Characterization of genetically defined sporadic and hereditary type 1 papillary renal cell carcinoma cell lines.

Renal cell carcinoma (RCC) is not a single disease but is made up of several different histologically defined subtypes that are associated with distinct genetic alterations which require subtype specific management and treatment. Papillary renal cell carcinoma (pRCC) is the second most common subtype after conventional/clear cell RCC (ccRCC), representing ~20% of cases, and is subcategorized into type 1 and type 2 pRCC. It is important for preclinical studies to have cell lines that accurately represent each specific RCC subtype. This study characterizes seven cell lines derived from both primary and metastatic sites of type 1 pRCC, including the first cell line derived from a hereditary papillary renal carcinoma (HPRC)-associated tumor. Complete or partial gain of chromosome 7 was observed in all cell lines and other common gains of chromosomes 16, 17, or 20 were seen in several cell lines. Activating mutations of MET were present in three cell lines that all demonstrated increased MET phosphorylation in response to HGF and abrogation of MET phosphorylation in response to MET inhibitors. CDKN2A loss due to mutation or gene deletion, associated with poor outcomes in type 1 pRCC patients, was observed in all cell line models. Six cell lines formed tumor xenografts in athymic nude mice and thus provide in vivo models of type 1 pRCC. These type 1 pRCC cell lines provide a comprehensive representation of the genetic alterations associated with pRCC that will give insight into the biology of this disease and be ideal preclinical models for therapeutic studies.

The evolution of single cell-derived colorectal cancer cell lines is dominated by the continued selection of tumor-specific genomic imbalances, despite random chromosomal instability.

Intratumor heterogeneity is a major challenge in cancer treatment. To decipher patterns of chromosomal heterogeneity, we analyzed six colorectal cancer cell lines by multiplex interphase FISH (miFISH). The mismatch-repair-deficient cell lines DLD-1 and HCT116 had the most stable copy numbers, whereas aneuploid cell lines (HT-29, SW480, SW620 and H508) displayed a higher degree of instability. We subsequently assessed the clonal evolution of single cells in two colorectal carcinoma cell lines, SW480 and HT-29, which both have aneuploid karyotypes but different degrees of chromosomal instability. The clonal compositions of the single cell-derived daughter lines, as assessed by miFISH, differed for HT-29 and SW480. Daughters of HT-29 were stable, clonal, with little heterogeneity. Daughters of SW480 were more heterogeneous, with the single cell-derived daughter lines separating into two distinct populations with different ploidy (hyper-diploid and near-triploid), morphology, gene expression and tumorigenicity. To better understand the evolutionary trajectory for the two SW480 populations, we constructed phylogenetic trees which showed ongoing instability in the daughter lines. When analyzing the evolutionary development over time, most single cell-derived daughter lines maintained their major clonal pattern, with the exception of one daughter line that showed a switch involving a loss of APC. Our meticulous analysis of the clonal evolution and composition of these colorectal cancer models shows that all chromosomes are subject to segregation errors, however, specific net genomic imbalances are maintained. Karyotype evolution is driven by the necessity to arrive at and maintain a specific plateau of chromosomal copy numbers as the drivers of carcinogenesis.

Novel mouse model recapitulates genome and transcriptome alterations in human colorectal carcinomas.

Human colorectal carcinomas are defined by a nonrandom distribution of genomic imbalances that are characteristic for this disease. Often, these imbalances affect entire chromosomes. Understanding the role of these aneuploidies for carcinogenesis is of utmost importance. Currently, established transgenic mice do not recapitulate the pathognonomic genome aberration profile of human colorectal carcinomas. We have developed a novel model based on the spontaneous transformation of murine colon epithelial cells. During this process, cells progress through stages of pre-immortalization, immortalization and, finally, transformation, and result in tumors when injected into immunocompromised mice. We analyzed our model for genome and transcriptome alterations using ArrayCGH, spectral karyotyping (SKY), and array based gene expression profiling. ArrayCGH revealed a recurrent pattern of genomic imbalances. These results were confirmed by SKY. Comparing these imbalances with orthologous maps of human chromosomes revealed a remarkable overlap. We observed focal deletions of the tumor suppressor genes Trp53 and Cdkn2a/p16. High-level focal genomic amplification included the locus harboring the oncogene Mdm2, which was confirmed by FISH in the form of double minute chromosomes. Array-based global gene expression revealed distinct differences between the sequential steps of spontaneous transformation. Gene expression changes showed significant similarities with human colorectal carcinomas. Pathways most prominently affected included genes involved in chromosomal instability and in epithelial to mesenchymal transition. Our novel mouse model therefore recapitulates the most prominent genome and transcriptome alterations in human colorectal cancer, and might serve as a valuable tool for understanding the dynamic process of tumorigenesis, and for preclinical drug testing. © 2016 Wiley Periodicals, Inc.

Genomic and metabolic characterization of a chromophobe renal cell carcinoma cell line model (UOK276).

Chromophobe renal cell carcinoma (ChRCC) represents 5% of all RCC cases and frequently demonstrates multiple chromosomal losses and an indolent pattern of local growth, but can demonstrate aggressive features and resistance to treatment in a metastatic setting. Cell line models are an important tool for the investigation of tumor biology and therapeutic drug efficacy. Currently, there are few ChRCC-derived cell lines and none is well characterized. This study characterizes a novel ChRCC-derived cell line model, UOK276. A large ChRCC tumor with regions of sarcomatoid differentiation was used to establish a spontaneously immortal cell line, UOK276. UOK276 was evaluated for chromosomal, mutational, and metabolic aberrations. The UOK276 cell line is hyperdiploid with a modal number of 49 chromosomes per cell, and evidence of copy-neutral loss of heterozygosity, as opposed to the classic pattern of ChRCC chromosomal losses. UOK276 demonstrated a TP53 missense mutation, expressed mutant TP53 protein, and responded to treatment with a small-molecule therapeutic agent, NSC319726, designed to reactivate mutated TP53. Xenograft tumors grew in nude mice and provide an in vivo animal model for the investigation of potential therapeutic regimes. The xenograft pathology and genetic analysis suggested that UOK276 was derived from the sarcomatoid region of the original tumor. In summary, UOK276 represents a novel in vitro and in vivo cell line model for aggressive, sarcomatoid-differentiated, TP53 mutant ChRCC. This preclinical model system could be used to investigate the novel biology of aggressive, sarcomatoid ChRCC and evaluate the new therapeutic regimes.

ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans.

The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M(+) B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors.

Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells.

Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research.

Spontaneous transformation of murine epithelial cells requires the early acquisition of specific chromosomal aneuploidies and genomic imbalances.

Human carcinomas are defined by recurrent chromosomal aneuploidies, which result in a tissue-specific distribution of genomic imbalances. In order to develop models for these genome mutations and to determine their role in tumorigenesis, we generated 45 spontaneously transformed murine cell lines from normal epithelial cells derived from bladder, cervix, colon, kidney, lung, and mammary gland. Phenotypic changes, chromosomal aberrations, centrosome number, and telomerase activity were assayed in control uncultured cells and in three subsequent stages of transformation. Supernumerary centrosomes, binucleate cells, and tetraploidy were observed as early as 48 hr after explantation. In addition, telomerase activity increased throughout progression. Live-cell imaging revealed that failure of cytokinesis, not cell fusion, promoted genome duplication. Spectral karyotyping demonstrated that aneuploidy preceded immortalization, consisting predominantly of whole chromosome losses (4, 9, 12, 13, 16, and Y) and gains (1, 10, 15, and 19). After transformation, focal amplifications of the oncogenes Myc and Mdm2 were frequently detected. Fifty percent of the transformed lines resulted in tumors on injection into immunocompromised mice. The phenotypic and genomic alterations observed in spontaneously transformed murine epithelial cells recapitulated the aberration pattern observed during human carcinogenesis. The dominant aberration of these cell lines was the presence of specific chromosomal aneuploidies. We propose that our newly derived cancer models will be useful tools to dissect the sequential steps of genome mutations during malignant transformation, and also to identify cancer-specific genes, signaling pathways, and the role of chromosomal instability in this process.

Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines.

In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer.

A virus causes cancer by inducing massive chromosomal instability through cell fusion.

Chromosomal instability (CIN) underlies malignant properties of many solid cancers and their ability to escape therapy, and it might itself cause cancer [1, 2]. CIN is sustained by deficiencies in proteins, such as the tumor suppressor p53 [3-5], that police genome integrity, but the primary cause of CIN in sporadic cancers remains uncertain [6, 7]. The primary suspects are mutations that deregulate telomere maintenance, or mitosis, yet such mutations have not been identified in the majority of sporadic cancers [6]. Alternatively, CIN could be caused by a transient event that destabilizes the genome without permanently affecting mechanisms of mitosis or proliferation [5, 8]. Here, we show that an otherwise harmless virus rapidly causes massive chromosomal instability by fusing cells whose cell cycle is deregulated by oncogenes. This synergy between fusion and oncogenes "randomizes" normal diploid human fibroblasts so extensively that each analyzed cell has a unique karyotype, and some produce aggressive, highly aneuploid, heterogeneous, and transplantable epithelial cancers in mice. Because many viruses are fusogenic, this study suggests that viruses, including those that have not been linked to carcinogenesis, can cause chromosomal instability and, consequently, cancer by fusing cells.

Integrative genomics reveals mechanisms of copy number alterations responsible for transcriptional deregulation in colorectal cancer.

To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations.

The UOK 257 cell line: a novel model for studies of the human Birt-Hogg-Dubé gene pathway.

The establishment, characterization, and tumorigenicity of a new epithelial cell line (UOK 257) derived from human renal carcinoma of an individual with Birt-Hogg-Dubé (BHD) syndrome are reported. Unlike other established renal tumor cell lines from sporadic renal cell carcinoma, this is the first established renal tumor cell line of BHD, an inheritable neoplastic syndrome. The isolated tumor cells display loss of contact inhibition in vitro, and produce subcutaneous tumors in mouse xenografts. Histopathologic, ultrastructural, and cytogenetic characterizations of the established tumor cells are reported. Cytogenetic analysis using spectral karyotyping on UOK 257 cells revealed 17p loss and a near-triploid and aneuploid karyotype with multiple fluorescence in situ hybridization analysis using a locus-specific gene probe for MYC. The result demonstrates that the established tumor cells consist of two cell populations, one containing four and one containing five copies of the MYC oncogene.

The ancestral carnivore karyotype (2n = 38) lives today in ringtails.

Chromosome painting was used to investigate the conservation of high-resolution longitudinal 4',6-diamidino-2-phenylindole (DAPI)/G bands in Carnivore chromosomes. Cat (Felis catus) and raccoon dog (Nyctereutes procyonoides) painting probes were hybridized to the ringtail (Bassaricus astutus), dwarf mongoose (Helogale parvula), and Malagasy civet (Fossa fossa) to identify homologous chromosome elements. The patterns of chromosome segment homology among Carnivore species allowed us to reconstruct and propose the disposition of a high-resolution banded ancestral carnivore karyotype (ACK). Three bi-armed chromosomes consistently found among Caniformia species are represented as 6 homologous acrocentric chromosomes among Feliformia species of Carnivora. However, reexamination of the most basal of Feliformia species, the African palm civet Nandinia, revealed the presence of the 3 heretofore Caniformia bi-armed chromosomes. Because these 3 bi-armed chromosomes are found in both Caniformia and Feliformia lineages, they are presumed ancestral for all Carnivora, suggesting that the ACK chromosome number would be 38, rather than the previously supposed 42. Banded chromosomes of the ACK are used to evaluate the consistency between recently determined molecular phylogenetic relationships and postulated cytogenetic dynamics in the same Carnivore species.

SMYD5 Controls Heterochromatin and Chromosome Integrity during Embryonic Stem Cell Differentiation.

Epigenetic regulation of chromatin states is thought to control gene expression programs during lineage specification. However, the roles of repressive histone modifications, such as trimethylated histone lysine 20 (H4K20me3), in development and genome stability are largely unknown. Here, we show that depletion of SET and MYND domain-containing protein 5 (SMYD5), which mediates H4K20me3, leads to genome-wide decreases in H4K20me3 and H3K9me3 levels and derepression of endogenous LTR- and LINE-repetitive DNA elements during differentiation of mouse embryonic stem cells. SMYD5 depletion resulted in chromosomal aberrations and the formation of transformed cells that exhibited decreased H4K20me3 and H3K9me3 levels and an expression signature consistent with multiple human cancers. Moreover, dysregulated gene expression in SMYD5 cancer cells was associated with LTR and endogenous retrovirus elements and decreased H4K20me3. In addition, depletion of SMYD5 in human colon and lung cancer cells results in increased tumor growth and upregulation of genes overexpressed in colon and lung cancers, respectively. These findings implicate an important role for SMYD5 in maintaining chromosome integrity by regulating heterochromatin and repressing endogenous repetitive DNA elements during differentiation. Cancer Res; 77(23); 6729-45. ©2017 AACR.

Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas.

BACKGROUND: Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas. METHODS: Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and ß-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability. RESULTS: Sequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-ß-catenin complex, markedly depleted ß-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage. CONCLUSIONS: A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.

Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs.

Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations' impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease.

UOK 262 cell line, fumarate hydratase deficient (FH-/FH-) hereditary leiomyomatosis renal cell carcinoma: in vitro and in vivo model of an aberrant energy metabolic pathway in human cancer.

Energy deregulation and abnormalities of tumor cell metabolism are critical issues in understanding cancer. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is an aggressive form of RCC characterized by germline mutation of the Krebs cycle enzyme fumarate hydratase (FH), and one known to be highly metastatic and unusually lethal. There is considerable utility in establishing preclinical cell and xenograft models for study of disorders of energy metabolism, as well as in development of new therapeutic approaches targeting of tricarboxylic acid (TCA) cycle enzyme-deficient human cancers. Here we describe a new immortalized cell line, UOK 262, derived from a patient having aggressive HLRCC-associated recurring kidney cancer. We investigated gene expression, chromosome profiles, efflux bioenergetic analysis, mitochondrial ultrastructure, FH catabolic activity, invasiveness, and optimal glucose requirements for in vitro growth. UOK 262 cells have an isochromosome 1q recurring chromosome abnormality, i(1)(q10), and exhibit compromised oxidative phosphorylation and in vitro dependence on anaerobic glycolysis consistent with the clinical manifestation of HLRCC. The cells also display glucose-dependent growth, an elevated rate of lactate efflux, and overexpression of the glucose transporter GLUT1 and of lactate dehydrogenase A (LDHA). Mutant FH protein was present primarily in edematous mitochondria, but with catalytic activity nearly undetectable. UOK 262 xenografts retain the characteristics of HLRCC histopathology. Our findings indicate that the severe compromise of oxidative phosphorylation and rapid glycolytic flux in UOK 262 are an essential feature of this TCA cycle enzyme-deficient form of kidney cancer. This tumor model is the embodiment of the Warburg effect. UOK 262 provides a unique in vitro and in vivo preclinical model for studying the bioenergetics of the Warburg effect in human cancer.

A sequence-based survey of the complex structural organization of tumor genomes.

BACKGROUND: The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using end sequencing profiling, which relies on paired-end sequencing of cloned tumor genomes. RESULTS: In the present study brain, breast, ovary, and prostate tumors, along with three breast cancer cell lines, were surveyed using end sequencing profiling, yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization confirmed translocations and complex tumor genome structures that include co-amplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms revealed candidate somatic mutations and an elevated rate of novel single nucleotide polymorphisms in an ovarian tumor. CONCLUSION: These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than was previously appreciated and that genomic fusions, including fusion transcripts and proteins, may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.

Spectral karyotyping demonstrates genetically unstable skin-homing T lymphocytes in cutaneous T-cell lymphoma.

We initially established cell lines from skin biopsies from four patients (MF8, MF18, MF19 and MF31) in early stages of cutaneous T-cell lymphoma (CTCL) in 1999. After 3 weeks of culture, skin-homing T lymphocytes were stimulated with phytohaemagglutinin. Metaphase spreads were analysed using spectral karyotyping (SKY), a molecular cytogenetic technique. MF18 and MF19 had predominantly normal karyotypes. MF8 had recurrent numerical aberrations resulting in two T lymphocyte clones: one with trisomy 21 (12/20 cells) and the other with monosomy chromosome 22 (3/20 cells). MF8 also exhibited a clonal deletion, del(5)(p15.1), as well as multiple non-clonal structural aberrations. MF31 had a clonal deletion, del(17)(p12) and other non-clonal deletions involving chromosomes 2, 5, 10, 11. MF18 had a single abnormal cell that contained two reciprocal translocations t(1;2)(q32;p21) and t(4;10)(p15.2;q24). In 2001, three of the original patients had new skin biopsies taken and cell lines were established. SKY analysis revealed the continued presence of a T-cell clone in MF8 with trisomy 21 (4/20 cells). Additionally, a new clone was seen with a del(18)(p11.2) (17/20 cells). MF31 had only one aberrant cell with a del(17)(p12). MF18 had a clonal deletion, [del(1)(p36.1) in 3/20 cells] and non-clonal aberrations involving chromosomes 3, 4, 5, 6, 12, 13, 17 and 18. Thus, three of four patients continued to show numerous numerical and structural aberrations, both clonal and non-clonal, with only MF8 having a recurring T lymphocyte clone (+21). Our findings demonstrate high genetic instability among skin-homing T lymphocytes even in early stages of CTCL. We did not see genetic instability or evidence of clones in cell lines from a patient with atopic dermatitis and one with psoriasis.

Spectral karyotyping analysis of human and mouse chromosomes.

Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.

Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation.

Despite recent emerging evidence suggesting that cancer stem cells subsist in a variety of tumors, it is not yet fully elucidated whether postnatal stem cells are directly involved in tumorigenesis. We used murine bone marrow-derived mesenchymal stem cells (BMMSCs) as a model to test a hypothesis that tumorigenesis may originate from spontaneous mutation of stem cells. In this study, we demonstrated that murine BMMSCs, after numerous passages, obtained unlimited population doublings and proceeded to a malignant transformation state, resulting in fibrosarcoma formation in vivo. Transformed BMMSCs colonized to multiple organs when delivered systemically through the tail vein. Fibrosarcoma cells formed by transformed BMMSCs contained cancer progenitors, which were capable of generating colony clusters in vitro and fibrosarcoma in vivo by the second administration. The mechanism by which BMMSCs transformed to malignant cells was associated with accumulated chromosomal abnormalities, gradual elevation in telomerase activity, and increased c-myc expression. Moreover, BMMSCs and their transformed counterpart, fibrosarcoma-forming cells, demonstrated different sensitivity to anti-cancer drugs. BMMSCs/fibrosarcoma transformation system may provide an ideal system to elucidate the mechanism of how stem cells become cancer cells and to screen anti-sarcoma drugs.