Telomere capture stabilizes chromosome breakage.

Terminal deletions are found frequently in both malignancies and clinically recognizable deletion syndromes in man. Little is known, particularly in cancer, of the specific mechanisms which lead to the generation of deleted chromosomes or the process by which these broken chromosomes are stabilized. We demonstrate that several examples of apparent terminal deletions are, in fact, subtelomeric translocations which were not detectable using conventional cytogenetics. The unexpectedly high frequency of this phenomenon and the diversity of partner chromosomes involved in the subtelomeric translocations is consistent with a model in which telomere capture can stabilize chromosome breakage in man.

Identification of cryptic sites of DNA sequence amplification in human breast cancer by chromosome microdissection.

We have performed microdissection of 16 putative homogeneously staining regions (hsrs) from nine different breast cancer cell lines in order to determine their chromosomal origin and composition. As expected, the most commonly amplified chromosomal band-region was 17q12 (containing ERBB2). However, regions not containing known oncogenes were also identified, including 13q31 (5/9 cases) and 20q12-13.2 (4/9 cases). The chromosomal composition of the integrated amplified DNA within each hsr was determined and in 13/16 cases (81%), hsrs were shown to be composed of two or more chromosomal regions. These studies shed light on the mechanism of formation of hsrs, and identify chromosomal regions likely to harbour genes amplified in breast cancer.

Rapid generation of region specific probes by chromosome microdissection and their application.

The strategy presented here to identify unequivocally cryptic chromosomal rearrangements has relevance to both prenatal and postnatal cytogenetic analysis as well as the analysis of tumour-associated chromosome rearrangements. Microdissection and in vitro amplification of specific chromosomal regions are performed, followed by labelling for fluorescent in situ hybridization (FISH) to normal metaphase chromosomes (Micro-FISH). Micro-FISH probes have been used successfully to determine the derivation of chromosome segments unidentifiable by standard chromosome banding analysis. Micro-FISH probes (created in less than 24 hours) now make it possible to identify explicitly the chromosome constitution of virtually all cytologically visible chromosome rearrangements.

Use of a cDNA microarray to analyse gene expression patterns in human cancer.

The development and progression of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. Microarrays of cDNA provide a powerful tool for studying these complex phenomena. The tumorigenic properties of a human melanoma cell line, UACC-903, can be suppressed by introduction of a normal human chromosome 6, resulting in a reduction of growth rate, restoration of contact inhibition, and suppression of both soft agar clonogenicity and tumorigenicity in nude mice. We used a high density microarray of 1,161 DNA elements to search for differences in gene expression associated with tumour suppression in this system. Fluorescent probes for hybridization were derived from two sources of cellular mRNA [UACC-903 and UACC-903(+6)] which were labelled with different fluors to provide a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene. The fluorescence signals representing hybridization to each arrayed gene were analysed to determine the relative abundance in the two samples of mRNAs corresponding to each gene. Previously unrecognized alterations in the expression of specific genes provide leads for further investigation of the genetic basis of the tumorigenic phenotype of these cells.

Mutations in the human Jagged1 gene are responsible for Alagille syndrome.

Alagille syndrome (AGS) is an autosomal-dominant disorder characterized by intrahepatic cholestasis and abnormalities of heart, eye and vertebrae, as well as a characteristic facial appearance. Identification of rare AGS patients with cytogenetic deletions has allowed mapping of the gene of 20p12. We have generated a cloned contig of the critical region and used fluorescent in situ hybridization on cells from patients with submicroscopic deletions to narrow the candidate region to only 250 kb. Within this region we identified JAG1, the human homologue of rat Jagged1, which encodes a ligand for the Notch receptor. Cell-cell Jagged/Notch interactions are known to be critical for determination of cell fates in early development, making this an attractive candidate gene for a developmental disorder in humans. Determining the complete exon-intron structure of JAG1 allowed detailed mutational analysis of DNA samples from non-deletion AGS patients, revealing three frame-shift mutations, two splice donor mutations and one mutation abolishing RNA expression from the altered allele. We conclude that AGS is caused by haploinsufficiency of JAG1.

Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks.

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.

AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer.

Members of the recently recognized SRC-1 family of transcriptional coactivators interact with steroid hormone receptors to enhance ligand-dependent transcription. AIB1, a member of the SRC-1 family, was cloned during a search on the long arm of chromosome 20 for genes whose expression and copy number were elevated in human breast cancers. AIB1 amplification and overexpression were observed in four of five estrogen receptor-positive breast and ovarian cancer cell lines. Subsequent evaluation of 105 unselected specimens of primary breast cancer found AIB1 amplification in approximately 10 percent and high expression in 64 percent of the primary tumors analyzed. AIB1 protein interacted with estrogen receptors in a ligand-dependent fashion, and transfection of AIB1 resulted in enhancement of estrogen-dependent transcription. These observations identify AIB1 as a nuclear receptor coactivator whose altered expression may contribute to development of steroid-dependent cancers.

Interlobular and intralobular mammary stroma: genotype may not reflect phenotype.

BACKGROUND: The normal growth and function of mammary epithelial cells depend on interactions with the supportive stroma. Alterations in this communication can lead to the progression or expansion of malignant growth. The human mammary gland contains two distinctive types of fibroblasts within the stroma. The epithelial cells are surrounded by loosely connected intralobular fibroblasts, which are subsequently surrounded by the more compacted interlobular fibroblasts. The different proximity of these fibroblasts to the epithelial cells suggests distinctive functions for these two subtypes. In this report, we compared the gene expression profiles between the two stromal subtypes. METHODS: Fresh normal breast tissue was collected from reduction mammoplasty patients and immediately placed into embedding medium and frozen on dry ice. Tissue sections were subjected to laser capture microscopy to isolate the interlobular from the intralobular fibroblasts. RNA was prepared and subjected to microarray analysis using the Affymetrix Human Genome U133 GeneChip. Data was analyzed using the Affy and Limma packages available from Bioconductor. Findings from the microarray analysis were validated by RT-PCR and immunohistochemistry. RESULTS: No statistically significant difference was detected between the gene expression profiles of the interlobular and intralobular fibroblasts by microarray analysis and RT-PCR. However, for some of the genes tested, the protein expression patterns between the two subtypes of fibroblasts were significantly different. CONCLUSION: This study is the first to report the gene expression profiles of the two distinct fibroblast populations within the human mammary gland. While there was no significant difference in the gene expression profiles between the groups, there was an obvious difference in the expression pattern of several proteins tested. This report also highlights the importance of studying gene regulation at both the transcriptional and post-translational level.

Spotting the target: microarrays for disease gene discovery.

Microarray technologies enable genome-scale expression measurements. Already proved to be of value for the functional analysis of individual genes and biological processes, the application of expression profiling to disease gene discovery is now growing in importance and practicality.

HT1080/DR4: a P-glycoprotein-negative human fibrosarcoma cell line exhibiting resistance to topoisomerase II-reactive drugs despite the presence of a drug-sensitive topoisomerase II.

HT1080/DR4 (DR4) is a doxorubicin-resistant human fibrosarcoma line that exhibits 150-fold cross-resistance to etoposide but does not overexpress P-glycoprotein (one mechanism of multiple drug resistance). We examined another possible mechanism that could explain resistance to both doxorubicin and etoposide: a quantitative or qualitative alteration in topoisomerase II, the putative nuclear target of these agents. The amount of immunoreactive topoisomerase II present in whole-cell lysates and nuclear extracts was three- to 10-fold lower in DR4 than in HT1080 cells. However, the topoisomerase II in nuclear extracts from both lines was sensitive to the effects of amsacrine (AMSA) and etoposide. Following treatment with AMSA, etoposide, and 5-iminodaunorubicin, topoisomerase II-mediated DNA cleavage in DR4 cells and nuclei was reduced compared with cleavage in HT1080 parent cells and nuclei. The difference between the HT1080 and DR4 lines in AMSA- and 5-iminodaunorubicin-induced cleavage was similar in cells and nuclei and could be due to the lower amount of DR4 topoisomerase II. By contrast, the difference between the HT1080 and DR4 lines in etoposide-induced DNA cleavage was much greater in cells than in nuclei. This finding suggested that cytosolic factors, removed from isolated nuclei, could influence the susceptibility of intact cells to the cytotoxic and DNA-cleaving actions of etoposide. The specific activities of several antioxidant enzymes, components of the cell's defense against free-radical damage that may be produced by doxorubicin or etoposide, were significantly different in HT1080 and DR4 cytosolic extracts. These differences may constitute an additional mechanism of resistance. Regardless, the magnitude of the resistance of DR4 to doxorubicin and etoposide cannot be explained solely on the basis of a topoisomerase II-related mechanism.

Amplification of multiple genes from chromosomal region 12q13-14 in human malignant gliomas: preliminary mapping of the amplicons shows preferential involvement of CDK4, SAS, and MDM2.

We have investigated 234 tumors of the central nervous system for amplification of 9 different loci from 12q13-14 and report that about 15% of the anaplastic astrocytomas and glioblastomas show amplification at this chromosomal region. The genes most frequently amplified were CDK4 and SAS (18 of 19 cases). MDM2 was coamplified with CDK4 and SAS in 11 tumors while one glioblastoma showed only MDM2 amplification. Some amplicons additionally included GADD153 (9 cases), GLI (6 cases), A2MR (3 cases), and the anonymous locus D12S8 (2 cases). Either MDM2 or CDK4 and SAS showed the highest amplification level in each individual amplicon and amplification of these genes was consistently accompanied by strong overexpression. Our results thus suggest CDK4, SAS, and MDM2 as main targets for the amplification; however, the possibility exists that all amplicons share a common amplified region between MDM2 and CDK4/SAS which might contain one or more as yet unidentified genes.

Multidrug resistance in mitoxantrone-selected HL-60 leukemia cells in the absence of P-glycoprotein overexpression.

A multidrug-resistant variant of the human HL-60 promyelocytic leukemia cell line (HL-60/MX2) has been isolated in vitro by subculturing these cells in progressively increasing concentrations of mitoxantrone. The MX2 cells are cross-resistant to etoposide, teniposide, bisantrene, dactinomycin, 4'-(9-acridinylamino)methanesulfon-m-anisidide, and the anthracyclines daunorubicin and doxorubicin but retain sensitivity to the Vinca alkaloids melphalan and mitomycin C. In addition, the MX2 cells display slight collateral sensitivity to bleomycin. Despite being 30-35-fold less sensitive to mitoxantrone, net [14C]mitoxantrone accumulation at 60 min was reduced by only 10% in the mitoxantrone-resistant cells compared to the parental line. Furthermore, at later time points, e.g., 120 and 180 min, mitoxantrone accumulation in the MX2 cells exceeded that in HL-60 cells by 8.5 and 6.4%, respectively. No significant differences were observed between the sensitive and resistant cell lines in the initial (first 60 s) accumulation of mitoxantrone, and only minor (3-6%) enhancement of mitoxantrone efflux was detected in the resistant cell type. Monoclonal antibodies to P-glycoprotein had no detectable reactivity with membrane vesicles from either the sensitive or resistant cell types as determined by standard immunoblotting techniques. The mitoxantrone-resistant cells displayed a reciprocal translocation [rcpt(1;3)-(q21;p23)] not found in the sensitive parent, but there were no demonstrable double minute chromosomes or homogeneous staining regions in cells from either line. Thus, these mitoxantrone-resistant human leukemia cells display many features which are atypical for the "classic" multidrug resistance phenotype and should provide a useful model for the study of multidrug resistance which is not mediated by P-glycoprotein.

Hybrid selection of transcribed sequences from microdissected DNA: isolation of genes within amplified region at 20q11-q13.2 in breast cancer.

In human breast carcinomas, increased copy number of DNA sequences derived from the long arm of chromosome 20 (20q) has been commonly observed by both chromosome microdissection and comparative genomic hybridization. This chromosomal region is likely to contain one or more genes that are the biological targets of this amplification event. We describe here the utilization of a chromosome microdissection-hybrid selection strategy to isolate transcribed sequences from microdissected homogeneously staining regions encompassing 20q. Using this strategy, we have isolated three novel amplified genes (termed AIB1, AIB3, and AIB4) from a cDNA library constructed from the 20q amplified breast cancer cell line BT-474. These three genes were mapped to 20q11 (AIB3 and AIB4) and 20q12 (AIB1) by fluorescence in situ hybridization. Our results indicate an unsuspected complexity to the amplification pattern of 20q in breast cancer and provide probes that will be useful for further characterization of tumor specimens.

Microdissection based cloning of a translocation breakpoint in a human malignant melanoma.

Chromosome translocations in human malignancies have identified the genomic location of several important growth-regulatory sequences (e.g., cellular oncogenes and suppressor genes). Melanomas are characterized by recurring chromosome alterations, including deletion or translocations of the long arm of chromosome 6 (6q). This report details our efforts to clone the t(1;6)(q21;q14) breakpoint in a malignant melanoma to further our understanding of the biology of these tumors. The strategy utilized combined microdissection of the translocation chromosome, development and characterization of a DNA microclone library, isolation of cosmids and YACs from the breakpoint region, ordering of clones by two-color metaphase/interphase fluorescence in situ hybridization, and finally, identification of a YAC spanning the translocation breakpoint. By analogy to other tumor systems, molecular examination of the chromosome 6 breakpoint may provide insight into the pathobiology of this important neoplasm.

Refined mapping of 12q13-q15 amplicons in human malignant gliomas suggests CDK4/SAS and MDM2 as independent amplification targets.

We have reported previously that about 15% of anaplastic astrocytomas and glioblastomas show amplification and overexpression of one or more genes from chromosomal segment 12q13-q15 (G. Reifenberger et al., Cancer Res., 54, 4299-4303, 1994). The genes most frequently amplified and overexpressed were CDK4 (with coamplification of SAS) and MDM2. Because individual malignant gliomas showed CDK4/SAS amplification but no MDM2 amplification and vice versa, the possibility remained of a common amplification target gene located between CDK4 and MDM2. We have addressed this question by performing a detailed amplicon mapping of a series of 24 primary malignant gliomas and two glioblastoma cell lines with 12q13-q15 amplification. All tumors and cell lines were analyzed at eight gene loci and six anonymous loci from 12q13-q15, including seven loci located between CDK4 and MDM2. These studies revealed two centers of amplification, one at CDK4/SAS and the other at MDM2. A number of loci located close to either MDM2 or CDK4/SAS, including the genes GADD153, GLI, RAP1B, A2MR, and IFNG, were found to be coamplified in some tumors but not overexpressed consistently. All amplicons were discontinuous between CDK4/SAS and MDM2. Our results thus exclude a common amplification target between CDK4/SAS and MDM2 and provide additional evidence that these genes represent two independent targets of selection.

SAS amplification in soft tissue sarcomas.

Gene amplification is an important mechanism of increased gene expression in a number of human solid tumors. We have recently identified and cloned sequences from a novel DNA amplification unit in malignant fibrous histiocytoma. The amplified sequences are derived from chromosome 12q13-14 and encode a gene designated SAS (sarcoma amplified sequence). In the present study, a series of soft tissue sarcomas was studied to characterize further the phenomenon of SAS amplification. Seven of 22 (32%) malignant fibrous histiocytomas and three liposarcomas contained SAS amplification. Strikingly, all of the tumors with SAS amplification occurred in central sites (i.e., in the abdominal or inguinal regions) rather than in the extremities (i.e., in the arms of legs). These observations demonstrate that SAS amplification occurs with a significant frequency in mesenchymal tumors and is particularly associated with abdominal disease.

Construction of a representative cDNA library from prostatic intraepithelial neoplasia.

We report the construction of a plasmid-based cDNA library made from microdissected cells derived from prostatic intraepithelial neoplasia. Total RNA was extracted and converted to blunt-ended, double-stranded cDNA by oligo(dT)-mediated reverse transcription followed by linker addition. A linker-specific primer with UDG-compatible ends was used to amplify the cDNA and the resulting PCR product was subcloned. A total of 154 clones were sequenced and results indicated that 81.5% of the clones derived from either known genes, anonymous expressed sequence tags, or novel transcripts with very little redundancy of screened clones. These results demonstrate the feasibility of constructing complex representative cDNA libraries from specific microdissected cell populations that represent microscopic precursor stages of cancer progression. This method should facilitate identification of transcripts specifically expressed in cells of a distinct histological origin and tumorigenic stage.

Reversion of monochromosome-mediated suppression of tumorigenicity in malignant melanoma by retroviral transduction.

We have developed a general strategy to reverse monochromosome suppression of the malignant phenotypes by retroviral transduction. Our approach involved the introduction of a retroviral expression vector-carried cDNA library into a chromosome 6-suppressed melanoma subline UACC-903(+6) [J. M. Trent et al., Science (Washington DC), 247: 568-571, 1990]. The cDNA library was constructed from polyadenylated RNA isolated from the suppressed UACC-903(+6) cells, packaged into high-titer amphotropic retrovirus particles, and transduced into UACC-903(+6) cells. Revertant his(R) transductants were selected by isolating colony-forming cells in soft agar. A total of 121 large (> 150 microm) colonies was picked from soft agar culture with 18 of 121 (15%) established as permanent sublines. The revertant sublines demonstrated 7-58% cloning efficiency upon plating in agar, in contrast to <0.05% for the UACC-903(+6) subline. All 18 revertant sublines, termed SRS1-SRS18 (for "selection of revertants for suppression"), displayed a reduced population-doubling time, with 9 of 18 showing focus formation in monolayer similar to the parental (nonsuppressed) cell line. Preliminary evidence for reversion of the suppressed phenotype by injection of cells into athymic nude mice has been completed for one revertant subline. Southern analysis has demonstrated integration of the retroviral vector sequence in all 18 sublines. This approach should facilitate the identification of genes involved in the tumorigenic phenotype of malignant melanoma, and is readily adaptable to other model systems.

Gene expression profiling of alveolar rhabdomyosarcoma with cDNA microarrays.

Several forms of human sarcoma, lymphoma, and leukemia are characterized by somatically acquired chromosome translocations that result in fusion genes that encode chimeric transcription factors with oncogenic properties. We have used cDNA microarrays containing 1238 cDNAs to investigate the gene expression profile of a group of seven alveolar rhabdomyosarcoma (ARMS) cell lines characterized by the presence of the PAX3-FKHR fusion gene. Using the method of multidimensional scaling to represent the relationships among the cell lines in two-dimensional Euclidean space, we determined that ARMS cells show a consistent pattern of gene expression, which allows the cells to be clustered together. By searching across the seven ARMS cell lines, we found that 37 of 1238 genes were most consistently expressed in ARMS relative to a reference cell line. Only three of these genes have been previously reported to be expressed in ARMS. Among these 37 were genes related to both primary (PAX3-FKHR) and secondary (CDK4) genetic alterations in ARMS. These results in ARMS demonstrate the potential of cDNA microarray technology to elucidate tumor-specific gene expression profiles in human cancers.