Horizontal transfer of tumor DNA to endothelial cells in vivo.

Tumor endothelial cells have long been regarded as genomically stable and therefore less likely to develop resistance to antiangiogenic therapies. However, recent findings have challenged this notion. We have shown that DNA can be transferred between cells through phagocytosis of apoptotic bodies by adjacent viable cells. Propagation of the ingested DNA is prevented by the activation of the p53-p21 pathway. In this study, we examined whether concomitant transfer of tumor DNA with genes that inactivate the p53 pathway could overcome the barrier to tumor DNA propagation. Our results demonstrate that fibroblasts and endothelial cells are capable of acquiring and replicating tumor DNA when the apoptotic tumor cells contain the SV40 large T antigen. Analysis of the tumor stroma of xenotransplanted tumors in severe combined immunodeficient mice revealed that a sub-population of the endothelial cells contained tumor DNA. These cells maintained the ability to form functional vessels in an in vivo assay and concurrently express tumor-encoded and endothelial-specific genes.

Microdissection molecular copy-number counting (microMCC)--unlocking cancer archives with digital PCR.

Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate microMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffin-embedded clinical biopsies. Further, microMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of microMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and non-amplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics.

Evolutionarily plastic regions at human 3p21.3 coincide with tumor breakpoints identified by the "elimination test".

We have previously found with the microcell hybrid-based "elimination test" that human chromosome 3 transferred into murine or human tumor cells regularly lost certain 3p regions during tumor growth in SCID mice. The most common eliminated region, CER1, is approximately 2.4 Mb at 3p21.3. CER1 breakpoints were clustered in approximately 200-kb regions at both telomeric and centromeric borders. We have also shown, earlier, that tumor-related deletions often coincide with human/mouse synteny breakpoints on 3p12-p22. Here we describe the results of a comparative genomic analysis on the CER1 region in Caenorhabditis elegans, Drosophila melanogaster, Fugu rubripes, Gallus gallus, Mus musculus, Rattus norvegicus, and Canis familiaris. First, four independent synteny breaks were found within the CER1 telomeric breakpoint cluster region, comparing human, dog, and chicken genomes, and two independent synteny breaks within the CER1 centromeric breakpoint cluster region, comparing human, mouse, and chicken genomes, suggesting a nonrandom involvement of tumor breakpoint regions in chromosome evolution. Second, both CER1 breakpoint cluster regions show recent tandem duplications (seven Zn finger protein family genes at the telomeric and eight chemokine receptor genes at the centromeric side). Finally, all genes from these regions underwent horizontal evolution in mammals, with formation of new genes and expansion of gene families, which were displayed in the human genome as tandem gene duplications and pseudogene insertions. In contrast the CER1 middle region contained evolutionarily well-conserved solitary genes and a minimal amount of retroposed genes. The coincidence of evolutionary plasticity with CER1 breakpoints may suggest that regional structural instability is expressed in both evolutionary and cancer-associated chromosome rearrangements.

Human wig-1, a p53 target gene that encodes a growth inhibitory zinc finger protein.

We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein (Varmeh-Ziaie et al., 1997). Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53 expression in our BL41-ts p53 Burkitt lymphoma cells. Wig-1 is also induced in MCF7 cells following treatment with the DNA-damaging agent mitomycin C. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped wig-1 to human chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53-dependent growth regulatory pathway.

The position of t(11;22)(q23;q11) constitutional translocation breakpoint is conserved among its carriers.

The t(11;22)(q23;q11) translocation is the most common recurrent balanced translocation described in humans. Carriers are phenotypically normal and often go undetected until diagnosis as a result of infertility investigations or following the birth of chromosomally unbalanced offspring. Efficient diagnostics of t(11;22) is important for children born to carriers of the translocation and for prenatal and pre-implantation diagnosis. The translocation breakpoint on chromosome 22 is located within a region containing low copy repeats, and this site is one of the last unfilled gaps in the sequence of this chromosome. This autosome harbors multiple other low copy repeats, which have been entirely sequenced. We report a combined sequencing and fiber FISH breakpoint characterization in five translocation carriers. From one carrier a cosmid library was constructed, and two chimeric cosmids (cos4_der11 and cos6_der22) were sequenced, which showed that strong palindromes (or inverted repeats) occur on both chromosomes. The translocation breakpoints occur at the tip of both inverted repeats. The palindrome on chromosomes 22 and 11 is composed of 852 and 166 bases, respectively. Four additional carriers were studied using fiber FISH with a resolution limit of 2 kb. Analysis of breakpoints on the DNA sequence level, or at the level of fiber FISH, indicate that they occur at the same position on both chromosomes in all five carriers. Using cos6_der22, PAC 158L19 and BAC 3009A19, we demonstrate that FISH is an attractive alternative in molecular diagnostics of t(11;22), as PCR assays are not reliable, due to the presence of numerous copies of low copy repeats.

The LZTFL1 gene is a part of a transcriptional map covering 250 kb within the common eliminated region 1 (C3CER1) in 3p21.3.

Deletions on 3p have been described in a large number of human tumors, suggesting the presence of a tumor suppressor gene(s). Using the elimination test, we previously defined a 1-Mb segment from human 3p21.3 (C3CER1). Genomic sequencing allowed us to construct a transcription map covering 250 kb containing five genes. We have characterized a human leucine zipper containing gene, leucine zipper transcription factor-like 1 (LZTFL1), and its mouse orthologue (Lztfl1), which was also mapped to mouse chromosome 9F. The LZTFL1 gene has two transcript isoforms displaying alternative polyadenylation. We have localized the human orthologue of the yeast SAC1 (suppressor of actin) gene as well as characterized and mapped the mouse Sac1 gene. Furthermore, the XT3 gene was characterized, encoding a member of the Na(+)/Cl(-) neurotransmitter superfamily. It has been shown that the XT3 gene had an alternatively spliced brain-specific isoform, predicted to remove 1 of 12 putative transmembrane domains. The transcription map also includes the CC chemokine receptor 9 gene (CCR9) and the LIM domain containing gene 1 (LIMD1). This work partially defines the gene content of C3CER1 that is a prerequisite for delineation of its role in tumorigenesis.

Hgfr/Met oncogene acts as target for gene amplification in DMBA-induced rat sarcomas: free chromatin fluorescence in situ hybridization analysis of amplicon arrays in homogeneously staining regions.

Analysis of chromosome rearrangements in tumors is an important means for revealing genetic pathways underlying tumorigenesis and tumor progression. In five of 17 DMBA-induced rat sarcomas, cytogenetic analysis had disclosed homogeneously staining regions (hsrs), which are generally accepted to be cytogenetic signs of gene amplification. Using comparative genomic hybridization (CGH), regional increases in DNA copy number of the proximal part of rat chromosome (RNO) 4 were detected in four of the tumors harboring hsrs. Amplification of the Hgfr/Met oncogene, located at RNO4q21.2, was detected by fluorescence in situ hybridization (FISH) in five tumors. In four of them, a number of flanking genes located in the close vicinity of Hgfr/Met, including Cav1 (q21.1), Wnt2 (q21.2-q21.3), and Cftr (q21.3), also were amplified, though amplification was seen in a lower fraction of the cells than was Hgfr/Met amplification. In the fifth tumor (BL150T), Hgfr/Met was amplified in all cells and was the sole amplified gene of those tested. In addition, the Hgfr/Met FISH signals in BL150T were tightly clustered and formed compact and intense spots compared with the signals seen in the other four tumors. Application of the free chromatin FISH technique to BL150T showed that the genomic Hgfr/Met probe stained the extended chromatin fibers of up to 1.5 Mb with an almost uninterrupted signal, indicating that the BL150T amplicon was build up solely of Hgfr/Met gene sequences. Our results suggest that the Hgfr/Met oncogene is the primary target for amplification in a subset of rat DMBA sarcomas.

Similar regions of human chromosome 3 are eliminated from or retained in human/human and human/mouse microcell hybrids during tumor growth in severe combined immunodeficient (SCID) mice.

By passaging microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background through severe combined immunodeficient (SCID) mice (elimination test), we have previously defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a second eliminated region (ER2) at 3p21.1-p14 and a common retained region (CRR) at 3q26-qter. In the present work, chr3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chr3. Four MCHs were generated. Compared with KH39, they developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analyzed in comparison with corresponding MCHs by chr3 arm-specific painting, 19 fluorescent in situ hybridization (FISH) probes, and 27 polymorphic markers. Three MCHs that maintained the intact exogenous chr3 in vitro lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four tumors contained mixed cell populations that lacked either the exogenous or one endogenous KH39 derived 3p. In one MCH the exogenous chr3 showed deletions within CER1 and ER2 already in vitro. It remained essentially unchanged in 8/9 derived tumors. The third, exogenous copy of the 3q26-q27 region (part of CRR) was retained in 16/20 tumors. It can be concluded that the human/human MCH-based elimination test identifies similar eliminated and retained regions on chr3 as the human/murine MCH-based test.

Fine mapping of the constitutional translocation t(11;22)(q23;q11).

Translocation t(11;22)(q23;q11) is the most common constitutional reciprocal translocation in man. Balanced carriers are phenotypically normal, except for decreased fertility, an increased spontaneous abortion rate and a possible predisposition to breast cancer in some families. Here, we report the high resolution mapping of the t(11;22)(q23;q11) breakpoint. We have localised the breakpoint, by using fluorescence in situ hybidisation (FISH) walking, to a region between D11S1340 and WI-8564 on chromosome 11, and D22S134 and D22S264 on chromosome 22. We report the isolation of a bacterial artificial chromosome (BAC) clone spanning the breakpoint in 11q23. We have narrowed down the breakpoint to an 80-kb sequenced region on chromosome 11 and FISH analysis has revealed a variation of the breakpoint position between patients. In 22q11, we have sequenced two BACs (BAC2280L11 and BAC41C4) apparently mapping to the region; these contain low copy repeats (LCRs). Southern blot analysis with probes from BAC2280L11 has revealed different patterns between normal controls and translocation carriers, indicating that sequences similar/identical to these probes flank the translocation breakpoint. The occurrence of LCRs has previously been associated with genomic instability and "unclonable" regions. Hence, the presence of such repeats renders standard translocation breakpoint cloning techniques ineffective. Thus, we have used high resolution fiber-FISH to study this region in normal and translocation cases by using probes from 22q11, LCRs and 11q23. We demonstrate that the LCR containing the gap in 22q11 is probably substantially larger than the previous estimates of 100 kb. Using fiber-FISH, we have localised the breakpoint in 22q11 to approximately 20-40 kb from the centromeric border of the LCR (i.e. the telomeric end of AC006547) and have confirmed the breakpoint position on 11q23.

Combined LOH/CGH analysis proves the existence of interstitial 3p deletions in renal cell carcinoma.

We have recently developed an allele titration assay (ATA) to assess the sensitivity and influence of normal cell admixture in loss of heterozygosity (LOH) studies based on CA-repeat. The assay showed that these studies are biased by the size-dependent differential sensitivity of allele detection. Based on these data, we have set up new criteria for evaluation of LOH. By combining these new rules with comparative genome hybridization (CGH) we have shown the presence of interstitial deletions in renal cell carcinoma (RCC) biopsies and cell lines. At least three out of 11 analysed RCC cell lines and three out of 37 biopsies contain interstitial deletions on chromosome 3. Our study suggests the presence of several regions on human chromosome 3 that might contribute to tumor development by their loss: (i) 3p25-p26, around the VHL gene (D3S1317); (ii) 3p21. 3-p22 (between D3S1260 and D3S1611); (iii) 3p21.2 (around D3S1235 and D3S1289); (iv) 3p13-p14 (around D3S1312 and D3S1285). For the first time, AP20 region (3p21.3-p22) was carefully tested for LOH in RCC. It was found that the AP20 region is the most frequently affected area. Our data also suggest that another tumor suppressor gene is located near the VHL gene in 3p25-p26.

A 1-Mb PAC contig spanning the common eliminated region 1 (CER1) in microcell hybrid-derived SCID tumors.

We have developed an elimination test to identify chromosomal regions that contain tumor inhibitory genes. Monochromosomal human/mouse microcell hybrids are generated and passaged through SCID mice. Derived tumors are then analyzed for deletions on the transgenomic chromosome. Using this strategy, we have previously identified a 1.6-cM common eliminated region 1 (CER1) on human 3p21. 3. We now report that CER1 contains 14 markers that are deleted in 19 SCID-derived tumors. A 1-Mb PAC contig that spans CER1 was assembled. Five chemokine receptor genes (CCR1, CCR3, CCR2, CCR5, and CCR6) were localized in CER1 in a 225-kb cluster. The lactotransferrin gene (LTF, or lactoferrin, LF), which reportedly has tumor inhibitory activity, also maps to CER1. Our results create a basis for characterization and further functional testing of genes within CER1.

Analysis of NotI linking clones isolated from human chromosome 3 specific libraries.

We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences

TOM1 genes map to human chromosome 22q13.1 and mouse chromosome 8C1 and encode proteins similar to the endosomal proteins HGS and STAM.

The avian tom1 (target of myb 1) gene has been previously characterized from v-myb-transformed cells. We report here cloning of the human and mouse tom1 orthologs. Both genes are expressed ubiquitously, with the highest levels in skeletal muscle, brain, and intestines, as assessed by Northern blot and mRNA in situ hybridization. The N-terminal domain of the TOM1 protein shares similarity with HGS (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), which are associated with vesicular trafficking at the endosome. A putative coiled-coil domain was also detected in the central part of the TOM1 protein. This domain structure suggests that TOM1 is another member of a family of genes implicated in the trafficking regulation of growth-factor-receptor complexes that are destined for degradation in the lysosome. We also show that a human paralog of TOM1 (TOM1-like gene 1) exists. Furthermore, we provide a transcription map over a 190-kb contig of the TOM1 region. This map includes its distal neighbors HMOX1 and MCM5 and two proximal novel genes, one of which is a HMG-box-containing gene (HMG2L1), and the other of unknown function. Using a genomic PAC clone, we demonstrate that the mouse Tom1 and Hmox1 genes are part of an as yet undescribed syntenic group between mouse chromosome 8C1 and human chromosome 22q13.1.

The human LARGE gene from 22q12.3-q13.1 is a new, distinct member of the glycosyltransferase gene family.

Meningioma, a tumor of the meninges covering the central nervous system, shows frequent loss of material from human chromosome 22. Homozygous and heterozygous deletions in meningiomas defined a candidate region of >1 Mbp in 22q12.3-q13.1 and directed us to gene cloning in this segment. We characterized a new member of the N-acetylglucosaminyltransferase gene family, the LARGE gene. It occupies >664 kilobases and is one of the largest human genes. The predicted 756-aa N-acetylglucosaminyltransferase encoded by LARGE displays features that are absent in other glycosyltransferases. The human like-acetylglucosaminyltransferase polypeptide is much longer and contains putative coiled-coil domains. We characterized the mouse LARGE ortholog, which encodes a protein 97.75% identical with the human counterpart. Both genes reveal ubiquitous expression as assessed by Northern blot analysis and in situ histochemistry. Chromosomal mapping of the mouse gene reveals that mouse chromosome 8C1 corresponds to human 22q12.3-q13.1. Abnormal glycosylation of proteins and glycosphingolipids has been shown as a mechanism behind an increased potential of tumor formation and/or progression. Human tumors overexpress ganglioside GD3 (NeuAcalpha2,8NeuAcalpha2, 3Galbeta1,4Glc-Cer), which in meningiomas correlates with deletions on chromosome 22. It is the first time that a glycosyltransferase gene is involved in tumor-specific genomic rearrangements. An abnormal function of the human like-acetylglucosaminyltransferase protein may be linked to the development/progression of meningioma by altering the composition of gangliosides and/or by effect(s) on other glycosylated molecules in tumor cells.

A novel gene containing LIM domains (LIMD1) is located within the common eliminated region 1 (C3CER1) in 3p21.3.

Chromosomal deletions on 3p have been described in a large number of human tumors, suggesting the presence of a tumor suppressor gene(s). Using an experimental system, called the elimination test, we previously identified a 1 Mb segment, the common eliminated region 1 (C3CER1). C3CER1 was also covered by a PAC contig. Using the sequence of two overlapping PACs from C3CER1, we localized the human KIAA0028 cDNA, encoding the precursor of mitochondrial leucyl-tRNA synthetase. We also characterized a novel human LIM domain-containing gene (LIMD1) and its mouse ortholog (Limd1). LIM domains consist of a cysteine-rich consensus sequence containing two distinct zinc-binding subdomains, which mediate protein-protein interactions. The predicted protein sequences of the human and mouse genes reveal three LIM domains located at the C-terminal end, which indicates that they belong to the group 3 of the gene family encoding LIM motifs. We characterized the genomic structure of the human LIMD1 gene and assigned the mouse Limd1 gene to the chromosome 9F subtelomeric region. Both genes are ubiquitously expressed at the mRNA level. The LIM motif has been previously identified in many developmentally important factors from various eukaryotes. These factors have been shown to play a role in intracellular signaling, transcriptional regulation and cellular differentiation during development. The human C3CER1-located LIMD1 gene should therefore be further studied for its possible role in tumor suppression.

Human/mouse microcell hybrid based elimination test reduces the putative tumor suppressor region at 3p21.3 to 1.6 cM.

We have previously identified an approximately 7 cM long common eliminated region (CER), involving the 3p21.3 markers AP20R, D3S966, D3S3559, D3S1029, WI-7947, D3S2354, AFMb362wb9, and D3S32, in human chromosome 3/A9 mouse fibrosarcoma microcell hybrid (MCH) derived SCID mouse tumors. We now report the results of our more detailed analysis on 24 SCID mouse tumors derived from two MCH lines that originally carried intact human chromosomes 3. They were analyzed by fluorescence in situ hybridization (FISH) painting and PCR, using 24 markers covering the region between D3S1611 and D3S13235 at 3p22-p21.2. D3S32 and D3S2354 were regularly eliminated during in vivo tumor growth, whereas the other 22 markers, D3S1611, ACAA, D3S1260, WI-692, AP20R, D3S3521, D3S966, D3S1029, D3S643, WI-2420, MSTI. GNAI2, D3S1235, D3S1298, GLBI, WI-4193, D3S3658, D3S3559, D3S3678, WI-6400, WI-7947, and WI-10865, were regularly retained. We have defined a common eliminated region of approximately 1.6 cM (designated as CER1) inside the 7 cM CER described earlier. CER1 is flanked distally by D3S1029 and proximally by D3S643.

Differential elimination of 3p and retention of 3q segments in human/mouse microcell hybrids during tumor growth.

We have previously found that human chromosome 3 was fragmented in the course of in vivo tumor growth of monochromosomal human/mouse (A9 fibrosarcoma parent) microcell hybrids in SCID mice. Marker analysis of tumor cell lines has identified a regularly eliminated 7 cM segment on 3p21.3 referred to as the common eliminated region (CER). The same region is frequently affected by LOH in a variety of human carcinomas. The present study is a comparative chromosome painting, reverse painting, and PCR marker analysis of microcell hybrids (MCHs) that originally contained an intact chromosome 3 from two alternative donors, during and after four passages in SCID mice. We found regular elimination of 3p in parallel with preferential retention of 3q. In addition to CER on 3p, we can now define a common retained region (CRR) on 3q. It includes eight markers between D3S1282 (3q25-q26) and D3S1265 (3q27-qter) and spans approximately 43 cM. These observations are concordant with the frequent loss of corresponding 3p regions and the frequent retention, with occasional amplification, of 3q in several types of human tumors.

A micro-dissection approach for isolation of NotI linking clones from regions frequently deleted in RCC and SCLC.

We demonstrate that micro-dissection can be used for isolating NotI linking clones from the human 3p21-pter region. This approach is an improvement to positional cloning techniques, since NotI linking clones are directly linked with genes.

Assignment and ordering of twenty-three unique NotI-linking clones containing expressed genes including the guanosine 5'-monophosphate synthetase gene to human chromosome 3.

Twenty-three unique NotI-linking clones, mainly isolated from the NRL1 library, were mapped and ordered by fluorescence in situ hybridization to human chromosome 3. All these clones were partially sequenced around the NotI sites and thus represent sequence-tagged sites. The EMBL nucleotide database was then searched with sequences from the NotI-linking clones using the FASTA program. This search revealed that the NRL-090 clone (at 3q24) contains the gene encoding human guanosine 5'-monophosphate synthetase (GMPS-PEN). To our knowledge, this is the first localization of this gene. Clone NL1-320 (at 3p21.3) contains a gene encoding arginine tRNA (97.3% identity in 73 bp), while clones NRL-063, NRL-097 and NRL-143 contain expressed sequences with unknown functions. Other clones displayed 60-85% similarities to cDNAs, CpG islands and other genes.