggesting an association between altered HOX gene expression and kidney cancer. MATERIAL AND METHODS
Normal and cancerous kidney from untreated, non-selected patients with primary renal carcinomas were obtained at the department of Surgical Pathology, University of Florence. During radical nephrectomy, kidney was dissected and samples were taken from non-necrotic tumor tissue as well as from the area of the intact normal kidney closest to the tumor. The specimens were surgically removed, snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction. RNA extraction and analysis
Frozen tissues were pulverized by blender. Total RNA was extracted by the guanidinium thiocyanate technique (Chirgwin et al., 1979) and poly (A)+ selected by one passage on oligo 3To whom correspondence and reprint requests should be sent, at the International Institute of Genetics and Biophysics, Via Marconi 10,80125, Naples, Italy. Fax: 3918115936123-7257202. Received: January 10,1992 and in revised form March 21,1992.
HOX GENE EXPRESSION IN NORMAL AND NEOPLASTIC KIDNEY
(dT) cellulose columns. Poly (A)+ RNA was run on 1.25% agarose-formaldehyde gels, transferred to nylon (Schleicher and Schuell, Dassel, Germany NY13N) membranes by Northern capillary blotting, and hybridized to 10’ cpm of DNA probe labelled by nick-translation to a specific activity of 3-8 X lo8 dpm/kg. The probes contained the 3’ untranslated regions specific for each of the 38 HOX genes as previously reported (Stornaiuolo et al., 1990; Simeone et al., 1991; D’Esposito et al., 1991). Pre-hybridization and hybridization were carried out as described by Thomas (1980). After washing under stringent conditions (30 mM NaC1/3mM sodium citrate/0.2% SDS at 65”C), the blots were exposed for 1-7 days at -70°C to Kodak XR-5 films in an X-omatic intensifying screen cassette. RESULTS
Class-I homeobox genes within the 4 HOX loci can be aligned horizontally, according to their physical position on each chromosome, and vertically on the basis of the maximal sequence homology of their homeodomains. This alignment defines 13 paralogous groups (Simeone et al., 1991). Only 2 of these, namely groups 5 and 10, contain 4 HOX genes in all 4 loci (Fig. 1). We analyzed the expression of the 4 HOX gene clusters in normal human kidney and in tumor samples derived from patients with renal carcinomas. Poly (A)+ RNAs from normal kidney and from cancer specimens were hybridized by Northern blotting with probes containing the 3’ untranslated regions specific to each of the 38 HOX genes, organized in 4 large clusters, HOX1-HOX4 located on chromosomes 7, 17, 12 and 2, respectively (see Fig. 1). The pattern of HOXgene expression in normal human adult kidney is shown in Figure 1. Of the 38 HOX genes tested, 30 are actively expressed. In normal kidney, HOX genes are switched on or off in blocks containing a variable number of contiguous genes within each locus. Expression of the genes at the extreme 5’and 3’ ends of the HOX-1 locus, HOX-1J and HOX-IF, is undetectable, while 9 contiguous genes of the same locus, from HOX-11 to HOX-lK, are actively expressed. Furthermore, the entire HOX-2 locus is expressed, except for ~HOX-21,the gene nearest 3’. Moreover 3 contiguous genes, ,HOX-3G, HOX-3F, HOX-3H, at the 5’ end of the HOX-3 liocus, are silent, while a block of 6 genes (from HOX-31 to IYOX-~E)are actively expressed. Finally, the 2 genes at the extreme 5‘ end of the HOX-4 locus, HOX-41 and HOX-4H, are silent and 7 contiguous genes, at the 3‘ end, are all expressed, although the expression oSHOX-~G,the gene nearest 3‘ is very low. This complex pattern, illustrated in Figure 1, is specific to the kidney. The same analysis performed in normal colon, lung or liver shows different patterns of expression (data not shown) with different HOX genes switched on or off in these organs. HOX genes of the paralogous groups 5 and 10, which seem to represent boundaries that define genes 3‘ and 5’ displaying different expression patterns, are actively expressed in the normal kidney (Fig. 1). Major differences in the expression of HOX genes between normal kidney and renal carcinomas have been detected. Three HOX-2A transcripts, of 1.7 kb, 1.9 kb and over 8 kb, are present in normal kidney (Fig. 2a). The majority of kidney tumors tested (8/ 12) do not show detectable expression of this gene. However, 2 specimens have the same expression as normal kidney and 2 other biopsies show a specific expression pattern with the absence of the 1.9 molecular weight band in the only case of Wilms’ tumor studied, and 3 RNAs of high niolecular weight in the other biopsy. Two HOX-2E transcripts, of size 2.4 and 2.9 kb, are expressed in normal medullar kidney, whereas only the lower niRNA is detectable in cortical kidney (Fig. 2b). The tumor samples can be classified into 4 categories, according to the expression of this gene: 3/12 of the samples exhibit the same
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pattern as normal medullar kidney; 3/12 express high steadystate levels of the 2.9-kb transcript whereas the 2.4-kb species is undetectable. Conversely, in 2/12 tumors only the lower molecular weight transcript is evident; in 4/12 tumors no HOX-2E transcripts are detected. Specimens of this last category are all histologically classified as clear-cellkidney carcinomas. Expression of the HOX-3H gene is undetectable in normal kidney (Fig. 2c) and in 9/13 biopsies examined. In contrast, we have observed a 1.7-kb HOX-3H transcript in 3 other specimens (Fig. 2c). In the single Wilms’ tumor tested, 2 abundant HOX-3H transcripts of sizes 1.7 and 2.0 kb are observed. In normal kidney, a single HOX-1D transcript of size 1.9 kb is present (Fig. 1). Out of 13 biopsies (see Table I), 4 also express the same 1.9-kb transcript, and 7 tumor samples contain 2 HOX-1D transcripts of size 1.9 and 1.4 kb, with the higher band as a dominant one. These transcripts correspond in size to the mRNA species observed in other tissues for this HOX gene. In contrast, one biopsy (31) shows an intense 1.4-kb band as the dominant one over the higher molecular weight transcript, while in another specimen 2 novel classes of mRNAs of higher molecular weight are present. Of the genes in group 10, only the HOX-2F gene is expressed both in normal and in neoplastic kidney with no detectable differences in intensity or transcript size (data not shown). HOX 3E is abundantly expressed in normal kidney with 3 transcript classes of 1.8, 2.4 and 2.8 kb (Fig. 1). Table I summarizes HOX-3E expression in renal-cell carcinomas. In 9 out of 13 tumors, HOX-3E expression is similar to that observed in normal tissue. In one tumor, only the 1.8- and 2.4-kb bands are observed, while another tumor expresses only the 2.4- and 2.8-kb transcripts. A third tumor contains only the 1.8-kb transcript while, in the single Wilms’ tumor analyzed, the 2.4 transcript is the only one present. HOX-4B is expressed in normal kidney with 4 transcripts of size 5.4, 4.2, 2.8, 1.4 kb (Fig. 2d). The molecular mechanisms regulating the expression of this gene have been well elucidated (Cianetti et al., 1990). Two alternative promoters underlie the transcription of 2 classes of HOX-4B-specific mRNAs: the 5.4- and 2.8-kb transcripts are driven from a distal promoter while the 4.2- and 1.4-kb transcripts originate from a proximal promoter. The 2 promoters are differentially regulated in a tissue- and stage-specific manner and respond differentially to retinoic-acid induction (Mavilio et al., 1988). The expression of HOX-4B in renal carcinomas is shown in Figure 2d. On the basis of the expression of this gene, it is possible to group kidney cancer into 3 categories: (a) tumors exhibiting the same expression pattern as the normal kidney (7/13) with 4 major transcripts of 5 4 4 . 2 , 2.8 and 1.4 kb; (b) a category of tumors (3/13) with HOX-4B transcripts of size 5.4 and 2.8 kb, showing the transcript classes presumably originating from the distal promoter, and (c) 3 tumors in which HOX-4B expression is presumably due to the activity of the proximal promoter, with transcript sizes of 4.2 and 1.4 kb. It is interesting to note that normal kidney from the same patients showing the alternative transcripts presents the full spectrum of HOX-4B gene expression with the 4 transcript classes. To determine whether HOX genes are differentially expressed in different parts of the kidney, we have analyzed the expression of some of the HOX genes in the cortical and medullar kidney. As shown in Figure 2d, no differences in the expression patterns of the HOX-4B gene are observed; however, the levels of HOX-4B transcripts are higher in the medulla than in the cortical kidney. We have obtained the same results for the HOX-2A (Fig. 2a) and HOX-3H genes (Fig. 2).In contrast, the HOX-2E gene shows 2 mRNAs in the medullar kidney but only the lower one appears to be detectable in cortical kidney. Table I summarizes the expression patterns of the 6 HOX genes described above in 13 cancer specimens. In all cases,
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CILLO ETAL.
FIGURE 1 - HOX gene expression in normal human kidney. HOX genes are aligned horizontally according to their physical position on the chromosomes and vertically on the basis of maximal sequence homology of the homeodomain. Small stippled circles indicate homeodomains predicted in the scheme but not yet found. Northern blots of 5 pg of poly (A)+ RNA from normal kidney were hybridized to the probes indicated in the circles above each lane. Transcript sizes are given in kb. The 13 HD groups are indicated at the bottom.
kidney cancers display at least 2 HOX genes with altered expression, whereas HOX gene expression is identical in 4 different normal kidneys. The HOX-2A and HOX-2E genes are often altered in the same cancer biopsy. No correlation is observed between altered expression of the genes belonging to group 10 and the HOX-2A, HOX-2E and HOX-3H genes.
DISCUSSION
The kidney of vertebrates is an ideal model systeni for studying organogenesis, cell differentiation and neoplastic transformation (SaxCn, 1987). Transitory and vestigial pronephric and mesonephric structures are generated during early development, leading finally to the formation of the adult
FIGURE2 - Expression of HOX-2A ( a ) HOX-2E ( b ) HOX-3H (c) and HOX-4B ( d ) genes in normal kidney (total = NT, medullar = NM, cortical = NC) and in kidney cancer (the numbers of the different biopsies are indicated above each lane). Five micrograms of poly (A)+RNA from normal and neoplastic kidney were electrophoresed
through a 1.25% agarose gel, transferred to nitrocellulose filters and hybridized with probes representing the 3’ untranslated region of HOX-2A, 2E, 3H, 4B. Transcript size is indicated in kb. Control hybridization to a p-actin probe is shown.
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CILLO ETAL. TABLE I -VARIATION OF TRANSCRIPT CLASSES IN NEOPLASTIC VERSUS NORMAL KIDNEY Genes
Normal kidney Kidney tumors
HOX-2A
HOX-2E
HOX-311
> 8-1.9-1.7
2.9-2.4 2.9-2.4
-
-
n.5
-1
20 22 24
-
-
-
25 29 31
-
HOX-ID
HOX-3E
HOX-4B
Histology
1.9 2.8-2.4-1.8 5.4-4.2-2.8-1.4 1.9-1.4 (3.7-4.9) 2.8-2.4-1.8 5.4-4.2-2.8-1.4 Clear-cell carcinoma
(papillary type)
2.4
-
1.9-1.4 1.9 1.9
2.8-2.4-1.8 4.2-1.4 Clear-cell carcinoma 2.8-2.4 5.4-2.8 Clear-cell carcinoma 2.8-2.4-1.8 5.4-4.2-2.8-1.4 Renal-cell carcinoma
-
2.9
-
1.9-1.4
2.8-2.4-1.8 5.4-4.2-2.8-1.4 Renal-cell carcinoma
-
>8-1.9-1.7
2.9
1.7
1.9-1.4 1.9-1.4
2.8-2.4-1.8 5.4-2.8 Clear-cell carcinoma 1.8 5.4-4.2-2.8-1.4 Renal-cell carcinoma
32 33 36
>8-5-2.7-1.9
N.D. 2.9
1.7
1.9-1.4 1.9-1.4 1.9
2.4-1.8 5.4-4.2-2.8-1.4 Clear-cell carcinoma 2.8-2.4-1.8 5.4-4.2-2.8-1.4 Clear-cell carcinoma 2.8-2.4-1.8 4.2-1.4 Chromophilic-cell
37 38
>8-1.9-1.7
2.9-2.4 2.9-2.4 2.4
1.7 2-1.7
1.9-1.4 1.9-1.4 1.9
2.8-2.4-1.8 4.2-1.4 Clear-cell carcinoma 2.8-2.4-1.8 5.4-4.2-2.8-1.4 Clear-cell carcinoma 2.4 5.4-2.8 -
Wilms'
N.D.*
> 8-1.7
(sarcomatoid type) (mixed type)
(tubulo-papillary type)
carcinoma
Expression of HOX-2A, 2E, 3H, lD, 3E, 4B in normal kidney and in 13 kidney cancer biopsies is reported as size of transcripts (in kb) determined by Northern-blot analysis.-'( -) = undetectable expression.-*(N.D.) = not done.
kidney, the metanephros. The development of the metanephric kidney component, the branching epithelium of the ureter and the mesenchyme converted into epithelial elements, occurs in a synchronous manner that is strictly controlled, both temporally and spatially. The molecular mechanisms underlying the developmental decisions in kidney organogenesis are not at all understood. A gene of the paired box (PAX) family, a family of genes known to control normal embryonic development, is transiently expressed during the early stages of murine kidney organogenesis (Dressler et al., 1990). Here, we have analyzed the expression of the 4 HOX gene clusters in normal human kidney. Furthermore, we have compared the patterns ofHOXgene expression in normal and neoplastic kidney in order to determine whether this gene family might play a role in the malignant transformation of this organ. Our results show that normal adult human kidney exhibits a characteristic pattern of HOX gene expression with HOX genes switched on or off in blocks. These blocks contain a variable number of contiguous genes within each locus. Comparison between HOX gene expression in different normal human organs indicate very high expression of the whole panel of HOX genes in normal kidney with respect both to the number of HOXgenes turned on and to the abundance of the individual HOX transcripts. Since the homeoproteins are mostly involved, during normal development, in specifying positional information, the expression pattern of HOX genes we detected in normal adult human kidney may well represent the traces of the complex kidney organogenesis. It has been previously observed that the highly conserved organization of HOX genes in clusters reflects a regulatory hierarchy within this gene family. For example, in Drosophila, the physical order of the genes within the clusters correlates with the order in which they are expressed along the anteroposterior axis of the embryo (Ingham, 1988). This colinearity has also been observed in mammals (Akam, 1989). Our results suggest that this positional hierarchy of HOX gene expression is also maintained in the kidney. These observations are consistent with the idea that one or more upstream promoter elements account for the concerted expression of HOX genes in differentiating systems. Experimental evidence for a major promoter upstream of several HD-containing exons of the HOX-3 locus has been reported (Simeone et al., 1988). It is interesting to note that, although the 4 loci are abundantly expressed in normal kidney, in no case is the entire locus
expressed. Either the 5' or the 3' end genes, or both, are silent, indicating a particularly concerted transcriptional organization for these genes. In tumors of the kidney, the expression of some HOXgenes appears to be markedly altered when compared to that observed in normal kidney. HOXgenes can be turned on or off in tumors compared to normal kidney (HOX-3H, HOX-2A, HOX-2E). Other genes, although always expressed in normal and neoplastic tissue (HOX-lD, HOX-3E, HOX-4B), display different-sized classes of transcripts. These results indicate that HOX genes can be regulated at the level of transcription as well as at a post-transcriptional level, possibly by differential splicing. Homeobox genes within the 4 HOX loci can be aligned on the basis of the maximal sequence homology of their homeodomain. This alignment defines 13 homology groups. It has been observed that groups 5 and 10 represent a boundary that defines genes 3' and 5 ' , displaying different expression patterns in embryonal carcinoma cells (Stornaiuolo et al., 1990). Our data indicate that, in normal kidney, genes of these groups are highly expressed. Furthermore, in kidney cancer, they often display altered expression. For example, transcription of the HOX-4B gene in group 10 is initiated, in some cancer specimens, at either the distal or the proximal promoter, whereas normal kidney exhibits all 4 classes of HOX4B transcripts. These results suggest that neoplastic transformation in the kidney may be correlated with the inactivation of one of the promoters of this gene. In contrast, the HOX-2E gene in group 5 can be either altered in its expression in neoplastic kidney, or completely turned off. The inactivation of this gene is associated with clear-cell carcinoma of the kidney. In normal kidney, genes at the 5' end of group 5 show lower levels of expression. Groups 1 and 2 are silent. The HOX-3H gene in group 3 is the only HOXgene that is inactive in normal kidney and actively expressed in some kidney carcinomas. HOX-3H exhibits 2 very abundant transcripts in the only case of Wilms' tumor so far tested. There are 2 possible explanations for the differences in the patterns of HOX gene expression between normal and malignant kidney. First, the differences in HOXgene expression may reflect fundamental changes in the mechanisms of transcription of HOX genes, possibly reflecting the importance of these DNA-binding proteins in neoplastic transformation. Second, alterations in the pattern of HOX gene expression may reflect
HOX GENE EXPRESSION IN NORMAL AND NEOPLASTIC KIDNEY
corresponding changes between the heterogeneous cell populations present in normal kidney and the clonal cell populations that emerge in kidney tumors. An attempt to establish a correlation between HOX gene expression and clinical outcome of kidney cancer patients was hampered for several reasons in our study: (a) the low number of kidney cancer specimens (13) analyzed; (b) the small amount of RNA Poly (A)+ obtained from some of the biopsies; (c) the high number (38) of HOX genes to be screened for in each specimen; (d) the decision to test expression by Northern blotting in order to assess quantitative as well as qualitative differences in the expression of HOX genes. Consequently, we have aimed at establishing the general trend of HOX gene expression in normal and neoplastic kidney in order to identify the relevant genes to be tested in a further, more clinically oriented, analysis. In conclusion, our results suggest that HOX gene clusters display characteristic patterns of expression in normal kidney
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and provide an indication that the coordinate regulation of HOXgenes may play an important role in kidney organogenesis. The alteration of HOXgene expression observed in kidney cancers seems to indicate that transcription factors play an important role in cancer evolution. Further work is needed to assess the correlation between altered HOX gene expression and cancer. The identification of target genes that are activated or repressed by the HOX genes will facilitate our understanding of the molecular mechanisms involved in neoplastic transformation. ACKNOWLEDGEMENTS
We thank Dr. A. Bernstein for critical reading of the manuscript. This work was supported by the Italian Association for Cancer Research (AIRC), the Consiglio Nazionale delle Ricerche (CNR) progetti finalizzati Biotecnologia e Biostrumentazione and Ingegneria Genetica.
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Science, 253,79-82 (1991). tially activated by retinoic acid in embryonal carcinoma cells according IIoEY, T., WARRIOR, R., MANAK,J. and LEVINE,M., DNA-binding to their position within the four loci. Cell Difl Devel., 31, 119-127 activities of the Drosophila melanogaster even-skipped protein are (1990). mediated by its homeo domain and influenced by protein context. Mol. THOMAS,P.S., Hybridization of denatured RNA and small DNA cell. Biol., 8,45984607 (1988). fragments transferred to nitrocellulose. Proc. nut. Acad. Sci. (Wash.), INGHAM, P.W., The molecular genetics of embryonic pattern forma- 77,5201-5205 (1980). tion in Drosophila. Nature (Lond.), 335,25-34 (1988). VARMUS, H.E., Oncogenes and transcriptional control. Science, 238, XAMPS,M.P., MURRE,C., SUN,X-H. and BALTIMORE, D., A new 1337-1339 (1987). homeobox gene contributes the DNA binding domain of the t(1; 19) WEINBERG, R.A., Oncogenes, antioncogenes, and the molecular bases translocation protein in pre-B ALL. Cell, 60,547-555 (1990). of multistep carcinogenesis. Cancer Res., 49,3713-3721 (1989).
PLOIcatdon of the lnrernationa Unton Against Cancer
HOX GENE EXPRESSION IN NORMAL AND NEOPLASTIC HUMAN KIDNEY C. C I L L O ~P.~BARBA’, ~, G. FRESCHI~, G. BUCCIARELLI~, M.C. MAGLI’and E. BONCINELLI~ ‘InternationalInstitute of Genetics and Biophysics, Naples; and 2Cattedradi Patologia Chirurgica e Propedeutica Clinica, Universita di Firenze, Florence, Italy.
As a consequence of transformation, cancer cells generally lose some of their differentiative properties. Thus, alterations interfering with the genetic mechanisms required to maintain embryonic determination could lead to tumorigenesis. Homeobox genes are a network of genes encoding nuclear proteins containing DNA-binding homeodomains that are highly conserved throughout evolution. They are expressed in a stage-relatedfashion in the developingembryo and, in adult life, in normal tissues. In mice and humans, homeobox genes of the HOX family are organized in 4 clusters on different chrornosomes which have presumably evolved by duplication of a primordial gene cluster. Strikingly, the order of genes within each cluster is also highly conserved throughout evolution, suggesting that the physical organization of HOX genes might be essential for their expression. Recent reports indicate that homeobox mutant mice display morphologicalabnormalities or show neoplasticalterations, and that growth factors can turn on homeobox genes. We have studied the expression of the Antennapedia-like HOX genes in normal human kidney and in renal carcinomas. The great majority of the HOX genes analyzed are expressed in a peculiar manner in normal kidney: blocks of genes, even entire HOX loci, are coordinately regulated. Alterations in HOX gene expression in renal carcinoma can be observed in 2 genes of the HOX-2 locus, HOX-2A and HOX-2E, which are actively expressed in normal kidney and silent in cancer biopsies. The HOX-3H gene is not expressed in normal kidney whereas the HOX-3H transcripts are present in renal carcinomas. Homeobox genes within the 4 HOX loci can be aligned on the basis of the maximal sequence homology of their homeodomains: this alignment defines I 3 paralogous gene groups. In renal carcinomas, genes of group I 0 (HOX- ID, 2F, 3E, 48) display a marked difference in their transcript classes when compared to those of normal kidney. Our findings suggest an association between altered HOX gene expression and kidney cancer. o 1992 Wiley-Liss,Inc. Cancer is a complex, multi-step process involving the aberrant expression of genes that regulate cell growth. The biochemical function of proto-oncogenes allows the pathway of cell growth to be divided into several important steps (Weinberg, 1989). One of these entails the trans-activation of target genes involved in cell proliferation (Varmus, 1987). The products of several nuclear oncogenes (i.e. fos, jun, erb A)(Distel et al., 1987; Bohmann et al., 1987; Green and Chambon, 1986) as well as the tumor-suppressor gene Rb (Chellappan et al., 1991), are DNA-binding proteins with transactivating activity. Thus, the role played by proteins that can exert effects on gene expression is becoming increasingly important to an understanding of the molecular basis of neoplasia. Homeobox-containing genes regulate a number of developmental processes. Genes of this family, although different from one another, contain a common sequence of 183 nucleotides which encodes a 61 amino-acid domain called the homeodomain (HD) (Gehring and Hiromi, 1986). The homeodomain is a DNA-binding domain capable of recognizing specific sequences by virtue of a helix-turn-helix structural motif. On the basis of structural similarities and direct evidence that Drosophila homeodomain proteins are capable of binding DNA sequences and modulating transcriptional activity, it is generally accepted that homeodomain proteins are transcriptional regulators (Hoey et al., 1988; Han et al., 1989). The homeobox was originally identified in the homeotic genes
responsible for the determination of segment identity in Drosophila development (Levine et al., 1984). Subsequently, homeobox-containing genes have been found in a number of evolutionarily distant organisms including nematodes and vertebrates (Akam, 1989; Scott et al., 1989). Most homeobox genes analyzed so far belong to class I, for which Antennapedia (Antp) is the prototype (Gehring and Hiromi, 1986). Mammalian class-I homeobox (HOX) genes are organized in 4 clusters on chromosomes 2, 7, 12 and 17 (Acampora et al., 1989; Kappen et al., 1989). HOX genes are expressed during mammalian embryogenesis with a complex pattern of positional and temporal specificity (Simeone et al., 1986). Every HOX gene is expressed in the embryonic central nervous system (CNS) (Mavilioet al., 1986). In adult life, some of the HOX genes are expressed in normal tissues (Acampora et al., 1989). The hypothesis of an association between genes that control transcription and the oncogenic process has recently been strengthened by a number of independent observations. First, constitutive expression of the HOX 2.4 gene can be oncogenic in mice (Perkins et af., 1990). Second, thepbx homeobox gene in the t(1; 19) translocation of pre-B acute leukemias is aberrantly expressed (Kamps et al., 1990). Third, altered expression of the tcl-3 (HOX11) gene in the t(10; 14) translocation has been reported in some T-cell leukemias (Hatano et al., 1991). Fourth, the coordinate regulation of HOX genes may play an important role in human hemopoietic differentiation (Magli et al., 1991). Given this association between HOX genes, developmental processes and oncogenesis, we wished to determine whether the physical organization of HOX genes reflects a regulatory network involved in kidney organogenesis and/or in its neoplastic transformation. In this study we have analyzed the expression of a panel of 38 HOX genes in normal adult human kidney and in renal-cell carcinomas. Some of the HOX genes tested show marked differences in expression in cancer specimens when compared to normal kidney, suggesting an association between altered HOX gene expression and kidney cancer. MATERIAL AND METHODS
Normal and cancerous kidney from untreated, non-selected patients with primary renal carcinomas were obtained at the department of Surgical Pathology, University of Florence. During radical nephrectomy, kidney was dissected and samples were taken from non-necrotic tumor tissue as well as from the area of the intact normal kidney closest to the tumor. The specimens were surgically removed, snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction. RNA extraction and analysis
Frozen tissues were pulverized by blender. Total RNA was extracted by the guanidinium thiocyanate technique (Chirgwin et al., 1979) and poly (A)+ selected by one passage on oligo 3To whom correspondence and reprint requests should be sent, at the International Institute of Genetics and Biophysics, Via Marconi 10,80125, Naples, Italy. Fax: 3918115936123-7257202. Received: January 10,1992 and in revised form March 21,1992.
HOX GENE EXPRESSION IN NORMAL AND NEOPLASTIC KIDNEY
(dT) cellulose columns. Poly (A)+ RNA was run on 1.25% agarose-formaldehyde gels, transferred to nylon (Schleicher and Schuell, Dassel, Germany NY13N) membranes by Northern capillary blotting, and hybridized to 10’ cpm of DNA probe labelled by nick-translation to a specific activity of 3-8 X lo8 dpm/kg. The probes contained the 3’ untranslated regions specific for each of the 38 HOX genes as previously reported (Stornaiuolo et al., 1990; Simeone et al., 1991; D’Esposito et al., 1991). Pre-hybridization and hybridization were carried out as described by Thomas (1980). After washing under stringent conditions (30 mM NaC1/3mM sodium citrate/0.2% SDS at 65”C), the blots were exposed for 1-7 days at -70°C to Kodak XR-5 films in an X-omatic intensifying screen cassette. RESULTS
Class-I homeobox genes within the 4 HOX loci can be aligned horizontally, according to their physical position on each chromosome, and vertically on the basis of the maximal sequence homology of their homeodomains. This alignment defines 13 paralogous groups (Simeone et al., 1991). Only 2 of these, namely groups 5 and 10, contain 4 HOX genes in all 4 loci (Fig. 1). We analyzed the expression of the 4 HOX gene clusters in normal human kidney and in tumor samples derived from patients with renal carcinomas. Poly (A)+ RNAs from normal kidney and from cancer specimens were hybridized by Northern blotting with probes containing the 3’ untranslated regions specific to each of the 38 HOX genes, organized in 4 large clusters, HOX1-HOX4 located on chromosomes 7, 17, 12 and 2, respectively (see Fig. 1). The pattern of HOXgene expression in normal human adult kidney is shown in Figure 1. Of the 38 HOX genes tested, 30 are actively expressed. In normal kidney, HOX genes are switched on or off in blocks containing a variable number of contiguous genes within each locus. Expression of the genes at the extreme 5’and 3’ ends of the HOX-1 locus, HOX-1J and HOX-IF, is undetectable, while 9 contiguous genes of the same locus, from HOX-11 to HOX-lK, are actively expressed. Furthermore, the entire HOX-2 locus is expressed, except for ~HOX-21,the gene nearest 3’. Moreover 3 contiguous genes, ,HOX-3G, HOX-3F, HOX-3H, at the 5’ end of the HOX-3 liocus, are silent, while a block of 6 genes (from HOX-31 to IYOX-~E)are actively expressed. Finally, the 2 genes at the extreme 5‘ end of the HOX-4 locus, HOX-41 and HOX-4H, are silent and 7 contiguous genes, at the 3‘ end, are all expressed, although the expression oSHOX-~G,the gene nearest 3‘ is very low. This complex pattern, illustrated in Figure 1, is specific to the kidney. The same analysis performed in normal colon, lung or liver shows different patterns of expression (data not shown) with different HOX genes switched on or off in these organs. HOX genes of the paralogous groups 5 and 10, which seem to represent boundaries that define genes 3‘ and 5’ displaying different expression patterns, are actively expressed in the normal kidney (Fig. 1). Major differences in the expression of HOX genes between normal kidney and renal carcinomas have been detected. Three HOX-2A transcripts, of 1.7 kb, 1.9 kb and over 8 kb, are present in normal kidney (Fig. 2a). The majority of kidney tumors tested (8/ 12) do not show detectable expression of this gene. However, 2 specimens have the same expression as normal kidney and 2 other biopsies show a specific expression pattern with the absence of the 1.9 molecular weight band in the only case of Wilms’ tumor studied, and 3 RNAs of high niolecular weight in the other biopsy. Two HOX-2E transcripts, of size 2.4 and 2.9 kb, are expressed in normal medullar kidney, whereas only the lower niRNA is detectable in cortical kidney (Fig. 2b). The tumor samples can be classified into 4 categories, according to the expression of this gene: 3/12 of the samples exhibit the same
893
pattern as normal medullar kidney; 3/12 express high steadystate levels of the 2.9-kb transcript whereas the 2.4-kb species is undetectable. Conversely, in 2/12 tumors only the lower molecular weight transcript is evident; in 4/12 tumors no HOX-2E transcripts are detected. Specimens of this last category are all histologically classified as clear-cellkidney carcinomas. Expression of the HOX-3H gene is undetectable in normal kidney (Fig. 2c) and in 9/13 biopsies examined. In contrast, we have observed a 1.7-kb HOX-3H transcript in 3 other specimens (Fig. 2c). In the single Wilms’ tumor tested, 2 abundant HOX-3H transcripts of sizes 1.7 and 2.0 kb are observed. In normal kidney, a single HOX-1D transcript of size 1.9 kb is present (Fig. 1). Out of 13 biopsies (see Table I), 4 also express the same 1.9-kb transcript, and 7 tumor samples contain 2 HOX-1D transcripts of size 1.9 and 1.4 kb, with the higher band as a dominant one. These transcripts correspond in size to the mRNA species observed in other tissues for this HOX gene. In contrast, one biopsy (31) shows an intense 1.4-kb band as the dominant one over the higher molecular weight transcript, while in another specimen 2 novel classes of mRNAs of higher molecular weight are present. Of the genes in group 10, only the HOX-2F gene is expressed both in normal and in neoplastic kidney with no detectable differences in intensity or transcript size (data not shown). HOX 3E is abundantly expressed in normal kidney with 3 transcript classes of 1.8, 2.4 and 2.8 kb (Fig. 1). Table I summarizes HOX-3E expression in renal-cell carcinomas. In 9 out of 13 tumors, HOX-3E expression is similar to that observed in normal tissue. In one tumor, only the 1.8- and 2.4-kb bands are observed, while another tumor expresses only the 2.4- and 2.8-kb transcripts. A third tumor contains only the 1.8-kb transcript while, in the single Wilms’ tumor analyzed, the 2.4 transcript is the only one present. HOX-4B is expressed in normal kidney with 4 transcripts of size 5.4, 4.2, 2.8, 1.4 kb (Fig. 2d). The molecular mechanisms regulating the expression of this gene have been well elucidated (Cianetti et al., 1990). Two alternative promoters underlie the transcription of 2 classes of HOX-4B-specific mRNAs: the 5.4- and 2.8-kb transcripts are driven from a distal promoter while the 4.2- and 1.4-kb transcripts originate from a proximal promoter. The 2 promoters are differentially regulated in a tissue- and stage-specific manner and respond differentially to retinoic-acid induction (Mavilio et al., 1988). The expression of HOX-4B in renal carcinomas is shown in Figure 2d. On the basis of the expression of this gene, it is possible to group kidney cancer into 3 categories: (a) tumors exhibiting the same expression pattern as the normal kidney (7/13) with 4 major transcripts of 5 4 4 . 2 , 2.8 and 1.4 kb; (b) a category of tumors (3/13) with HOX-4B transcripts of size 5.4 and 2.8 kb, showing the transcript classes presumably originating from the distal promoter, and (c) 3 tumors in which HOX-4B expression is presumably due to the activity of the proximal promoter, with transcript sizes of 4.2 and 1.4 kb. It is interesting to note that normal kidney from the same patients showing the alternative transcripts presents the full spectrum of HOX-4B gene expression with the 4 transcript classes. To determine whether HOX genes are differentially expressed in different parts of the kidney, we have analyzed the expression of some of the HOX genes in the cortical and medullar kidney. As shown in Figure 2d, no differences in the expression patterns of the HOX-4B gene are observed; however, the levels of HOX-4B transcripts are higher in the medulla than in the cortical kidney. We have obtained the same results for the HOX-2A (Fig. 2a) and HOX-3H genes (Fig. 2).In contrast, the HOX-2E gene shows 2 mRNAs in the medullar kidney but only the lower one appears to be detectable in cortical kidney. Table I summarizes the expression patterns of the 6 HOX genes described above in 13 cancer specimens. In all cases,
894
CILLO ETAL.
FIGURE 1 - HOX gene expression in normal human kidney. HOX genes are aligned horizontally according to their physical position on the chromosomes and vertically on the basis of maximal sequence homology of the homeodomain. Small stippled circles indicate homeodomains predicted in the scheme but not yet found. Northern blots of 5 pg of poly (A)+ RNA from normal kidney were hybridized to the probes indicated in the circles above each lane. Transcript sizes are given in kb. The 13 HD groups are indicated at the bottom.
kidney cancers display at least 2 HOX genes with altered expression, whereas HOX gene expression is identical in 4 different normal kidneys. The HOX-2A and HOX-2E genes are often altered in the same cancer biopsy. No correlation is observed between altered expression of the genes belonging to group 10 and the HOX-2A, HOX-2E and HOX-3H genes.
DISCUSSION
The kidney of vertebrates is an ideal model systeni for studying organogenesis, cell differentiation and neoplastic transformation (SaxCn, 1987). Transitory and vestigial pronephric and mesonephric structures are generated during early development, leading finally to the formation of the adult
FIGURE2 - Expression of HOX-2A ( a ) HOX-2E ( b ) HOX-3H (c) and HOX-4B ( d ) genes in normal kidney (total = NT, medullar = NM, cortical = NC) and in kidney cancer (the numbers of the different biopsies are indicated above each lane). Five micrograms of poly (A)+RNA from normal and neoplastic kidney were electrophoresed
through a 1.25% agarose gel, transferred to nitrocellulose filters and hybridized with probes representing the 3’ untranslated region of HOX-2A, 2E, 3H, 4B. Transcript size is indicated in kb. Control hybridization to a p-actin probe is shown.
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CILLO ETAL. TABLE I -VARIATION OF TRANSCRIPT CLASSES IN NEOPLASTIC VERSUS NORMAL KIDNEY Genes
Normal kidney Kidney tumors
HOX-2A
HOX-2E
HOX-311
> 8-1.9-1.7
2.9-2.4 2.9-2.4
-
-
n.5
-1
20 22 24
-
-
-
25 29 31
-
HOX-ID
HOX-3E
HOX-4B
Histology
1.9 2.8-2.4-1.8 5.4-4.2-2.8-1.4 1.9-1.4 (3.7-4.9) 2.8-2.4-1.8 5.4-4.2-2.8-1.4 Clear-cell carcinoma
(papillary type)
2.4
-
1.9-1.4 1.9 1.9
2.8-2.4-1.8 4.2-1.4 Clear-cell carcinoma 2.8-2.4 5.4-2.8 Clear-cell carcinoma 2.8-2.4-1.8 5.4-4.2-2.8-1.4 Renal-cell carcinoma
-
2.9
-
1.9-1.4
2.8-2.4-1.8 5.4-4.2-2.8-1.4 Renal-cell carcinoma
-
>8-1.9-1.7
2.9
1.7
1.9-1.4 1.9-1.4
2.8-2.4-1.8 5.4-2.8 Clear-cell carcinoma 1.8 5.4-4.2-2.8-1.4 Renal-cell carcinoma
32 33 36
>8-5-2.7-1.9
N.D. 2.9
1.7
1.9-1.4 1.9-1.4 1.9
2.4-1.8 5.4-4.2-2.8-1.4 Clear-cell carcinoma 2.8-2.4-1.8 5.4-4.2-2.8-1.4 Clear-cell carcinoma 2.8-2.4-1.8 4.2-1.4 Chromophilic-cell
37 38
>8-1.9-1.7
2.9-2.4 2.9-2.4 2.4
1.7 2-1.7
1.9-1.4 1.9-1.4 1.9
2.8-2.4-1.8 4.2-1.4 Clear-cell carcinoma 2.8-2.4-1.8 5.4-4.2-2.8-1.4 Clear-cell carcinoma 2.4 5.4-2.8 -
Wilms'
N.D.*
> 8-1.7
(sarcomatoid type) (mixed type)
(tubulo-papillary type)
carcinoma
Expression of HOX-2A, 2E, 3H, lD, 3E, 4B in normal kidney and in 13 kidney cancer biopsies is reported as size of transcripts (in kb) determined by Northern-blot analysis.-'( -) = undetectable expression.-*(N.D.) = not done.
kidney, the metanephros. The development of the metanephric kidney component, the branching epithelium of the ureter and the mesenchyme converted into epithelial elements, occurs in a synchronous manner that is strictly controlled, both temporally and spatially. The molecular mechanisms underlying the developmental decisions in kidney organogenesis are not at all understood. A gene of the paired box (PAX) family, a family of genes known to control normal embryonic development, is transiently expressed during the early stages of murine kidney organogenesis (Dressler et al., 1990). Here, we have analyzed the expression of the 4 HOX gene clusters in normal human kidney. Furthermore, we have compared the patterns ofHOXgene expression in normal and neoplastic kidney in order to determine whether this gene family might play a role in the malignant transformation of this organ. Our results show that normal adult human kidney exhibits a characteristic pattern of HOX gene expression with HOX genes switched on or off in blocks. These blocks contain a variable number of contiguous genes within each locus. Comparison between HOX gene expression in different normal human organs indicate very high expression of the whole panel of HOX genes in normal kidney with respect both to the number of HOXgenes turned on and to the abundance of the individual HOX transcripts. Since the homeoproteins are mostly involved, during normal development, in specifying positional information, the expression pattern of HOX genes we detected in normal adult human kidney may well represent the traces of the complex kidney organogenesis. It has been previously observed that the highly conserved organization of HOX genes in clusters reflects a regulatory hierarchy within this gene family. For example, in Drosophila, the physical order of the genes within the clusters correlates with the order in which they are expressed along the anteroposterior axis of the embryo (Ingham, 1988). This colinearity has also been observed in mammals (Akam, 1989). Our results suggest that this positional hierarchy of HOX gene expression is also maintained in the kidney. These observations are consistent with the idea that one or more upstream promoter elements account for the concerted expression of HOX genes in differentiating systems. Experimental evidence for a major promoter upstream of several HD-containing exons of the HOX-3 locus has been reported (Simeone et al., 1988). It is interesting to note that, although the 4 loci are abundantly expressed in normal kidney, in no case is the entire locus
expressed. Either the 5' or the 3' end genes, or both, are silent, indicating a particularly concerted transcriptional organization for these genes. In tumors of the kidney, the expression of some HOXgenes appears to be markedly altered when compared to that observed in normal kidney. HOXgenes can be turned on or off in tumors compared to normal kidney (HOX-3H, HOX-2A, HOX-2E). Other genes, although always expressed in normal and neoplastic tissue (HOX-lD, HOX-3E, HOX-4B), display different-sized classes of transcripts. These results indicate that HOX genes can be regulated at the level of transcription as well as at a post-transcriptional level, possibly by differential splicing. Homeobox genes within the 4 HOX loci can be aligned on the basis of the maximal sequence homology of their homeodomain. This alignment defines 13 homology groups. It has been observed that groups 5 and 10 represent a boundary that defines genes 3' and 5 ' , displaying different expression patterns in embryonal carcinoma cells (Stornaiuolo et al., 1990). Our data indicate that, in normal kidney, genes of these groups are highly expressed. Furthermore, in kidney cancer, they often display altered expression. For example, transcription of the HOX-4B gene in group 10 is initiated, in some cancer specimens, at either the distal or the proximal promoter, whereas normal kidney exhibits all 4 classes of HOX4B transcripts. These results suggest that neoplastic transformation in the kidney may be correlated with the inactivation of one of the promoters of this gene. In contrast, the HOX-2E gene in group 5 can be either altered in its expression in neoplastic kidney, or completely turned off. The inactivation of this gene is associated with clear-cell carcinoma of the kidney. In normal kidney, genes at the 5' end of group 5 show lower levels of expression. Groups 1 and 2 are silent. The HOX-3H gene in group 3 is the only HOXgene that is inactive in normal kidney and actively expressed in some kidney carcinomas. HOX-3H exhibits 2 very abundant transcripts in the only case of Wilms' tumor so far tested. There are 2 possible explanations for the differences in the patterns of HOX gene expression between normal and malignant kidney. First, the differences in HOXgene expression may reflect fundamental changes in the mechanisms of transcription of HOX genes, possibly reflecting the importance of these DNA-binding proteins in neoplastic transformation. Second, alterations in the pattern of HOX gene expression may reflect
HOX GENE EXPRESSION IN NORMAL AND NEOPLASTIC KIDNEY
corresponding changes between the heterogeneous cell populations present in normal kidney and the clonal cell populations that emerge in kidney tumors. An attempt to establish a correlation between HOX gene expression and clinical outcome of kidney cancer patients was hampered for several reasons in our study: (a) the low number of kidney cancer specimens (13) analyzed; (b) the small amount of RNA Poly (A)+ obtained from some of the biopsies; (c) the high number (38) of HOX genes to be screened for in each specimen; (d) the decision to test expression by Northern blotting in order to assess quantitative as well as qualitative differences in the expression of HOX genes. Consequently, we have aimed at establishing the general trend of HOX gene expression in normal and neoplastic kidney in order to identify the relevant genes to be tested in a further, more clinically oriented, analysis. In conclusion, our results suggest that HOX gene clusters display characteristic patterns of expression in normal kidney
897
and provide an indication that the coordinate regulation of HOXgenes may play an important role in kidney organogenesis. The alteration of HOXgene expression observed in kidney cancers seems to indicate that transcription factors play an important role in cancer evolution. Further work is needed to assess the correlation between altered HOX gene expression and cancer. The identification of target genes that are activated or repressed by the HOX genes will facilitate our understanding of the molecular mechanisms involved in neoplastic transformation. ACKNOWLEDGEMENTS
We thank Dr. A. Bernstein for critical reading of the manuscript. This work was supported by the Italian Association for Cancer Research (AIRC), the Consiglio Nazionale delle Ricerche (CNR) progetti finalizzati Biotecnologia e Biostrumentazione and Ingegneria Genetica.
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