Article Figures & Data
Figures
- Figure 1.
Characterization of the nephroblastoma collection. A, the frequency (%) of total (blue columns) and defective (red columns) proviruses integrated in individual clonal tumors. Integrated proviruses were detected in 90 tumors by Southern blot hybridization. B, histologic picture (×100 magnification) of MAV-2-infected nontumor renal cortex (0) and chicken nephroblastoma samples representing classes I to III. 0, differentiated nephron segments: proximal tubule (PT), distal tubule (DT), and glomerule (Gl). I, tumor comprised of differentiated PT, Gl, and besides cystically dilated tubules (CDT), and clusters of unorganized cells (UOC). II, tumor with prevalence of UOC and “nests of pseudonephrogenesis” (NPG). III, tumor composed exclusively of NPG and bundles of UOC. C, classification of nephroblastomas according to proportions of typical histopathologic attributes. The representation of each tumor class in the studied nephroblastoma collection is expressed in percentages. D, the relationship between tumor class (x-axis) and tumor size (y-axis, log scale). Fifty arbitrarily selected tumors were analyzed.
- Figure 2.
Schematic representations and typical results of PCR-based methods used in this work. A, inverse PCR: the integrated provirus (complete MAV-2 genome) containing long terminal repeats (blank boxes) and sequences coding for gag, pol, and env viral genes (thick line) is flanked by a host DNA (thin line). Combined BstY and BclI digestion generates fragments with compatible cohesive ends containing (a) the 5′-end of the provirus linked to a left-flanking fragment and (b) the 3′-end of the provirus linked to a right-flanking fragment. Self-ligation generates circular DNAs, which are then linearized by ApaLI. Using LTR3 and LTR2 oligonucleotide primers, DNAs are amplified (PCR products 1 and 2). Example of electrophoretic separation of amplified DNAs obtained from nine clonal tumors (bottom). In the majority of cases, each VIS is represented by two distinct PCR fragments. B, LTR-RACE: the integrated provirus [complete MAV-2 genome as in (A)] is transcribed from the R site within the left LTR. mRNAs (sketched above the MAV-2 genome) are terminated either at the right LTR termination signal (75% of transcripts) or at a termination signal in a downstream host sequence (25% of transcripts). Some transcripts are spliced by joining the splice donor site (SD) of the gag sequence and a splice acceptor site (SA) in env or within a downstream host gene. All these transcripts are converted to cDNAs using SMART RACE kit (Clontech). Nested amplification with SMART RACE kit using LTR1 and subsequently LTR2 primers yields PCR products: env RACE, MAV2-host RACE, and readthrough RACE. Examples of electrophoretic separation of first round PCR products (tumors 030.2b and 030.1d) and individual isolated and nested PCR reamplified products (tumors 435.1b and 813.1a; bottom).
- Figure 3.
Provirus insertions into plag1, twist, and foxP1 and expression of these genes in chicken nephroblastomas. A, positions of all VIS detected within plag1, twist, and foxP1 gene loci are indicated by arrows above the sketched genes. Arrows, the direction of transcription driven by integrated proviruses (transcription of host genes proceeds from the left to the right). The numbers at arrows specify tumor clones in which the integration was found. Thick bars and rectangles, exons; *, positions of initiation ATGs. Shaded and open rectangles, coding and noncoding exons in plag1 and twist genes, respectively. The precise genomic locations of MAV-2 proviruses in the plag1 locus are known only for tumors 304.1a, 813.1a, and 789.1c. The structure of chimeric virus-plag1 mRNAs is identical in all tumors listed in the box; the proviral gag donor sequence is spliced to the second plag1 exon. B, a typical example of Northern blot analysis of plag1, twist, and foxP1 expression. RNAs from 53 selected samples were separated by electrophoresis and blots were stained with methylene blue to reveal the amount and integrity of RNA in samples (18S) and subsequently hybridized with specific probes for chicken plag1, twist, and foxP1 genes. Open arrows, samples harboring integration within each gene. Normal mRNA sizes are marked on the left (kb).
- Figure 4.
Analysis of cVIS in c-Ha-ras, nov, and sprouty2 loci. A, precise structure of MAV-c-Ha-ras chimeric mRNAs. Dashed lines, the borders of c-Ha-ras exons; shaded boxes, host genomic sequences between the MAV right LTR (integration site) and the splice acceptor site; arrows, the direction of transcription driven by integrated proviruses; *, initiation ATGs. X03578 is the accession number of the previously described VIS. Northern blot analysis of c-Ha-ras expression in nine selected tumors including samples 389.2a and 821.1a (right). Methylene blue staining of 18S rRNA is shown. B, positions of VIS in the sketched nov locus. Northern blot analysis of nov expression in the tumors (including sample 435.1b) and control tissues (right). Methylene blue staining of 18S rRNA or glyceraldehyde-3-phosphate dehydrogenase hybridization is shown. X59284 is the accession number of the previously described VIS. C, positions of VIS in the sketched sprouty2 locus. Northern blot analysis of sprouty2 expression in the tumors (including samples 344.2a and 326.1c) and control tissues (right). Methylene blue staining of 18S rRNA is shown. Symbols and marks used in (B) and (C) are the same as in Fig. 3.
- Figure 5.
Northern blot analysis of twist, plag1, and nov in human renal tumors. RNAs from 16 Wilms tumors (WT), clear cell sarcoma of kidney (CCSK), and renal cell carcinomas were sequentially hybridized with the nov, twist, plag1, and GAPDH probes. mRNA sizes are indicated (kb).
Tables
- Table 1.
Candidate nephroblastoma-associated loci inferred from the analysis of VIS
Candidate locus Candidate genes Samples Evidence Nal 1-17 WCC939.1a, WCBO30.1d 1 Nal 1-19 Hyal-2 WCB789.1b, WCB818.2a 1 Nal 1-85 POU2/OTF1/OCT1 BL101.1a 2, 5 Nal 1-106 WCB819.2c, WCB030.2a 1 Nal 1-145 spry-2 BL344.2a, BL326.1c 1, 2, 4 Nal 1-187 WCB813.1a 5 Nal 2-28 twist BL102.2b, BL107.1a, BL122.1a, BL395.1a, WCB030.1b ( 10) 1, 2, 3, 4 Nal 2-88 dynein BL395.1b, WCC036.2a 2 Nal 2-104 WCB039.1d, WCC939.1a 1 Nal 2-110 plag1 BL304.1a, BL338.2a, BL384.2a, BL410.2c, WCB789.1c, WCB813.1a 1, 2, 4 Nal 2-118 atbf1 BL326.1c, BL819.1a 2 Nal 2-132 nov BL435.1b, X59284 ( 13) 2, 3, 4 Nal 2-145a kiaa0196, flj32440 WCC037.2b, WCC042.2a 1, 4 Nal 2-145b enc1, fbx032 WCB030.2c, WCC042.1b, WCC939.1c 1 Nal 3-30 LOC116228 WCB030.1e, BL378.2a 1 Nal 5-13 c-Ha-ras BL389.2a, BL821.1a, X03578 ( 21) 1, 2, 3 Nal 6-28 WCB822.2b, WCC850.1c 1 Nal 12-15 foxP1 WCC036.2a, WCC037.1a, WCC814.1d, WCC85O.1a, WCB826.1a 1, 2, 4 -
NOTE: Candidate loci were selected as described in Results, based on the following criteria: 1, multiple VISs within 20 kbp; 2, multiple hits in a defined large gene locus (VIS distance >20 kbp); 3, previously described VIS (accession nos. and the corresponding references are listed in the third column along with our samples); 4, experimentally confirmed deregulated expression of the candidate gene (overexpression or presence of transcripts with an altered coding sequence); 5, single clonal retroviral integration in the sample (single integration confirmed both by Southern blot and inverse PCR analyses). The criteria that led to the selection of candidate loci are summarized in the fourth column.
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Supplementary Data Pajer, et al.
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