Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

Joos, Stefan; Scherthan, Harry; Speicher, Michael R.; Schlegel, Jürgen; Cremer, Thomas; Lichter, Peter

doi:10.1007/bf00202475

Cited by 120 publications

(71 citation statements)

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“…In order to examine the physical relationship between the map position of the hTR locus and the region ampli®ed in these tumours, genomic DNA isolated from two of the tumours (one cervix and the lung sample), was used as complex in situ probes, and reverse in situ hybridisation back onto normal metaphase chromosomes carried out as a double hybridisation with di erentially labelled hTR probe (Hoare et al, 1977;Joos et al, 1993). Figure 1d (lung), and f (cervix) show typical intensity plots for uorescence along the pro®le of chromosome 3, generating a visual representation of the hybridisation site for hTR relative to the genomic sequences ampli®ed in the carcinomas.…”

Section: Ampli®cation Of the Htr Gene In Solid Tumoursmentioning

confidence: 99%

“…Reverse in situ hybridisation (REVISH) using genomic DNA as a probe (Hoare et al, 1997) Co-localisation of tumour amplicons with the position of the hTR locus on chromosome 3 was carried out by cohybridising labelled tumour DNA with the cloned hTR probe (Hoare et al, 1997;Joos et al, 1993). Analysis of digitised images was carried out using IPLab Spectrum software with SmartCapture extensions from Digital Scienti®c Ltd (Cambridge UK).…”

Section: Fish Analysismentioning

confidence: 99%

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Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer

et al. 1997

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Telomere length is maintained by the enzyme, telomerase, which has been linked to cellular immortality and tumour progression. However, the reasons for the high levels of telomerase found in human tumours are unknown. We have mapped the human telomerase RNA gene, (hTR), to chromosome 3q26.3 and show the hTR gene to be ampli®ed in four carcinomas, (2/33 cervix, 1/31 head and neck, 1/9 lung). In addition, increased copy numbers of the hTR locus was also observed in 97% of tumours. By in situ hybridisation, the histological distribution of high levels of hTR expression could be demonstrated in a lung tumour and its metastasis with hTR ampli®cation. These results are the ®rst report of genetic alterations involving a known component of telomerase in human cancer. Indeed, it is also the ®rst report of the ampli®cation of a speci®c locus within the chromosome 3q region frequently subject to copy number gains in human tumours. In addition, we also show for the ®rst time the histological distribution of the RNA component of telomerase in human tumours.

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Section: Ampli®cation Of the Htr Gene In Solid Tumoursmentioning

confidence: 99%

Section: Fish Analysismentioning

confidence: 99%

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer

et al. 1997

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“…It is apparent from the REVISH studies shown here, as well as published data from a number of others, that the overall picture of genetic changes detected by REVISH in any one sample is complex (Joos et al, 1993;Houldsworth and Chaganti, 1994;Kallioniemi et al, 1993;Van Ommen et al, 1995). It is, therefore, important when investigating acquired drug resistance that changes specifically associated with the drug-resistant test sample or cell line can be identified among the genetic alterations common to both the parental and resistant lines.…”

Section: Discussionmentioning

confidence: 72%

“…When analysing genetic changes for example, screening is limited to the use of gene-or region-specific probes. Recently, the molecular cytogenetic techniques of reverse in situ hybridization (REVISH) and its more advanced relative, comparative genomic hybridization (CGH), have been developed for the rapid global detection and mapping of genetic imbalances in tumour genomes (Kallioniemi et al, 1992(Kallioniemi et al, , 1993Joos et al, 1993;Houldsworth and Chaganti, 1994;Lichter et al, 1995;Mitelman, 1995;Van Ommen et al, 1995). In REVISH, genomic DNA from the tumour is used as a complex probe and hybridized to normal metaphase chromosomes (Mitelman, 1995).…”

mentioning

confidence: 99%

“…In REVISH, genomic DNA from the tumour is used as a complex probe and hybridized to normal metaphase chromosomes (Mitelman, 1995). Genomic sequences amplified in the tumour are then detected as an increased intensity of signal at the normal chromosomal position from which the amplified sequences are derived (Joos et al, 1993;Lichter et al, 1995). For more accurate analysis of both loss and gain of genetic material, CGH is then required.…”

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confidence: 99%

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Identification of genetic changes associated with drug resistance by reverse in situ hybridization

Hoare

Freeman²,

Coutts³

et al. 1997

Br J Cancer

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Summary The molecular cytogenetic techniques of comparative genomic hybridization (CGH) and reverse in situ hybridization (REVISH) allow the entire genomes of tumours to be screened for genetic changes without the requirement for specific probes or markers. In order to define the ability of REVISH to detect and map regions of amplification associated with drug resistance, we investigated a panel of cell lines selected for resistance to doxorubicin and intrinsic sensitivity to topoisomerase 11-inhibitory drugs. We have defined a modified REVISH protocol, which involves double hybridizations with genomic DNA from the test cell lines and chromosome-specific whole chromosome paints to identify the chromosomes to which the amplicons localize. Sites of amplification are then mapped by fractional length measurements (Flpter), using published genome databases. Our findings show that amplification of the topoisomerase lla gene is readily detected and mapped, as is amplification of the MDR and MRP loci. Interestingly, REVISH detected a new amplicon in the doxorubicin-resistant lung cancer cell line, GLC4-ADR, which mapped to chromosome 1 q. REVISH is therefore ideally suited to characterize genetic changes specific for drug resistance within a background of genetic anomalies associated with tumour progression.Keywords: molecular cytogenetics; fluorescence in situ hybridization; chromosome painting; drug resistance; gene amplification; gene mapping; reverse in situ hybridization Many tumours respond to a range of cytotoxic agents. However, resistance often develops (van der Zee et al, 1995;Harrison, 1995). Understanding the mechanisms of resistance may provide new therapeutic options (Kastan et al, 1995;FroelichAmmon and Osheroff, 1995). At the cellular level, a number of resistance mechanisms can potentially operate. These mechanisms include drug efflux via membrane pumps, such as p-glycoprotein or multidrug resistance protein (MRP), drug metabolism, including inactivation or failure to activate a prodrug, an alteration in abundance of the target protein, for example topoisomerase II (topoll) enzyme, mutation of target protein and inactivation of pathways leading to cell death, such as apoptotic signalling (Booser and Hortobagyi, 1994;Harrison, 1995;Kastan et al, 1995;van der Zee et al, 1995). It is likely that in any one particular tumour, response to therapy is dependent on concurrent expression of multiple mechanisms. Even for extensively studied drugs, such as etoposide, which is a known topoll inhibitor, resistance is a complex issue (Su et al, 1992;Takano et al, 1992;Booser and Hortobagyi, 1994;Chen and Liu, 1994;Pommier et al, 1994;Sinha, 1995). The recent association of reduced kinesin expression with etoposide resistance, as identified by a genetic suppressor element approach, highlights the usefulness and requirement for new approaches to define potential components of the drug resistance repertoire (Gudkov et al, 1994;Roninson et al, 1995).A major drawback to many of the conventional approaches used to investig...

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Comparative genomic hybridization as a tool in tumour cytogenetics

James

1999

J. Pathol.

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The quality of cytogenetic analysis of solid tumours has greatly improved in the past decade, but a number of technical difficulties remain which limit the characterization of solid tumour chromosomes by conventional cytogenetics alone. The identification of regions of chromosomal abnormality has been aided by the introduction of molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH). Of these, a recently developed approach, comparative genomic hybridization (CGH), has had a particular impact on the cytogenetic analysis of solid tumours. It incorporates the sensitivity of in situ techniques and overcomes many of the drawbacks of conventional cytogenetic analysis. This review first outlines the CGH method, giving details for the preparation of DNA probes and target human metaphase chromosomes together with information on the in situ technique and data handling criteria used in our laboratory. It then presents an overview of some of the current applications of CGH, together with a discussion of future directions in the field. Copyright © 1999 John Wiley & Sons, Ltd.

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Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

Cited by 120 publications

References 23 publications

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer

Identification of genetic changes associated with drug resistance by reverse in situ hybridization

Comparative genomic hybridization as a tool in tumour cytogenetics

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