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.

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.

Molecular cytogenetic characterization of early and late renal cell carcinomas in von Hippel-Lindau disease.

Deletions of 3p25, gains of chromosomes 7 and 10, and isochromosome 17q are known cytogenetic aberrations in sporadic renal cell carcinoma (RCC). In addition, a majority of RCCs have loss of heterozygosity (LOH) of the Von Hippel-Lindau (VHL) gene located at chromosome band 3p25. Patients who inherit a germline mutation of the VHL gene can develop multifocal RCCs and other solid tumors, including malignancies of the pancreas, adrenal medulla, and brain. VHL tumors follow the two-hit model of tumorigenesis, as LOH of VHL, a classic tumor suppressor gene, is the critical event in the development of the neoplastic phenotype. In an attempt to define the cytogenetic aberrations from early tumors to late RCC further, we applied spectral karyotyping (SKY) to 23 renal tumors harvested from 6 unrelated VHL patients undergoing surgery. Cysts and low-grade solid lesions were near-diploid and contained 1-2 reciprocal translocations, dicentric chromosomes, and/or isochromosomes. A variety of sole numerical aberrations included gains of chromosomes 1, 2, 4, 7, 10, 13, 21, and the X chromosome, although no tumors had sole numerical losses. Three patients shared a breakpoint at 2p21-22, and three others shared a dicentric chromosome 9 or an isochromosome 9q. In contrast to the near-diploidy of the low-grade lesions, a high-grade lesion and its nodal metastasis were markedly aneuploid, revealed loss of VHL by fluorescence in situ hybridization (FISH), and contained recurrent unbalanced translocations and losses of chromosome arms 2q, 3p, 4q, 9p, 14q, and 19p as demonstrated by comparative genomic hybridization (CGH). By combining SKY, CGH, and FISH of multiple tumors from the same VHL kidney, we have begun to identify chromosomal aberrations in the earliest stages of VHL-related renal cell tumors. Our current findings illustrate the cytogenetic heterogeneity of different VHL lesions from the same kidney, which supports the multiclonal origins of hereditary RCCs. Published 2001 Wiley-Liss, Inc.

Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms.

Jumping translocations (JTs) and segmental jumping translocations (SJTs) are unbalanced translocations involving a donor chromosome arm or chromosome segment that has fused to multiple recipient chromosomes. In leukemia, where JTs have been predominantly observed, the donor segment (usually 1q) preferentially fuses to the telomere regions of recipient chromosomes. In this study, spectral karyotyping (SKY) and FISH analysis revealed 188 JTs and SJTs in 10 cell lines derived from carcinomas of the bladder, prostate, breast, cervix, and pancreas. Multiple JTs and SJTs were detected in each cell line and contributed to recurrent unbalanced whole-arm translocations involving chromosome arms 5p, 14q, 15q, 20q, and 21q. Sixty percent (113/188) of JT breakpoints occurred within centromere or pericentromeric regions of the recipient chromosomes, whereas only 12% of the breakpoints were located in the telomere regions. JT breakpoints of both donor and recipient chromosomes coincided with numerous fragile sites as well as viral integration sites for human DNA viruses. The JTs within each tumor cell line promoted clonal progression, leading to the acquisition of extra copies of the donated chromosome segments that often contained oncogenes (MYC, ABL, HER2/NEU, etc.), consequently resulting in tumor-specific genomic imbalances. Published 2001 Wiley-Liss, Inc.

Isolation, genomic organization, and expression analysis of Men1, the murine homolog of the MEN1 gene.

The mouse homolog of the human MEN1 gene, which is defective in a dominant familial cancer syndrome, multiple endocrine neoplasia type 1 (MEN1), has been identified and characterized. The mouse Men1 transcript contains an open reading frame encoding a protein of 611 amino acids which has 97% identity and 98% similarity to human menin. Sequence of the entire Men1 gene (9.3 kb) was assembled, revealing 10 exons, with exon 1 being non-coding; a polymorphic tetranucleotide repeat was located in the 5'- flanking region. The exon-intron organization and the size of the coding exons 2-9 were well conserved between the human and mouse genes. Fluorescence in situ hybridization localized the Men1 gene to mouse Chromosome (Chr) 19, a region known to be syntenic to human Chr 11q13, the locus for the MEN1 gene. Northern analysis indicated two messages-2.7 kb and 3.1 kb-expressed in all stages of the embryo analyzed and in all eight adult tissues tested. The larger transcript differs from the smaller by the inclusion of an unspliced intron 1. Whole-mount in situ hybridization of 10.5-day and 11.5-day embryos showed ubiquitous expression of Men1 RNA. Western analysis with antibodies raised against a conserved C-terminal peptide identified an approximately 67-kDa protein in the lysates of adult mouse brain, kidney, liver, pancreas, and spleen tissues, consistent with the size of human menin. The levels of mouse menin do not appear to fluctuate during the cell cycle.

Specific chromosomal aberrations and amplification of the AIB1 nuclear receptor coactivator gene in pancreatic carcinomas.

To screen pancreatic carcinomas for chromosomal aberrations we have applied molecular cytogenetic techniques, including fluorescent in situ hybridization, comparative genomic hybridization, and spectral karyotyping to a series of nine established cell lines. Comparative genomic hybridization revealed recurring chromosomal gains on chromosome arms 3q, 5p, 7p, 8q, 12p, and 20q. Chromosome losses were mapped to chromosome arms 8p, 9p, 17p, 18q, 19p, and chromosome 21. The comparison with comparative genomic hybridization data from primary pancreatic tumors indicates that a specific pattern of chromosomal copy number changes is maintained in cell culture. Metaphase chromosomes from six cell lines were analyzed by spectral karyotyping, a technique that allows one to visualize all chromosomes simultaneously in different colors. Spectral karyotyping identified multiple chromosomal rearrangements, the majority of which were unbalanced. No recurring reciprocal translocation was detected. Cytogenetic aberrations were confirmed using fluorescent in situ hybridization with probes for the MDR gene and the tumor suppressor genes p16 and DCC. Copy number increases on chromosome 20q were validated with a probe specific for the nuclear receptor coactivator AIB1 that maps to chromosome 20q12. Amplification of this gene was identified in six of nine pancreatic cancer cell lines and correlated with increased expression.

Spectral karyotyping, a 24-colour FISH technique for the identification of chromosomal rearrangements.

Spectral karyotyping (SKY) is a new fluorescence in situ hybridisation (FISH) technique that refers to the molecular cytogenetic analysis of metaphase preparations by means of spectral microscopy. For SKY of human metaphase chromosomes, 24 chromosome-specific painting probes are used in just one FISH experiment. The probes are labelled by degenerate oligonucleotide-primed PCR using three fluorochromes and two haptens. Each probe is differentially labelled with one, two, three or four fluorescent dyes, resulting in a unique spectral signature for every chromosome. After in situ hybridisation and immunodetection, a spectral image is acquired using a conventional fluorescence light microscope equipped with a custom-designed triple-bandpass filter and the SpectraCube, which is able to retrieve spectral information for every pixel in a digital CCD image. The 24-colour display and chromosome classification are based on the unique emission spectra of the chromosomes. Together with chromosome banding information from an inverted DAPI or a G-banded metaphase, a comprehensive overview of chromosomal aberrations is presented.

Molecular and cytogenetic studies of an X;autosome translocation in a patient with premature ovarian failure and review of the literature.

We have identified a patient with premature ovarian failure (POF) and a balanced X;autosome translocation: 46,X,t(X;6)(q13.3 or q21;p12) using high-resolution cytogenetic analysis and FISH. BrdU analysis showed that her normal X was late-replicating and translocated X earlier-replicating which is typical of balanced X;autosome rearrangements. Molecular studies were done to characterize the breakpoint on Xq and to determine the parental origin. PCR probes of tetranucleotide and dinucleotide repeat polymorphisms, and genomic probes were used to study DNA from the patient, her chromosomally normal parents and brother, and somatic cell hybrids containing each translocation chromosome. The translocation is paternally derived and is localized to Xq13.3-proximal Xq21.1, between PGK1 and DXS447 loci, a distance of 0.1 centimorgans. A "critical region" for normal ovarian function has been proposed for Xq13-q26 [Sarto et al., Am J Hum Genet 25:262-270, 1973; Phelan et al., Am J Obstet Gynecol 129:607-613, 1977; Summitt et al., BD:OAS XIV(6C):219-247, 1978] based on cytogenetic and clinical studies of patients with X;autosome translocations. Few cases have had molecular characterization of the breakpoints to further define the region. While translocations in the region may lead to ovarian dysfunction by disrupting normal meiosis or by a position effect, two recent reports of patients with premature ovarian failure and Xq deletions suggest that there is a gene (POF1) localized to Xq21.3-q27 [Krauss et al., N Engl J Med 317:125-131, 1987; Davies et al., Cytogenet Cell Genet 58:853-966, 1991] or within Xq26.1-q27 [Tharapel et al., Am J Hum Genet 52:463-471, 1993] responsible for POF.(ABSTRACT TRUNCATED AT 250 WORDS)

A chromosome 17q de novo paracentric inversion in a patient with campomelic dysplasia; case report and etiologic hypothesis.

The campomelic syndrome is a skeletal dysplasia with a characteristic pattern of deformity involving the proximal and distal extremities, pelvic and shoulder girdles, thoracic cage and palate. Respiratory compromise often leads to death in early infancy. Etiology has not been determined although evidence suggests genetic heterogeneity in patients with campomelia. Cytogenetic analysis in the past have revealed an unexpectedly high incidence of a 46, XY karyotype in phenotypic females. We report here on a patient with a typical case of campomelic dysplasia in whom a de novo paracentric inversion of chromosome 17q was identified. Review of the genetic map of the inverted region identified potential "structural" genes including the Hox-2-homeobox gene and the collagen gene, COLIA1, which may be involved in the pathogenesis of campomelic syndrome.