Cancer driver-passenger distinction via sporadic human and dog cancer comparison: a proof-of-principle study with colorectal cancer.

Herein we report a proof-of-principle study illustrating a novel dog-human comparison strategy that addresses a central aim of cancer research, namely cancer driver-passenger distinction. We previously demonstrated that sporadic canine colorectal cancers (CRCs) share similar molecular pathogenesis mechanisms as their human counterparts. In this study, we compared the genome-wide copy number abnormalities between 29 human and 10 canine sporadic CRCs. This led to the identification of 73 driver candidate genes (DCGs), altered in both species, and with 27 from the whole genome and 46 from dog-human genomic rearrangement breakpoint (GRB) regions, as well as 38 passenger candidate genes (PCGs), altered in humans only and located in GRB regions. We noted that DCGs significantly differ from PCGs in every analysis conducted to assess their cancer relevance and biological functions. Importantly, although PCGs are not enriched in any specific functions, DCGs possess significantly enhanced functionality closely associated with cell proliferation and death regulation, as well as with epithelial cell apicobasal polarity establishment/maintenance. These observations support the notion that, in sporadic CRCs of both species, cell polarity genes not only contribute in preventing cancer cell invasion and spreading, but also likely serve as tumor suppressors by modulating cell growth. This pilot study validates our novel strategy and has uncovered four new potential cell polarity and colorectal tumor suppressor genes (RASA3, NUPL1, DENND5A and AVL9). Expansion of this study would make more driver-passenger distinctions for cancers with large genomic amplifications or deletions, and address key questions regarding the relationship between cancer pathogenesis and epithelial cell polarity control in mammals.

MicroRNA-24 regulates XIAP to reduce the apoptosis threshold in cancer cells.

MicroRNAs have been implicated as important mediators of cancer cell homeostasis, and accumulating data suggest compelling roles for them in the apoptosis pathway. X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor and an important barrier to apoptotic cell death, but the mechanisms that determine the diverse range of XIAP expression seen in cancer remains unclear. In this study, we present evidence that miR-24 directly targets the 3'UTR of the XIAP messenger RNA (mRNA) to exert translational repression. Using a heuristic algorithm of bioinformatics analysis and in vitro screening, we identified miR-24 as a candidate regulator of XIAP expression. Array comparative genomic hybridization and spectral karyotype analysis reveal that genomic copy number loss at the miR-24 locus is concordant with the loss of endogenous miR-24 in cancer cells. Using a luciferase construct of the XIAP 3'UTR, we showed that miR-24 specifically coordinates to the XIAP mRNA. Interference with miR-24's binding of the critical seed region, resulting from site-directed mutagenesis of the 3'UTR, significantly abrogated miR-24's effects on XIAP expression. Moreover, miR-24 overexpression can overcome apoptosis resistance in cancer cells via downregulation of XIAP expression, and the resulting cancer cell death induced by tumor necrosis factor-related apoptosis-inducing ligand is executed by the canonical caspase-mediated apoptosis pathway. In summary, our data suggest a novel mechanism by which miR-24 directly modulates XIAP expression level and consequently the apoptosis threshold in cancer cells.

Short telomeres result in chromosomal instability in hematopoietic cells and precede malignant evolution in human aplastic anemia.

In cell and animal models, telomere erosion promotes chromosomal instability via breakage-fusion-bridge cycles, contributing to the early stages of tumorigenesis. However, evidence involving short telomeres in cancer development in humans is scarce, epidemiological and indirect. Here we directly implicate telomere shortening as a critical molecular event for malignant evolution in aplastic anemia (AA). Patients' telomere lengths at diagnosis of AA, while comparable to age-matched controls, inversely correlated with the probability of developing a cytogenetically abnormal clone. A significantly increased number of telomere signal-free chromosomal ends and chromosomal numerical and structural abnormalities were observed in bone marrow cells of patients with shorter telomeres in comparison with patients with longer telomeres and healthy subjects. The proportion of monosomy-7 cells in the bone marrow at diagnosis of AA inversely correlated with telomere length, years before the emergence of an autonomous and clinically detectable abnormal clone. Marrow cells of clinically healthy individuals carrying loss-of-function telomerase mutations and with extremely short telomeres also showed chromosomal instability in vitro. These results provide the first clinical direct evidence in humans that short telomeres in hematopoietic cells are dysfunctional, mediate chromosomal instability and predispose to malignant transformation in a human disease.

CASZ1, a candidate tumor-suppressor gene, suppresses neuroblastoma tumor growth through reprogramming gene expression.

Neuroblastoma (NB) is a common childhood malignant tumor of the neural crest-derived sympathetic nervous system. In NB the frequent loss of heterozygosity (LOH) on chromosome 1p raises the possibility that this region contains tumor-suppressor genes whose inactivation contributes to tumorigenesis. The human homolog of the Drosophila neural fate determination gene CASZ1, a zinc-finger transcription factor, maps to chromosome 1p36.22, a region implicated in NB tumorigenesis. Quantitative real-time PCR analysis showed that low-CASZ1 expression is significantly correlated with increased age (≥18 months), Children's Oncology Group high-risk classification, 1p LOH and MYCN amplification (all P<0.0002) and decreased survival probability (P=0.0009). CASZ1 was more highly expressed in NB with a differentiated histopathology (P<0.0001). Retinoids and epigenetic modification agents associated with regulation of differentiation induced CASZ1 expression. Expression profiling analysis revealed that CASZ1 regulates the expression of genes involved in regulation of cell growth and developmental processes. Specific restoration of CASZ1 in NB cells induced cell differentiation, enhanced cell adhesion, inhibited migration and suppressed tumorigenicity. These data are consistent with CASZ1 being a critical modulator of neural cell development, and that somatically acquired disruption of normal CASZ1 expression contributes to the malignant phenotype of human NB.

Rb inactivation accelerates neoplastic growth and substitutes for recurrent amplification of cIAP1, cIAP2 and Yap1 in sporadic mammary carcinoma associated with p53 deficiency.

Genetically defined mouse models offer an important tool to identify critical secondary genetic alterations with relevance to human cancer pathogenesis. We used newly generated MMTV-Cre105Ayn mice to inactivate p53 and/or Rb strictly in the mammary epithelium, and to determine recurrent genomic changes associated with deficiencies of these genes. p53 inactivation led to formation of estrogen receptor-positive raloxifene-responsive mammary carcinomas with features of luminal subtype B. Rb deficiency was insufficient to initiate carcinogenesis but promoted genomic instability and growth rate of neoplasms associated with p53 inactivation. Genome-wide analysis of mammary carcinomas identified a recurrent amplification at chromosome band 9A1, a locus orthologous to human 11q22, which contains protooncogenes cIAP1 (Birc2), cIAP2 (Birc3) and Yap1. It is interesting that this amplicon was preferentially detected in carcinomas carrying wild-type Rb. However, all three genes were overexpressed in carcinomas with p53 and Rb inactivation, likely due to E2F-mediated transactivation, and cooperated in carcinogenesis according to gene knockdown experiments. These findings establish a model of luminal subtype B mammary carcinoma, identify critical role of cIAP1, cIAP2 and Yap1 co-expression in mammary carcinogenesis and provide an explanation for the lack of recurrent amplifications of cIAP1, cIAP2 and Yap1 in some tumors with frequent Rb deficiency, such as mammary carcinoma.

Genetic instability and mammary tumor formation in mice carrying mammary-specific disruption of Chk1 and p53.

Checkpoint kinase 1 (Chk1) is a key element in the DNA-damage response pathway that is required for maintaining genomic stability. To study the potential role of Chk1 in mammary tumorigenesis, we disrupted it using a Cre/loxP system. We showed that although Chk1 heterozygosity caused abnormal development of the mammary gland, it was not sufficient to induce tumorigenesis. Simultaneous deletion of one copy of p53 failed to rescue the developmental defects; however, it synergistically induced mammary tumor formation in Chk1(+/-);MMTV-Cre animals with a median time to tumor latency of about 10 months. Chk1 deficiency caused a preponderance of abnormalities, including prolongation, multipolarity, misalignment, mitotic catastrophe and loss of spindle checkpoint, that are accompanied by reduced expression of several cell cycle regulators, including Mad2. On the other hand, we also showed that Chk1 deficiency inhibited mammary tumor formation in mice carrying a homozygous deletion of p53, uncovering a complex relationship between Chk1 and p53. Furthermore, inhibition of Chk1 with a specific inhibitor, SB-218078, or acute deletion of Chk1 using small hairpin RNA killed mammary tumor cells effectively. These data show that Chk1 is critical for maintaining genome integrity and serves as a double-edged sword for cancer: although its inhibition kills cancer cells, it also triggers tumorigenesis when favorable mutations are accumulated for cell growth.

Genetic reporter system for oncogenic Igh-Myc translocations in mice.

The Myc-deregulating chromosomal T(12;15)(Igh-Myc) translocation, the hallmark mutation of inflammation- and interleukin 6-dependent mouse plasmacytoma (PCT), is the premier model of cancer-associated chromosomal translocations because it is the only translocation in mice that occurs spontaneously (B lymphocyte lineage) and with predictably high incidence (approximately 85% of PCT), and has a direct counterpart in humans: Burkitt lymphoma t(8;14)(q24;q32) translocation. Here, we report on the development of a genetic system for the detection of T(12;15)(Igh-Myc) translocations in plasma cells of a mouse strain in which an enhanced green fluorescent protein (GFP)-encoding reporter gene has been targeted to Myc. Four of the PCTs that developed in the newly generated translocation reporter mice, designated iGFP(5'Myc), expressed GFP consequent to naturally occurring T(12;15) translocation. GFP expression did not interfere with tumor development or the deregulation of Myc on derivative 12 of translocation, der (12), because the reporter gene was allocated to the reciprocal product of translocation, der (15). Although the described reporter gene approach requires refinement before T(12;15) translocations can be quantitatively detected in vivo, including in B lymphocyte lineage cells that have not yet completed malignant transformation, our findings provide proof of principle that reporter gene tagging of oncogenes in gene-targeted mice can be used to elucidate unresolved questions on the occurrence, distribution and trafficking of cells that have acquired cancer-causing chromosomal translocations of great relevance for humans.

BCR/ABL induces chromosomal instability after genotoxic stress and alters the cell death threshold.

Earlier reports have suggested that the BCR/ABL oncogene, associated with chronic myeloid leukemia, induces a mutator phenotype; however, it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However, although BCR/ABL-expressing cell lines have an equivalent cell survival, they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples, normal CD34+ progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells, but not normal progenitor cells, consistent with error-prone DNA repair. Taken together, these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair.

Ki-67 expression predicts locoregional recurrence in stage I oral tongue carcinoma.

Oral tongue squamous cell carcinoma (OTSCC) is an aggressive cancer associated with poor prognosis. Methods for determining the aggressiveness of OTSCC from analysis of the primary tumour specimen are thus highly desirable. We investigated whether genomic instability and proliferative activity (by means of Ki-67 activity) could be of clinical use for prediction of locoregional recurrence in 76 pretreatment OTSCC paraffin samples (stage I, n=22; stage II, n=33; stage III, n=8; stage IV, n=13). Eleven surgical tumour specimens were also analysed for remnants of proliferative activity after preoperative radiotherapy. Ninety-seven percent of cases (n=72) were characterised as being aneuploid as measured by means of image cytometry. Preoperative radiotherapy (50-68 Gy) resulted in significant reduction of proliferative activity in all patients for which post-treatment biopsies were available (P-value=0.001). Proliferative activity was not associated with response to radiation in stage II patients. However, we report a significant correlation between high proliferation rates and locoregional recurrences in stage I OTSCC patients (P-value=0.028). High-proliferative activity is thus related to an elevated risk of recurrence after surgery alone. We therefore conclude that Ki-67 expression level is a potentially useful clinical marker for predicting recurrence in surgically treated stage I OTSCC.

Undifferentiated pelvic adenocarcinomas: diagnostic potential of protein profiling and multivariate analysis.

BACKGROUND AND AIMS: Despite improved techniques, the determination of tumor origin in poorly differentiated adenocarcinomas still remains a challenge for the pathologist. Here we report the use of protein profiling combined with principal component analysis to improve diagnostic decision-making in tumor samples, in which standard pathologic investigations cannot present reliable results. MATERIALS AND METHODS: A poorly differentiated adenocarcinoma of unknown origin located in the pelvis, infiltrating the sigmoid colon as well as the ovary, served as a model to evaluate our proteomic approach. Firstly, we characterized the protein expression profiles from eight advanced colon and seven ovarian adenocarcinomas using two-dimensional gel electrophoresis (2-DE). Qualitative and quantitative patterns were recorded and compared to the tumor of unknown origin. Based on these protein profiles, match sets from the different tumors were created. Finally, a multivariate principal component analysis was applied to the entire 2-DE data to disclose differences in protein patterns between the different tumors. RESULTS: Over 89% of the unknown tumor sample spots could be matched with the colon standard gel, whereas only 63% of the spots could be matched with the ovarian standard. In addition, principal component analysis impressively displayed the clustering of the unknown case within the colon cancer samples, whereas this case did not cluster at all within the group of ovarian adenocarcinomas. CONCLUSION: These results show that 2-DE protein expression profiling combined with principal component analysis is a sensitive method for diagnosing undifferentiated adenocarcinomas of unknown origin. The described approach can contribute greatly to diagnostic decision-making and, with further technical improvements and a higher throughput, become a powerful tool in the armentarium of the pathologist.

Molecular cytogenetic characterization of pancreas cancer cell lines reveals high complexity chromosomal alterations.

Karyotype analysis can provide clues to significant genes involved in the genesis and growth of pancreas cancer. The genome of pancreas cancer is complex, and G-band analysis cannot resolve many of the karyotypic abnormalities seen. We studied the karyotypes of 15 recently established cell lines using molecular cytogenetic tools. Comparative genomic hybridization (CGH) analysis of all 15 lines identified genomic gains of 3q, 8q, 11q, 17q, and chromosome 20 in nine or more cell lines. CGH confirmed frequent loss of chromosome 18, 17p, 6q, and 8p. 14/15 cell lines demonstrated loss of chromosome 18q, either by loss of a copy of chromosome 18 (n = 5), all of 18q (n = 7) or portions of 18q (n = 2). Multicolor FISH (Spectral Karyotyping, or SKY) of 11 lines identified many complex structural chromosomal aberrations. 93 structurally abnormal chromosomes were evaluated, for which SKY added new information to 67. Several potentially site-specific recurrent rearrangements were observed. Chromosome region 18q11.2 was recurrently involved in nine cell lines, including formation of derivative chromosomes 18 from a t(18;22) (three cell lines), t(17;18) (two cell lines), and t(12;18), t(15;18), t(18;20), and ins(6;18) (one cell line each). To further define the breakpoints involved on chromosome 18, YACs from the 18q11.2 region, spanning approximately 8 Mb, were used to perform targeted FISH analyses of these lines. We found significant heterogeneity in the breakpoints despite their G-band similarity, including multiple independent regions of loss proximal to the already identified loss of DPC4 at 18q21.

Haploinsufficiency of Parp1 accelerates Brca1-associated centrosome amplification, telomere shortening, genetic instability, apoptosis, and embryonic lethality.

The breast tumor associated gene-1 (BRCA1) and poly(ADP-ribose) polymerase-1 (PARP1) are both involved in DNA-damage response and DNA-damage repair. Recent investigations have suggested that inhibition of PARP1 represents a promising chemopreventive/therapeutic approach for specifically treating BRCA1- and BRCA2-associated breast cancer. However, studies in mouse models reveal that Parp1-null mutation results in genetic instability and mammary tumor formation, casting significant doubt on the safety of PARP1 inhibition as a therapy for the breast cancer. To study the genetic interactions between Brca1 and Parp1, we interbred mice carrying a heterozygous deletion of full-length Brca1 (Brca1(+/Delta11)) with Parp1-null mice. We show that Brca1(Delta11/Delta11);Parp1(-/-) embryos die before embryonic (E) day 6.5, whereas Brca1(Delta11/Delta11) embryos die after E12.5, indicating that absence of Parp1 dramatically accelerates lethality caused by Brca1 deficiency. Surprisingly, haploinsufficiency of Parp1 in Brca1(Delta11/Delta11) embryos induces a severe chromosome aberrations, centrosome amplification, and telomere dysfunction, leading to apoptosis and accelerated embryonic lethality. Notably, telomere shortening in Brca1(Delta11/Delta11);Parp1(+/-) MEFs was correlated with decreased expression of Ku70, which plays an important role in telomere maintenance. Thus, haploid loss of Parp1 is sufficient to induce lethality of Brca1-deficient cells, suggesting that partial inhibition of PARP1 may represent a practical chemopreventive/therapeutic approach for BRCA1-associated breast cancer.

Frequent gain of the human telomerase gene TERC at 3q26 in cervical adenocarcinomas.

The level of genomic amplification of the human telomerase gene TERC, which maps to chromosome band 3q26, was determined in primary cervical adenocarcinomas. Interphase nuclei prepared from archival material of 12 primary cervical adenocarcinomas, eight of which were human papillomavirus positive, were hybridised with a triple colour probe set specific for centromeres of chromosomes 3 and 7 and the TERC gene. We observed high proportions of nuclei with increased absolute copy numbers for TERC in all tumours (mean 3.3; range 2.3-5.2). Amplification of the human telomerase gene TERC is a consistent aberration in cervical adenocarcinomas. Therefore, application of our probe set may provide an objective genetic test for the assessment of glandular cells in Pap smears and hence for the diagnosis of cervical adenocarcinomas.

Gain of chromosome 3q is an early and consistent genetic aberration in carcinomas of the vulva.

The aim was to determine whether specific gains of chromosome 3q and laminin-5gamma2-chain expression can improve early detection of invasive capacity in precancerous and squamous cell carcinoma of the vulva (VSCC). Six VSCC and three precancerous lesions were studied. Multicolor fluorescence in situ hybridization (FISH) probe sets were applied to nuclei suspensions prepared from archival material using the Hedley method. The probe panel consists of the centromers of chromosome 7, chromosome 3, and the TERC gene residing on the long arm of chromosome 3. Laminin-5gamma2-chain immunohistochemical analysis was performed on corresponding specimens and was expressed only in the VSCC. The genome-specific FISH analysis revealed 3q amplification in 43% of the nuclei analyzed for the VSCC and 22% of the nuclei for the precancerous lesions. Low-level 3q amplifications were found in precancerous lesions with an average fold increase of 1.15 for 3q. The invasive lesions showed higher average fold increases for 3q, averaging 1.32. Laminin-5gamma2-chain protein was expressed only in VSCC, whereas 3q gains were observed both in precancerous lesions and in VSCC, indicating that gain of chromosome 3q is an early and consistent event during carcinogenesis of VSCC.

Characterisation of breast fine-needle aspiration biopsies by centrosome aberrations and genomic instability.

Recent studies have suggested that aneuploidy in malignant tumours could be a consequence of centrosome aberrations. Using immunofluorescence analysis with an antibody against gamma-tubulin and DNA image cytometry, we measured centrosome aberrations and DNA ploidy patterns in fine-needle aspiration biopsies (FNABs) of 58 breast lesions. Benign lesions did not show any centrosome aberrations. DNA diploid carcinomas showed a mean percentage of cells with centrosomal defects of 2.1%. The aneuploid invasive carcinomas could be divided into two subgroups by their significantly (P=0.0003) different percentage of cells with centrosome aberrations (2.0 and 10.3%, respectively) and their significantly (P=0.0003) different percentage of cells with nonmodal DNA content values determined by the Stemline Scatter Index (SSI), a measure of genomic instability. The percentage of cells with centrosome aberrations demonstrated a positive, linear correlation with the corresponding SSI (r=0.82, P<0.0001) and loss of tissue differentiation (r=0.78, P<0.0001). Our results indicate the percentage of cells with centrosome aberrations as being sufficient to divide the investigated tumours into three significantly different groups: benign lesions with no centrosomal aberrations, and two malignant tumour types with mean values of 2.1 and 9.6% of centrosomal defects, respectively. Together, these results demonstrate that centrosome aberrations correlate with genomic instability and loss of tissue differentiation. Furthermore, this study shows the feasibility of centrosomal analysis in FNAB of the breast and suggests centrosomal aberrations as possessing diagnostic and prognostic value.

Sequential proteome alterations during genesis and progression of colon cancer.

Changes in the proteome of colon mucosal cells accompany the transition from normal mucosa via adenoma and invasive cancer to metastatic disease. Samples from 15 patients with sporadic sigmoid cancers were analyzed. Proteins were separated by two-dimensional gel electrophoresis. Relative differences in expression levels between normal tissue, adenoma, carcinoma and metastasis were evaluated in both intra- and inter-patient comparisons. Up- and down-regulated proteins (> twofold) during development to cancer or metastasis were excised and submitted to peptide mass fingerprinting and MS/MS sequence analysis, facilitated by the use of a compact disc workstation. In total, 112 protein spots were found to be differentially regulated, of which 72 were determined as to protein identity, 46 being up-regulated toward the progression of cancer, and 26 down-regulated. Several of the identifications correlate with proteins of the cell cycle, cytoskeleton or metabolic pathways. The pattern changes now identified have the potential for design of marker panels for assistance in diagnostics and therapeutic strategies in colorectal cancer.

Gene expression profiles in human non-small and small-cell lung cancers.

Suppression subtractive hybridisation (SSH) was performed comparing normal bronchial epithelial cells with a lung squamous cell carcinoma (SCC) and a metastatic small-cell lung carcinoma (SCLC). The sequence analysis of four cDNA libraries revealed 869 individual sequences. Of these, 342 were tested using northern blots of lung cancer cell lines representing the three major subtypes (SCC, adenocarcinoma, SCLC) which confirmed the differential expression of 236 cDNAs. The extended analysis of 31 randomly chosen fragments confirmed the validity of the approach to identify genes associated with lung cancer development. Additionally, five novel full-length cDNA were isolated encoding the microtubule-associated proteins 1A/1B light chain 3, the epithelial V-like antigen 1 (EVA1), the GTP-binding protein SAR1, a new member of the S100-type calcium binding protein family and a new homeobox-containing gene.

[Interphase cytogenetics with DNA-probes for chromosome 8 to detect circulating tumor cells in breast cancer patients]. / Interphasezytogenetik mit DNA-Sonden für Chromosom 8 zur Detektion zirkulierender Tumorzellen beim Mammakarzinom.

The detection of micrometastases in the bone marrow or peripheral blood of cancer patients is increasingly used for a more sensitive tumor staging and prognostication. The potential value of the currently used techniques for the detection of epithelial antigens by RT-PCR or immunohistochemistry in respect of specificity is currently controversially discussed. In the present study we demonstrate a new approach which enables the direct visualization of the tumor specific alteration of chromosome 8 in circulating tumor cells. We have therefore studied breast cancer patients with various tumor stages and tried to determine the frequency of circulating tumor cells in the peripheral blood by using interphase cytogenetics for chromosome 7 and 8. Imprints of primary breast cancers and cytospins with circulating tumor cells of corresponding patients were studied in a blinded fashion. The blood samples were generated by immunomagnetic enrichment of circulating tumor cells from peripheral blood by ferrofluid and centrifugation onto cover slips. These cytospins were then hybridized with centromer probes 7 and 8. After analyzing 27 patients with benign as well as malignant breast tumors we can demonstrate that the chromosomal pattern between malignant tumor and corresponding circulating tumor cells is identical. Furthermore, the detection of circulating tumor cells directly correlates with the primary tumor stage. We did not find any cells with chromosome 8 alterations in the patients with benign disease. Surprisingly, even in early breast cancers (T1N0) interphase cytogenetics identified circulating tumor cells in 2 out of 4 patients. In conclusion, interphase cytogenetics represent a non-invasive, sensitive and specific assay for the direct visualization of circulating tumor cells in the peripheral blood. The prognostic value of these findings remains to be further evaluated in larger prospective studies.

The consequences of chromosomal aneuploidy on gene expression profiles in a cell line model for prostate carcinogenesis.

Here we report the genetic characterization of immortalized prostate epithelial cells before and after conversion to tumorigenicity using molecular cytogenetics and microarray technology. We were particularly interested to analyze the consequences of acquired chromosomal aneuploidies with respect to modifications of gene expression profiles. Compared with nontumorigenic but immortalized prostate epithelium, prostate tumor cell lines showed high levels of chromosomal rearrangements that led to gains of 1p, 5, 11q, 12p, 16q, and 20q and losses of 1pter, 11p, 17, 20p, 21, 22, and Y. Of 5700 unique targets on a 6.5K cDNA microarray, approximately 3% were subject to modification in expression levels; these included GRO-1, -2, IAP-1,- 2, MMP-9, and cyclin D1, which showed increased expression, and TRAIL, BRCA1, and CTNNA, which showed decreased expression. Thirty % of expression changes occurred in regions the genomic copy number of which remained balanced. Of the remainder, 42% of down-regulated and 51% of up-regulated genes mapped to regions present in decreased or increased genomic copy numbers, respectively. A relative gain or loss of a chromosome or chromosomal arm usually resulted in a statistically significant increase or decrease, respectively, in the average expression level of all of the genes on the chromosome. However, of these genes, very few (e.g., 5 of 101 genes on chromosome 11q), and in some instances only two genes (MMP-9 and PROCR on chromosome 20q), were overexpressed by > or =1.7-fold when scored individually. Cluster analysis by gene function suggests that prostate tumorigenesis in these cell line models involves alterations in gene expression that may favor invasion, prevent apoptosis, and promote growth.