Comparison of 177Lu-octreotate and 177Lu-octreotide for treatment in human neuroblastoma-bearing mice.

Background: Patients with high-risk neuroblastoma (NB) have a 5-year event-free survival of less than 50 %, and novel and improved treatment options are needed. Radiolabeled somatostatin analogs (SSTAs) could be a treatment option. The aims of this work were to compare the biodistribution and the therapeutic effects of 177Lu-octreotate and 177Lu-octreotide in mice bearing the human CLB-BAR NB cell line, and to evaluate their regulatory effects on apoptosis-related genes. Methods: The biodistribution of 177Lu-octreotide in mice bearing CLB-BAR tumors was studied at 1, 24, and 168 h after administration, and the absorbed dose was estimated to tumor and normal tissues. Further, animals were administered different amounts of 177Lu-octreotate or 177Lu-octreotide. Tumor volume was measured over time and compared to a control group given saline. RNA was extracted from tumors, and the expression of 84 selected genes involved in apoptosis was quantified with qPCR. Results: The activity concentration was generally lower in most tissues for 177Lu-octreotide compared to 177Lu-octreotate. Mean absorbed dose per administered activity to tumor after injection of 1.5 MBq and 15 MBq was 0.74 and 0.03 Gy/MBq for 177Lu-octreotide and 2.9 and 0.45 Gy/MBq for 177Lu-octreotate, respectively. 177Lu-octreotide treatment resulted in statistically significant differences compared to controls. Fractionated administration led to a higher survival fraction than after a single administration. The pro-apoptotic genes TNSFS8, TNSFS10, and TRADD were regulated after administration with 177Lu-octreotate. Treatment with 177Lu-octreotide yielded regulation of the pro-apoptotic genes CASP5 and TRADD, and of the anti-apoptotic gene IL10 as well as the apoptosis-related gene TNF. Conclusion: 177Lu-octreotide gave somewhat better anti-tumor effects than 177Lu-octreotate. The similar effect observed in the treated groups with 177Lu-octreotate suggests saturation of the somatostatin receptors. Pronounced anti-tumor effects following fractionated administration merited receptor saturation as an explanation. The gene expression analyses suggest apoptosis activation through the extrinsic pathway for both radiopharmaceuticals.

Frequent MYC coamplification and DNA hypomethylation of multiple genes on 8q in 8p11-p12-amplified breast carcinomas.

Genetic and epigenetic (DNA methylation, histone modifications, microRNA expression) crosstalk promotes inactivation of tumor suppressor genes or activation of oncogenes by gene loss/hypermethylation or duplications/hypomethylation, respectively. The 8p11-p12 chromosomal region is a hotspot for genomic aberrations (chromosomal rearrangements, amplifications and deletions) in several cancer forms, including breast carcinoma where amplification has been associated with increased proliferation rates and reduced patient survival. Here, an integrative genomics screen (DNA copy number, transcriptional and DNA methylation profiling) performed in 229 primary invasive breast carcinomas identified substantial coamplification of the 8p11-p12 genomic region and the MYC oncogene (8q24.21), as well as aberrant methylation and transcriptional patterns for several genes spanning the 8q12.1-q24.22 genomic region (ENPP2, FABP5, IMPAD1, NDRG1, PLEKHF2, RRM2B, SQLE, TAF2, TATDN1, TRPS1, VPS13B). Taken together, our findings suggest that MYC activity and aberrant DNA methylation may also have a pivotal role in the aggressive tumor phenotype frequently observed in breast carcinomas harboring 8p11-p12 regional amplification.

Chromosome ideograms of the laboratory rat (Rattus norvegicus) based on high-resolution banding, and anchoring of the cytogenetic map to the DNA sequence by FISH in sample chromosomes.

A detailed banded ideogram representation of the rat chromosomes was constructed based on actual G-banded prometaphase chromosomes. The approach yielded 535 individual bands, a significant increase compared to previously presented ideograms. The new ideogram was adapted to the existing band nomenclature. The gene locus positions in the rat draft DNA sequence were compared to the chromosomal positions as determined by dual-color FISH, using rat (RNO) chromosomes 6 and 15 and a segment of RNO4 as sample regions. It was found that there was generally an excellent correlation in the chromosome regions tested between the relative gene position in the DNA molecules and the sub-chromosomal localization by FISH and subsequent information transfer on ideograms from measurements of chromosomal images. However, in the metacentric chromosome (RNO15), the correlation was much better in the short arm than in the long arm, suggesting that the centromeric region may distort the linear relationship between the chromosomal image and the corresponding DNA molecule.

Chromosomal alterations in 98 endometrioid adenocarcinomas analyzed with comparative genomic hybridization.

The aim of the present study was to investigate chromosomal alterations in a large set of homogeneous tumors, 98 endometrioid adenocarcinomas. We also wanted to evaluate differences in chromosomal alterations in the different groups of tumors in relation to stage, survival and invasive or metastatic properties of the tumors. Comparative genomic hybridization (CGH) was used to detect chromosomal alterations in tissue samples from 98 endometrioid adenocarcinomas. All chromosomes were involved in DNA copy number variations at least once in the tumor material, but certain changes were recurrent and rather specific. Among the specific changes, it was possible to identify 39 chromosomal regions displaying frequent DNA copy number alterations. The most frequent alteration was detected at 1q25-->q42, in which gains were found in 30 cases (30%). Gains at 19pter-->p13.1 were detected in 26 tumors (26%) and at 19q13.1-->q13.3 in 19 tumors (19%). Increased copy numbers were also detected at 8q (8q21-->q22 and 8q22-->qter), at a relatively high rate, in 17 cases (17%). Furthermore, gains at 10q21-->q23 and 10p were found in 14 (14%) and 13 cases (13%), respectively. The most common losses were found in the three regions 4q22-->qter, 16q21-->qter and 18q21-->qter, all of which were detected in eight of the 98 tumors (8%). We also detected differences between the tumors from deceased patients and from survivors. Gain at 1q25-->q42 was more commonly detected in the tumors from patients who died of cancer. We noted that the regions most affected differed in the different surgical stages (I-IV). The results of the CGH analysis identify specific chromosomal regions affected by copy number changes, appropriate objects for further genetic studies.

The biological effect of pentoxifylline on the survival of human head and neck cancer cells treated with continuous low and high dose-rate irradiation.

AIM: The aim of this study was to compare the radiosensitivity effect of the G2/M arrest-abrogating substance, pentoxifylline (PTX), with high dose-rate irradiation (HDRI) and low dose-rate irradiation (LDRI), during which DNA repair and cell proliferation occur. METHODS: Three squamous cell carcinoma cell lines, FaDu, RPMI 2650 and SCC-61, with differences in genomic imbalance and intrinsic radiosensitivity, were irradiated with 140 cGy/min (HDRI) and 0.7 cGy/min (LDRI) in the presence and absence of 2.0 mM PTX. The surviving fraction at 2.0 Gy (SF2) and cell-cycle phase distribution were assessed by DNA flow cytometry analysis and bromodeoxyuridine incorporation. RESULTS: With HDRI and LDRI the SF2 of FaDu cells decreased by 38.5% and 27.6%, respectively, while the corresponding figures for RPMI 2650 were 28.5% and 48.5%, and for SCC-61 were 44.2% and 28.6%. Increases in G2 populations were evident after both HDRI and LDRI of all cell lines. CONCLUSIONS: The enhancement in the cytotoxic effect of PTX was statistically significant after HDRI as well as after LDRI in all three cell lines. We therefore conclude that PTX in combination with LDRI is worth further study, both in vitro, for disclosing underlying mechanisms, and in vivo, to confirm the findings.

Chromosome assignment of Cd36 transgenes in two rat SHR lines by FISH and linkage mapping of transgenic insert in the SHR-TG19 line.

The chromosome position of the Cd36 insert was determined by FISH in two rat transgenic lines (SHR/Ola-TgN(EF1aCd36)10Ipcv (SHR-TG10) and SHR/Ola-TgN(EF1aCd36)19Ipcv (SHR-TG19). The Cd36 transgene construct labelled with digoxigenin-11-dNTP was used as a probe in the FISH analysis. In accord with the previous finding that the SHR-TG10 harbours 6-8 copies of the transgene, the signals from both metaphase and interphase nuclei of SHR-TG10 preparations were rather strong and the probe hybridized to both copies of chromosome 1 at band q55. The probe hybridization to SHR-TG19 metaphase preparations also showed homozygosity of the transgene with localization of both copies to chromosome 11 at band q11. The signals were distinct but much weaker compared to the SHR-TG10, which again is in accord with the fact that the SHR-TG19 line harbours only a single copy of the transgene. In order to look for a possible impact of the insertion site neighbourhood upon the transgene phenotypic effect, we performed linkage mapping of the transgene in the SHR-TG19 line. By linkage mapping, the placement of the transgene to the proximal part of RNO11 was confirmed, the critical interval being 4 cM between D11Rat20 and D11Rat21, in good agreement with the RH map. Within the close neighbourhood of the inserted Cd36 transgene, there are several genes known to be expressed in kidney, and so the influence of some regulatory sequences enhancing kidney expression of the Cd36 transgene can be envisaged.

A dual-color FISH gene map of the proximal region of rat Chromosome 4 and comparative analysis in human and mouse.

The development and refinement of the rat genome map is a prerequisite for a continued qualified and fruitful use of this model system for the study of complex traits. In two distinct rat cancer models, recurrent amplification affecting the proximal region of rat Chr 4 was detected. To further characterize this region, we turned to the evolutionarily conserved chromosome segments in human Chr 7 and mouse Chrs 5 and 6 to identify functional and positional candidate genes. By means of single- and dual-color FISH on metaphase, prometaphase, and interphase chromatin, 15 genes in rat Chr 4q11-q23 (Cdk5, Hgf, Dmtf1, Abcb1, Cyp51, Cdk6, Tac1, Asns, Cav1, Met, Wnt2, Cftr, Smoh, Braf, Arhgef5) were mapped and aligned. In the course of this work, six cancer-related rat genes were isolated de novo and partly sequenced. Ten loci were also mapped by FISH in the mouse. The map provides the framework for a more detailed genetic characterization of individual tumor amplicons, but may also be valuable for the analysis of this region in other rat models of human complex disease. In addition, our data facilitate the analysis of events in mammalian chromosomal evolution affecting the region. In a comparison with human sequence data, we found that there is considerable conservation in this region both in gene order and in distances between genes. There is a single evolutionary breakpoint between rat and mouse and two between rat and human. Since our analysis shows that the three breaks all occurred in different positions, they must be independent of one another. The data tend to support the notion that the genomic configuration in rat Chr 4 is ancestral compared with that in humans and mice.

Independent amplification of two gene clusters on chromosome 4 in rat endometrial cancer: identification and molecular characterization.

The BDII rat is genetically predisposed to hormone-dependent endometrial adenocarcinoma and was used to model human cancer. Tumors arising spontaneously in strain crosses involving BDII rats were analyzed by means of comparative genome hybridization. The most common aberration was amplification of the proximal region of rat chromosome 4, centered around bands q12-q22. The copy numbers of 15 cancer-related genes from the region were examined in tissue cultures of 11 endometrial carcinomas (10 endometrial adenocarcinomas and 1 endometrial squamous cell carcinoma) and one peritoneal mesothelioma. Amplification in rat chromosome 4 was detected in six tumors (50%), five of which carried two separate amplified regions, situated at 4q12-q13 and 4q21-q22, interrupted by a nonamplified segment at 4q13-q21.1. The genes Cdk6 (cyclin-dependent kinase 6) and Met (hepatocyte growth factor receptor) were located in the core of each amplified region and were amplified most recurrently and at the highest levels among the genes tested. Using fluorescence in situ hybridization on tumor metaphases, it was observed that the amplified Cdk6 and Met sequences were situated on typical homogeneously staining regions (HSRs). In three tumors, both genes were amplified in the same HSRs, whereas in two tumors, the amplified sequences of each gene were situated in separate HSRs. In addition, Cdk6 and Met amplification was consistently associated with a corresponding increase in gene expression, suggesting that the two genes might represent the targets for the amplifications. In the sixth tumor, which carried amplified sequences of Met but not of Cdk6, coexpression of Met and the normally silent hepatocyte growth factor gene (Hgf; the ligand of Met) was observed. This finding suggests that an autocrine signaling circuit might be operating in this particular tumor. Taken together, our findings suggest that up-regulation of Cdk6 and/or Met may contribute to the development of endometrial cancers in the BDII rat.

Between rat and mouse zoo-FISH reveals 49 chromosomal segments that have been conserved in evolution.

Mouse single chromosome paints were applied to rat prophase/prometaphase chromosomes to detect homologous chromosome regions. The analysis revealed 49 rat chromosomal regions ranging in size from whole chromosomes down to small bands near the limit of detection with this method, which was estimated to be 2-3 Mb. When all the painted regions were taken into account, the whole rat genome was covered with mouse-homologous regions, with the exception of small segments near the centromeres and the short arms of Chromosomes (Chrs) 3, 11, 12, and 13. These regions were shown to contain high levels of rat-specific repetitive DNA. The number of conserved segments between rat and mouse detected by our high-resolution zoo-FISH method was significantly higher than that reported in previous studies.

Mouse chromosome 19 and distal rat chromosome 1: a chromosome segment conserved in evolution.

Through a combination of radiation hybrid mapping and studies by FISH and zoo-FISH we have made a comparative investigation of the distal portion of rat chromosome 1 (RNO1) and the entire mouse chromosome 19 (MMU19). It was found that homologous segments of RNO1 and MMU19 are similar in banding morphology and in length as determined by several different methods, and that the gene order of the 46 genes studied appears to be conserved across the homologous segments in the two species. High-resolution zoo-FISH techniques showed that MMU19 probes highlight only a continuous segment on RNO1 (1q43-qter), with no detectable signals on other rat chromosomes. We conclude that these data suggest the evolutionary conservation of a chromosomal segment from a common rodent ancestor. This segment now constitutes the entire MMU19 and a large segment distally on RNO1q in the mouse and rat, respectively.

Amplification of Mycn, Ddx1, Rrm2, and Odc1 in rat uterine endometrial carcinomas.

The BDII rat is genetically predisposed to estrogen-dependent endometrial adenocarcinoma and represents a valuable model for this type of tumor. Tumors arising in strain crosses involving the BDII rats had previously been screened for DNA copy number changes using comparative genome hybridization (CGH). It was found that extra copies of the proximal region of rat chromosome (RNO) 6 commonly could be detected in these tumors. Based on RH-mapping data and comparative mapping with mouse and human, seven cancer-related genes were predicted to be situated in RNO6q14-q16. Rat PACs were isolated for the N-myc proto-oncogene (Mycn), apolipoprotein B (Apob), the DEAD box gene 1 (Ddx1), ornithine decarboxylase 1 (Odc1), proopiomelanocortin (Pomc1), ribonucleotide reductase, M2 polypeptide (Rrm2), and syndecan 1 (Sdc1). The localization of the genes to the region was verified by FISH (fluorescence in situ hybridization) mapping, and the detailed order among them was determined by dual-color FISH. By Southern blot analysis, it was found that the Mycn locus was highly amplified in two out of 10 cell cultures derived from the tumors. In one of them (designated RUT30), the amplification level of Mycn was estimated at 140x. Two other genes were coamplified (Ddx1 and Rrm2) at much lower levels. Similarly, in another culture (designated RUT2), Mycn was amplified more than 40x, whereas three of the other genes (Ddx1, Rrm2, and Odc1) were coamplified at lower levels. Using FISH on metaphase chromosomes from the cell cultures analyzed, the amplified sequences were shown to be located in typical HSRs. With competitive RT-PCR, distinct overexpression of Mycn and Ddx1 could be demonstrated in both RUT2 and RUT30. In addition, Mycn was overexpressed in two other tumors not exhibiting Mycn amplification. Taken together, our results suggest that overexpression of Mycn plays an important role in the development of endometrial cancer in the BDII rat. In humans, Mycn amplification has been reported mainly from tumors of neuronal origin. To our knowledge, this is the first report of Mycn amplification and overexpression in hormone-dependent tumors.

Analysis of genetic changes in rat endometrial carcinomas by means of comparative genomic hybridization.

Animals of the BDII inbred rat strain are known to be genetically predisposed to endometrial adenocarcinoma (EAC). Using them as models of human EACs, we studied tumors arising in F1 and F2 progeny from BDII animals crossed to animals from two other inbred strains, in which EACs were quite rare. In order to identify chromosomal regions exhibiting DNA copy number changes, comparative genomic hybridization (CGH) was applied in a series corresponding to 27 different solid tumors, most of which were classified as EACs, from these animals. The main findings from the study were that, although many different chromosomes were involved in copy number variation, some of the changes detected were recurrent and quite specific. Among specific changes found were gains in rat chromosome (RNO) regions 4q12 approximately q22, 6q14 approximately q16, and whole chromosome arms in some of the small metacentric chromosomes (e.g., RNO14, 16, and 18). RNO10 was involved in gain in the terminal and proximal regions. Each of these regions contains previously identified cancer-related genes representing possible candidates to be involved in the development of EAC. Furthermore, it was observed that there were clear differences in the pattern of copy number changes between tumors occurring in the two different crosses, and also between solid tumors and cell cultures. Endometrial cancer is the most common human gynecological cancer, but not much is known about specific genetic changes influencing this disease. Two genetic alterations that have been reported from human endometrial cancer are amplification of the ERBB2 gene and mutations in the 12 codon of the KRAS gene. One case of Erbb2 amplification was found but there were no Kras mutations in the rat material studied. We conclude that molecular genetic analysis of the rat BDII model will provide important new information about EAC in mammals.

Rat-mouse and rat-human comparative maps based on gene homology and high-resolution zoo-FISH.

The laboratory rat, Rattus norvegicus, and the laboratory mouse, Mus musculus, are key animal models in biomedical research. A deeper understanding of the genetic interrelationsships between Homo sapiens and these two rodent species is desirable for extending the usefulness of the animal models. We present comprehensive rat-human and rat-mouse comparative maps, based on 1090 gene homology assignments available for rat genes. Radiation hybrid, FISH, and zoo-FISH mapping data have been integrated to produce comparative maps that are estimated to comprise 83-100% of the conserved regions between rat and mouse and 66-82% of the conserved regions between rat and human. The rat-mouse zoo-FISH analysis, supported by data for individual genes, revealed nine previously undetected conserved regions compared to earlier reports. Since there is almost complete genome coverage in the rat-mouse comparative map, we conclude that it is feasible to make accurate predictions of gene positions in the rat based on gene locations in the mouse.

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.

Genomewide assessment of genetic alterations in DMBA-induced rat sarcomas: cytogenetic, CGH, and allelotype analyses reveal recurrent DNA copy number changes in rat chromosomes 1, 2, 4, and 7.

Rat sarcomas, induced by subcutaneous injections of 7,12-dimethylbenz[a]anthracene (DMBA), were studied with the objective of identifying critical chromosome regions associated with tumorigenesis. We employed three genomewide screening techniques-cytogenetics, CGH, and allelotyping-in 19 DMBA-induced sarcomas in F1 (BN/Han x LE/Mol) rats. The most conspicuous finding in the cytogenetic analysis was a high incidence of trisomy for rat chromosome 2 (RNO2). Signs of gene amplification (hsr) were also seen in several tumors. The CGH analysis revealed that gains in copy number were much more common than losses. The gains mostly affected RNO2 (10/19), RNO12q (7/19), and RNO19q (5/19), as well as the proximal part of RNO4 (8/19) and the distal part of RNO7 (7/19). Reduction in copy number was seen in RNO17 (2/19). For the allelotyping, we used 318 polymorphic microsatellite marker loci covering the entire genome. We identified regions of allelic imbalance affecting most of the rat chromosomes. The highest incidences of recurrent allelic imbalance were observed at loci in certain regions in RNO1, 2, 4, and 7 and at lower incidences in parts of RNO12, 16, 18, and 19. The combined results suggested that genetic alterations detected in RNO2 and RNO12 usually corresponded to complete or partial trisomy, whereas those in RNO1 and RNO7 seemed to involve regional deletions and/or gains. Furthermore, we could confirm that copy number gains occur proximally in RNO4, where a previous study showed amplification of the Met oncogene in a subset of these tumors.

Overexpression of the hepatocyte growth factor (HGF) receptor (Met) and presence of a truncated and activated intracellular HGF receptor fragment in locally aggressive/malignant human musculoskeletal tumors.

Enhanced hepatocyte growth factor (HGF) receptor (Met) signaling has been suggested to play an important role in the development and progression of various epithelial and nonepithelial tumors. N-terminally truncated forms of the HGF receptor have been shown to be constitutively activated and tumorigenic in animal experiments. In the present study, 102 benign and malignant human musculoskeletal tumors were examined for expression of the HGF receptor by Western blotting and/or immunohistochemistry. A clear predominance of HGF receptor expression was seen in malignant as compared to benign tumors (Western blotting, P < 0.001; immunohistochemistry, P < 0.02). For the first time we show HGF receptor expression in the following four tumor types: dermatofibrosarcoma protuberans, clear cell sarcoma of tendons, malignant primitive neuroectodermal tumor, and benign fibrous histiocytoma. In three cases of sarcoma with high HGF receptor expression by Western blotting, we found indications of a short 85-kd N-terminally truncated HGF receptor that was tyrosine phosphorylated and located in the cytoplasm. Although fragments of this length were seen in 18 of 65 tumors, most were not tyrosine-phosphorylated. Northern blotting revealed only the 7.5-kb full-length HGF receptor transcript, suggesting that the 85-kd fragment is generated by an alternative initiation of translation or by proteolytic cleavage. Southern blotting detected no amplification of the Hgfr/Met gene in the 35 tumors examined, in contrast to our recent report of Hgfr/Met gene amplification in 7, 12-dimethylbenz(a)anthracene (DMBA)-induced rat sarcomas. The present data suggest that the locally aggressive and malignant properties of human mesenchymal tumors maybe related, in part, to high levels of full-length HGF receptors, and in some cases to the occurrence of N-terminally truncated HGF receptors, activated independently of HGF.

A dual-color FISH framework map for the characterization of the Sai1 tumor suppression region on rat chromosome 5.

The analysis of cell hybrids between malignant mouse hepatoma cells and normal rat fibroblasts has previously demonstrated the critical role of a deletion in rat chromosome 5 (RNO5) that was related to an anchorage independent phenotype. Those hybrids that were anchorage independent displayed loss of the entire RNO5 or an interstitial deletion in RNO5. These findings suggested that a putative tumor suppressor gene, Sai1 (suppression of anchorage independence 1), was located within the deleted region. To explore the molecular basis of the tumor suppressor activity of the Sai1 region, we analyzed the RNO5q23-q36 region with several genes and microsatellite markers that could be assigned to the region, as well as with new markers derived by representational difference analysis (RDA) or by microdissection. Dual-color FISH was used to construct a detailed physical map of the entire RNO5. These new data can be used to connect the physical and linkage maps in the rat, as well as to identify the details of the comparative map with other mammalian species including humans and mice. Using as FISH reagents genomic YAC, P1, or phage lambda clones corresponding to RNO5 markers, the order and unique positions of 18 markers could be established. The map provided a framework for the detailed characterization of the deletion found in anchorage independent hybrids. All markers within the bands RNO5q31.3-q35 were shown to be lost, including known cancer-related genes such as Ifna (5q32), Cdkn2a, -b (5q32), Jun (5q34), and Cdkn2c (5q35). However, the aberration in the deletion chromosome turned out to be more complex than originally thought in that we detected the presence of a paracentric inversion in addition to a deletion. The inversion led to the juxtaposition of the gene markers Tal2 (5q24.1) and Cd30lg (5q24.3). The framework map will provide the basis for the detailed physical YAC clone contig mapping of this region, and facilitate the identification and characterization of the Sai1 locus.

Cytogenetic localization of the growth and reproduction complex (Grc) in the rat and in the mouse and its position in relation to RT1.EC and other loci in the rat MHC.

The segment of rat chromosome 20 (RNO20p12) that contains the classical loci of the major histocompatibility complex (MHC; RT1.A-RT1.E) also contains genes affecting growth, reproduction and susceptibility to chemical carcinogens (the Grc) and multiple genes encoding class I MHC antigens (the EC region). The relative positions of the MHC, Grc, and EC region have not been demonstrated explicitly, although they have been postulated from genetic mapping studies. The present study was undertaken to map these regions cytogenetically by several different approaches using cosmids specific for the Rps 18, Hspa1 and Bat1 genes. The order was shown to be: centromere-Rps 18-Hspa1-Bat1-EC-Grc.

Amplification and overexpression of the hepatocyte growth factor receptor (HGFR/MET) in rat DMBA sarcomas.

In the present study subcutaneous fibrosarcomas were induced by the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) in rats from F1 generation cross breedings of two different inbred strains. Comparative genomic hybridization (CGH) analysis, which allows detection of DNA sequence copy changes, was applied to one of the tumors and it was found that there were increased copy numbers of sequences at chromosome 4q12-q21 in this tumor. We have previously determined that the loci for the hepatocyte growth factor (Hgf) and hepatocyte growth factor receptor (Hgfr/Met), a protooncogene, are situated in this particular chromosome region. Using probes for the two genes in FISH (fluorescence in situ hybridization) and in Southern blots we found that the Hgfr/Met gene was amplified in five of the 19 sarcomas studied, and that the Hgf gene was coamplified in two of them. Northern and Western blots and tyrosine phosphorylation analysis showed that the HGF receptor was overexpressed and functional in all five tumors, as well as in two additional tumors. In summary, both amplification and overexpression of the Hgfr/Met gene was found in about 25% of DMBA-induced experimental rat sarcomas, and HGF receptor overexpression alone was seen in two additional tumors. Possibly this reflects an involvement in paracrine or autocrine stimulation of growth and invasiveness by HGF. Our finding could provide a rodent model system to increased knowledge about causality and therapy, which may be applicable to the sizeable fraction of human musculoskeletal tumors displaying MET overexpression.