Biomedical subjects

Thomas Ried

Publications and source records attributed to Thomas Ried.

At least 19 recordsLinked to original sources

PubMed2007

Gene expression profiling reveals a massive, aneuploidy-dependent transcriptional deregulation and distinct differences between lymph node-negative and lymph node-positive colon carcinomas.

To characterize patterns of global transcriptional deregulation in primary colon carcinomas, we did gene expression profiling of 73 tumors [Unio Internationale Contra Cancrum stage II (n = 33) and stage III (n = 40)] using oligonucleotide microarrays. For 30 of the tumors, expression profiles were compared with those from matched normal mucosa samples. We identified a set of 1,950 genes with highly significant deregulation between tumors and mucosa samples (P < 1e-7). A significant proportion of these genes mapped to chromosome 20 (P = 0.01). Seventeen genes had a >5-fold average expression difference between normal colon mucosa and carcinomas, including up-regulation of MYC and of HMGA1, a putative oncogene. Furthermore, we identified 68 genes that were significantly differentially expressed between lymph node-negative and lymph node-positive tumors (P < 0.001), the functional annotation of which revealed a preponderance of genes that play a role in cellular immune response and surveillance. The microarray-derived gene expression levels of 20 deregulated genes were validated using quantitative real-time reverse transcription-PCR in >40 tumor and normal mucosa samples with good concordance between the techniques. Finally, we established a relationship between specific genomic imbalances, which were mapped for 32 of the analyzed colon tumors by comparative genomic hybridization, and alterations of global transcriptional activity. Previously, we had conducted a similar analysis of primary rectal carcinomas. The systematic comparison of colon and rectal carcinomas revealed a significant overlap of genomic imbalances and transcriptional deregulation, including activation of the Wnt/beta-catenin signaling cascade, suggesting similar pathogenic pathways.

Adenocarcinoma↗

PubMed2007

Stage-specific alterations of the genome, transcriptome, and proteome during colorectal carcinogenesis.

To identify sequential alterations of the genome, transcriptome, and proteome during colorectal cancer progression, we have analyzed tissue samples from 36 patients, including the complete mucosa-adenoma-carcinoma sequence from 8 patients. Comparative genomic hybridization (CGH) revealed patterns of stage specific, recurrent genomic imbalances. Gene expression analysis on 9K cDNA arrays identified 58 genes differentially expressed between normal mucosa and adenoma, 116 genes between adenoma and carcinoma, and 158 genes between primary carcinoma and liver metastasis (P < 0.001). Parallel analysis of our samples by CGH and expression profiling revealed a direct correlation of chromosomal copy number changes with chromosome-specific average gene expression levels. Protein expression was analyzed by two-dimensional gel electrophoresis and subsequent mass spectrometry. Although there was no direct match of differentially expressed proteins and genes, the majority of them belonged to identical pathways or networks. In conclusion, increasing genomic instability and a recurrent pattern of chromosomal imbalances as well as specific gene and protein expression changes correlate with distinct stages of colorectal cancer progression. Chromosomal aneuploidies directly affect average resident gene expression levels, thereby contributing to a massive deregulation of the cellular transcriptome. The identification of novel genes and proteins might deliver molecular targets for diagnostic and therapeutic interventions.

Adult↗

PubMed2007

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.

Adult↗

PubMed2006

Combined breast ductal lavage and ductal endoscopy for the evaluation of the high-risk breast: a feasibility study.

BACKGROUND AND OBJECTIVES: Evaluation of the ductal epithelium of the breast at increased risk for breast cancer is needed to define the carcinogenic pathway, for risk assessment, and to improve selection of women for chemoprevention therapy. We studied the feasibility of combining breast ductal endoscopy with ductal lavage in the high-risk contralateral breast of women with ipsilateral breast cancer for the evaluation of high-risk ducts and acquisition of ductal epithelial cells for analysis. METHODS: Breast ducts were studied by ductal lavage and ductal endoscopy, and epithelial cell content studied cytologically and quantitatively. RESULTS: Twenty-five subjects and 44 ducts, including 22 (50.0%) which did not produce nipple aspirate fluid (NAF), were studied. Cellular atypia was present in five subjects. Ductal endoscopy was performed on 1 or more ducts in 24 subjects. Structural changes were noted in 63.6% of the ducts, most commonly fibrous stranding or bridging. Ductal sampling with endoscopic brush and coil sampling devices provided additional cellular samples of relatively pure ductal epithelial content (> or = 91% purity) in 8/11 subjects. CONCLUSIONS: Breast ductal endoscopy combined with ductal lavage represents a feasible approach for characterizing the ducts and ductal epithelium of the high-risk breast, especially in a research setting.

Adult↗

PubMed2006

Comparative genome hybridization reveals specific genomic imbalances during the genesis from benign through borderline to malignant ovarian tumors.

Ovarian cancer is one of the most common types of malignancy in women throughout the developed world. Despite recent therapeutic advances, long-term survival is poor because ovarian cancer is largely asymptomatic in its early stages. Comparative genomic hybridization (CGH) was applied to a series of 8 benign, 8 borderline, and 17 malignant ovarian to establish genomic imbalances associated with tumor progression. Benign and borderline tumors were characterized by losses at 1p32 approximately p11, 2q14 approximately q34, 4q13 approximately q34, 5q11 approximately q23, and 6q12 approximately q24, as well as gains of 6p and chromosome 12. Similar chromosomal changes were also detected in malignant tumors but included additional chromosomal changes: gains at 1q21 approximately q31, 2p, 3q, 5p, 7, 10p, 12p, 16p, 17, 19q, 20q, and 22q, as well as losses at X, 3p, 8p, 9, 11p, 13, 14, and 18. Some individual cases of benign and borderline tumors revealed no genetic alterations detectable by CGH, suggesting that these tumors may represent a subset of tumors that originate by an alternative mechanism of tumorigenesis. Furthermore, our findings reveal that borderline tumors are more similar to benign tumors than to malignant tumors with respect to their genetic profiles.

Chromosome Aberrations↗

PubMed2006

Tumor formation via loss of a molecular motor protein.

Aneuploidy has long been suggested to be causal in tumor formation. Direct testing of this hypothesis has been difficult because of the absence of methods to specifically induce aneuploidy. The chromosome-associated kinesin motor KIF4 plays multiple roles in mitosis, and its loss leads to multiple mitotic defects including aneuploidy. Here, we have taken advantage of the direct formation of aneuploidy in the absence of KIF4 to determine whether loss of a molecular motor and generation of aneuploidy during mitosis can trigger tumorigenesis. We find that embryonic stem cells genetically depleted of KIF4 support anchorage-independent growth and form tumors in nude mice. In cells lacking KIF4, mitotic spindle checkpoints and DNA-damage response pathways are activated. Down regulation or loss of KIF4 is physiologically relevant because reduced KIF4 levels are present in 35% of human cancers from several tissues. Our results support the notion that loss of a molecular motor leads to tumor formation and that aneuploidy can act as a primary trigger of tumorigenesis.

Aneuploidy↗

PubMed2006

Linking the human cytogenetic map with nucleotide sequence: the CCAP clone set.

We present the completed dataset and clone repository of the Cancer Chromosome Aberration Project (CCAP), an initiative developed and funded through the intramural program of the U.S. National Cancer Institute, to provide seamless linkage of human cytogenetic markers with the primary nucleotide sequence of the human genome. Spaced at 1-2 Mb intervals across the human genome, 1,339 bacterial artificial chromosome (BAC) clones have been localized to chromosomal bands through high-resolution fluorescence in situ hybridization (FISH) mapping. Of these clones, 99.8% can be positioned on the primary human genome sequence and 95% are placed at or close to their precise nucleotide starts and stops. This dataset can be studied and manipulated within generally available public Web sites. The clones are available from a commercial repository. The CCAP BAC clone set provides anchors for the interrogation of gene and sequence involvement in oncogenic and developmental disorders when the starting point is the recognition of a structural, numerical, or interstitial chromosomal aberration. This dataset also provides a current view of the quality and coherence of the available genome sequence and insight into the nucleotide and three-dimensional structures that manifest as Giemsa light and dark chromosomal banding patterns.

Base Composition↗

PubMed2006

SMC1 inhibition results in FRA3B expression but has no effect on its delayed replication.

Cellular processes involved in fragile site expression have been investigated by studying the effect on the replication pattern of the commonest fragile site FRA3B of RNA interference (RNAi)-mediated sister maintenance chromosome 1 (SMC1) inhibition in normal human fibroblasts. Replication timing of FRA3B in G2 was studied by bromodeoxyuridine (BrdU) labeling for the final 2h of cell culture whereas in the S phase was investigated by a fluorescence in situ hybridization (FISH)-based approach through the analysis of clones spanning the FRA3B region. Results showed that FRA3B is normally late replicated even though it is not expressed in untreated cells. On the other hand, SMC1 inhibition leads to FRA3B expression even if the percent of late replicated cells is comparable to control cells. These results obtained by analysing the commonest fragile site suggest that SMC1 plays a role in protecting late replicating regions from stresses occurring in the final steps of genome replication and that delayed replication is necessary but not sufficient for inducing fragile site expression.

Acid Anhydride Hydrolases↗

PubMed2006

Complex rearrangements involving der(8)t(8;20) and der(14)t(8;14)t(11;14), CCND1, and duplication of IgH constant region in acute plasmablastic leukemia.

We report on a rapidly fatal case of acute plasmablastic leukemia in a 72-year-old male from The Ukraine, who was 70 km away from Chernobyl at the time of the atomic accident in 1986. Spectral karyotyping and fluorescence in situ hybridization (FISH) studies of a bone marrow sample obtained at diagnosis revealed a hypodiploid karyotype with 45 chromosomes and two novel complex rearrangements, der(8)t(8;20)(p11.2;p?12) and der(14)t(8;14)(p?;p11.2)t(11;14)(q13;q32), with juxtaposition of CH (constant region of IgH) sequences to the oncogene CCND1 (translocated to 14q32). FISH analysis demonstrated that the CH on the der(14) was duplicated.

Aged↗

PubMed2006

Aneuploidy-dependent massive deregulation of the cellular transcriptome and apparent divergence of the Wnt/beta-catenin signaling pathway in human rectal carcinomas.

To identify genetic alterations underlying rectal carcinogenesis, we used global gene expression profiling of a series of 17 locally advanced rectal adenocarcinomas and 20 normal rectal mucosa biopsies on oligonucleotide arrays. A total of 351 genes were differentially expressed (P < 1.0e-7) between normal rectal mucosa and rectal carcinomas, 77 genes had a >5-fold difference, and 85 genes always had at least a 2-fold change in all of the matched samples. Twelve genes satisfied all three of these criteria. Altered expression of genes such as PTGS2 (COX-2), WNT1, TGFB1, VEGF, and MYC was confirmed, whereas our data for other genes, like PPARD and LEF1, were inconsistent with previous reports. In addition, we found deregulated expression of many genes whose involvement in rectal carcinogenesis has not been reported. By mapping the genomic imbalances in the tumors using comparative genomic hybridization, we could show that DNA copy number gains of recurrently aneuploid chromosome arms 7p, 8q, 13q, 18q, 20p, and 20q correlated significantly with their average chromosome arm expression profile. Taken together, our results show that both the high-level, significant transcriptional deregulation of specific genes and general modification of the average transcriptional activity of genes residing on aneuploid chromosomes coexist in rectal adenocarcinomas.

Adenocarcinoma↗

PubMed2006

From genome to proteome in tumor profiling: molecular events in colorectal cancer genesis.

Biomedical research has advanced rapidly in recent years with the sequencing of the human genome and the availability of technologies such as global gene and protein expression profiling using different chip platforms. However, this progress has not yet been transferred to the bedside. While detection of cancer at early stages is critical for curative treatment interventions, efficient diagnostic and therapeutic markers for the majority of malignancies still seem to be lacking. Comprehensive tumor profiling has therefore become a field of intensive research aiming at identifying biomarkers relevant for improved diagnostics and therapeutics. This chapter will demonstrate a genomic and proteomic approach while focusing on tumor profiling during colorectal cancer development.

Animals↗

PubMed2006

Increased serum levels of complement C3a anaphylatoxin indicate the presence of colorectal tumors.

BACKGROUND & AIMS: Late diagnosis of colorectal carcinoma results in a significant reduction of average survival times. Yet despite screening programs, about 70% of tumors are detected at advanced stages (International Union Against Cancer stages III/IV). We explored whether detection of malignant disease would be possible through identification of tumor-specific protein biomarkers in serum samples. METHODS: A discovery set of sera from patients with colorectal malignancy (n = 58) and healthy control individuals (n = 32) were screened for potential differences using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Candidate proteins were identified and their expression levels were validated in independent sample sets using a specific immunoassay (enzyme-linked immunosorbent assay). RESULTS: By using class comparison and custom-developed algorithms we identified several m/z values that were expressed differentially between the malignant samples and the healthy controls of the discovery set. Characterization of the most prominent m/z values revealed a member of the complement system, the stable form of C3a anaphylatoxin (ie, C3a-desArg). Based on a specific enzyme-linked immunosorbent assay, serum levels of complement C3a-desArg predicted the presence of colorectal malignancy in a blinded validation set (n = 59) with a sensitivity of 96.8% and a specificity of 96.2%. Increased serum levels were also detected in 86.1% of independently collected sera from patients with colorectal adenomas (n = 36), whereas only 5.6% were classified as normal. CONCLUSIONS: Complement C3a-desArg is present at significantly higher levels in serum from patients with colorectal adenomas (P < .0001) and carcinomas (P < .0001) than in healthy individuals. This suggests that quantification of C3a-desArg levels could ameliorate existing screening tests for colorectal cancer.

Adenoma↗

PubMed2006

Molecular cytogenetics: genomic imbalances in colorectal cancer and their clinical impact.

Chromosomal aberrations play a dominant role in colorectal carcinogenesis. The application of fluorescence in situ hybridization (FISH) based techniques such as comparative genomic hybridization (CGH) and spectral karyotyping (SKY) revealed that colorectal carcinomas are characterized by a specific pattern of chromosomal imbalances which sequentially accumulate during cancer progression. This review aims to summarize molecular cytogenetic studies, provides a background on the functional relevance of chromosomal aberrations for colorectal cancer progression and discusses their potential clinical impact.

Chromosome Aberrations↗

PubMed2005

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.

Animals↗

PubMed2005

Molecular and cytological features of the mouse B-cell lymphoma line iMycEmu-1.

BACKGROUND: Myc-induced lymphoblastic B-cell lymphoma (LBL) in iMycEmu mice may provide a model system for the study of the mechanism by which human MYC facilitates the initiation and progression of B cell and plasma cell neoplasms in human beings. We have recently shown that gene-targeted iMycEmu mice that carry a His6-tagged mouse Myc cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Emu, are prone to B cell and plasma cell tumors. The predominant tumor (approximately 50%) that arose in the iMycEmu mice on the mixed genetic background of segregating C57BL/6 and 129/SvJ alleles was LBL. The purpose of this study was to establish and characterize a cell line, designated iMycEmu-1, for the in-depth evaluation of LBL in vitro. METHODS: The morphological features and the surface marker expression profile of the iMycEmu-1 cells were evaluated using cytological methods and FACS, respectively. The cytogenetic make-up of the iMycEmu-1 cells was assessed by spectral karyotyping (SKY). The expression of the inserted MycHis gene was determined using RT-PCR and qPCR. Clonotypic immunoglobulin gene arrangements were detected by Southern blotting. The global gene expression program of the iMycEmu-1 cells and the expression of 768 "pathway" genes were determined with the help of the Mouse Lymphochip(c) and Superarray(c) cDNA micro- and macroarrays, respectively. Array results were verified, in part, by RT-PCR and qPCR. RESULTS: Consistent with their derivation from LBL, the iMycEmu-1 cells were found to be neoplastic IgMhighIgDlow lymphoblasts that expressed typical B-cell surface markers including CD40, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). The iMycEmu-1 cells harbored a reciprocal T(9;11) and three non-reciprocal chromosomal translocations, over-expressed MycHis at the expense of normal Myc, and exhibited gene expression changes on Mouse Lymphochip microarrays that were consistent with MycHis-driven B-cell neoplasia. Upon comparison to normal B cells using eight different Superarray cDNA macroarrays, the iMycEmu-1 cells showed the highest number of changes on the NFkappaB array. CONCLUSION: The iMycEmu-1 cells may provide a uniquely useful model system to study the growth and survival requirements of Myc-driven mouse LBL in vitro.

Animals↗

PubMed2005

Genetic and epigenetic modeling of the origins of multidrug-resistant cells in a human sarcoma cell line.

The origin of drug-resistant cells in human cancers has been a fundamental problem of cancer pharmacology. Two major contrasting hypotheses (genetics versus epigenetics) have been proposed to elucidate the mechanisms of acquired drug resistance. In this study, we answer these fundamental questions through investigation of the genetic and epigenetic pathways that control the origin of ABCB1 (MDR1) gene activation with acquired multidrug resistance in drug-sensitive human sarcoma (MES-SA cells). The genetic and epigenetic bases of this selected activation involve the initiation of transcription at a site 112 kb upstream of the ABCB1 proximal promoter (P1) in the drug-resistant cells. This activation was associated with a chromatin-remodeling process characterized by an increase in acetylated histone H3 within a 968-bp region 5' of the ABCB1 upstream promoter. These alterations provide both genetic and epigenetic susceptibility for ABCB1 expression in drug-resistant cells. Complete activation of the ABCB1 gene through the coding region was proposed by interactions of selected trans-alterations or epigenetic changes on the ABCB1 proximal promoter, which occurred during initial drug exposure. Thus, our data provide evidence for a major genomic alteration that changes the chromatin structure of the ABCB1 upstream promoter via acetylation of histone H3 initiating ABCB1 activation, further elucidating the genetic and epigenetic bases that determine chemotherapeutic response in drug-resistant derivatives of MES-SA cells.

Acetylation↗

PubMed2005

Atm heterozygosity cooperates with loss of Brca1 to increase the severity of mammary gland cancer and reduce ductal branching.

The role of homozygous ataxia telangiectasia mutated (ATM) mutations in familial and sporadic forms of cancer is well established, but the contribution of ATM heterozygosity to mammary gland and other cancers has been controversial. To test the effect of Atm heterozygosity on mammary gland cancer, mice with complete loss of exon 11 of Brca1 specifically in mammary epithelium (Brca1-MG-Deltaex11) were studied in either Atm heterozygous or Atm wild-type backgrounds. Targeted deletion of Brca1 in mammary epithelium resulted in carcinomas and adenocarcinomas of varying histology with long (>9 months) latency. Latency to tumorigenesis was found to be unchanged in the Brca1-MG-Deltaex11;Atm heterozygous mice compared with Brca1-MG-Deltaex11;Atm wild-type mice. However, the mice displayed variable tumor severity and differences in mammary tissue development. Mammary tumors from Brca1-MG-Deltaex11;Atm heterozygous mice were anaplastic and undifferentiated in all 20 tumors tested, whereas tumors from mice that were Brca1-MG-Deltaex11 but wild-type for Atm displayed variable histologic profiles, with some anaplastic tumors and other differentiated and less invasive tumor types. Previously reported developmental defects for Brca1-deficient mice were also observed in our model with and without Atm heterozygosity, but Brca1-MG-Deltaex11;Atm heterozygous mice displayed decreased ductal branching during puberty, a phenotype that was not observed in Brca1-MG-Deltaex11;Atm wild-type mice. Our results provide evidence that Atm heterozygosity influences severity of mammary gland tumors in the Brca1-MG-Deltaex11 tumor-prone mouse and suggest that this mutation leads to a newly characterized developmental defect during glandular maturation.

Animals↗

PubMed2005

Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoxantrone-selected SF295 human glioblastoma cells.

The human ABCG2 gene, located on chromosome 4, encodes an ATP-binding cassette half-transporter that has been shown to confer resistance to chemotherapeutic agents. Relatively little is known about the mechanisms controlling expression of ABCG2. In previous studies, we had shown that overexpression of ABCG2 can result from rearrangement or gene amplification involving chromosome 4. To better characterize the mechanisms of ABCG2 overexpression, SF295 glioblastoma cells were exposed to increasing amounts of mitoxantrone to generate the SF295 MX50, MX100, MX250, and MX500 sublines, maintained in mitoxantrone concentrations ranging from 50 to 500 nmol/L. Northern blot analysis confirmed overexpression of ABCG2 mRNA, and immunoblot analysis demonstrated increased protein expression in the selected cell lines. Efflux of BODIPY-prazosin confirmed a functional protein. ABCG2 gene amplification was observed in all resistant sublines, as determined by Southern blot analysis. Fluorescence in situ hybridization (FISH) revealed amplification of ABCG2 via double minute chromosomes (dmins) detected in metaphase chromosome spreads in the SF295 MX50 and MX100 sublines. At higher levels of drug selection, in the MX250 and MX500 sublines, fewer dmins were observed but homogeneously staining regions (hsr) were visible with FISH analysis, revealing reintegration of the ABCG2 gene into multiple chromosomes. Spectral karyotyping (SKY) demonstrated multiple clonal and nonclonal rearrangements of chromosome 4, including hsrs. These results suggest that amplification of ABCG2 occurred initially in the form of dmins, followed by chromosomal reintegration of the amplicon at multiple sites. This occurred with increasing drug-selection pressure, generating a more stable genotype.

ATP Binding Cassette Transporter, Subfamily G, Mem↗