Prognostic significance of cell cycle proteins and genomic instability in borderline, early and advanced stage ovarian carcinomas.

Disturbed cell cycle-regulating checkpoints and impairment of genomic stability are key events during the genesis and progression of malignant tumors. We analyzed 80 epithelial ovarian tumors of benign (n = 10) and borderline type (n = 18) in addition to carcinomas of early (n = 26) and advanced (n = 26) stages for the expression of Ki67, cyclin A and cyclin E, p21WAF-1, p27KIP-1 and p53 and correlated the results with the clinical course. Genomic instability was assessed by DNA ploidy measurements and, in 35 cases, by comparative genomic hybridization. Overexpression of cyclin A and cyclin E was observed in the majority of invasive carcinomas, only rarely in borderline tumors and in none of the benign tumors. Similarly, high expression of p53 together with undetectable p21 or loss of chromosome arm 17p were frequent events only in adenocarcinomas. Both borderline tumors and adenocarcinomas revealed a high number of chromosomal gains and losses. However, regional chromosomal amplifications were found to occur 13 times more frequently in the adenocarcinomas than in the borderline tumors. The expression pattern of low p27 together with high Ki67 was found to be an independent predictor of poor outcome in invasive carcinomas. The results provide a link between disturbed cell cycle regulatory proteins, chromosomal aberrations and survival in ovarian carcinomas.

Spectral karyotyping combined with locus-specific FISH simultaneously defines genes and chromosomes involved in chromosomal translocations.

Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the FISH probes could cover a wide range and still be used successfully. Some of the nucleotide-bound dyes used for the labeling, as Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from cancer gene expression studies. Published 2000 Wiley-Liss, Inc.

14-3-3 epsilon has no homology to LIS1 and lies telomeric to it on chromosome 17p13.3 outside the Miller-Dieker syndrome chromosome region.

Previously, we isolated several cDNA clones of the LIS1 gene implicated in Miller-Dieker syndrome. Analysis of the 5' end of one of the clones (8-1), which was originally thought to represent the 5' end of LIS1, indicates a striking similarity to mouse 14-3-3 epsilon. We have isolated a full-length cDNA of human 14-3-3 epsilon, for which sequence analysis reveals a strong nucleotide conservation with mouse 14-3-3 epsilon in both translated and untranslated regions (UTRs). Additionally, the predicted peptides of human, sheep, rat, and mouse 14-3-3 epsilon are identical. Using a 205-bp fragment common to LIS1 (8-1) and 14-3-3 epsilon as probe on adult and fetal multiple-tissue Northern blots, a -2-kb transcript is detected, identical to the pattern observed with a full-length 14-3-3 epsilon cDNA probe. LIS1-specific transcripts of approximately 7.5 and approximately 5 kb are not detected by the 0.2-kb probe, indicating that the similarity between the 5' sequence of LIS1 (8-1) and the 3' UTR of 14-3-3 epsilon is not the result of shared homology between the two genes. Instead, clone 8-1 is a chimera of 14-3-3 epsilon and LIS1 partial cDNAs, and therefore its 5' sequence does not represent the LIS1 5' end. Interestingly, we have mapped the 14-3-3 epsilon gene to the same chromosomal sub-band as LIS1 (17p13.3). However, 14-3-3 epsilon lies telomeric to LIS1 and outside the Miller-Dieker syndrome chromosome region but in a region frequently deleted in several types of cancer, and is a reasonable candidate tumor suppressor gene.

Identification of a novel paternally expressed transcript adjacent to snRPN in the Prader-Willi syndrome critical region.

Small nuclear ribonucleoprotein-associated polypeptide N (snRPN) and an anonymous transcript, PAR-5, are two of the paternally expressed transcripts mapped to the Prader-Willi syndrome critical region. Using long-range PCR, we have isolated the genomic interval between snRPN and PAR-5, identified a novel transcript in this region, and termed it PAR-SN. Northern analysis demonstrates that PAR-SN is expressed in brain, skeletal muscle, and heart. Like snRPN and PAR-5, PAR-SN is expressed exclusively from the paternal homolog in cultured lymphoblasts. Sequence analysis of the transcript revealed no significant open reading frame but did include a polymorphic dinucleotide repeat (CA)17.

A revision of the lissencephaly and Miller-Dieker syndrome critical regions in chromosome 17p13.3.

Miller-Dieker syndrome (MDS) is a multiple malformation syndrome characterized by classical lissencephaly and a characteristic facies. It is associated with visible or submicroscopic deletions within chromosome band 17p13.3. Lissencephaly without facial dysmorphism has also been observed and is referred to as isolated lissencephaly sequence (ILS). Apparently partial and non-overlapping deletions of the 5' or 3' end of a candidate gene LIS1 in one ILS and one MDS patient had suggested that MDS was a single gene disorder, and that LIS1 spans in excess of 400 kb. However, the originally presumed 5' end of LIS1 was found to belong to the 14-33 epsilon gene residing more distally on 17p13.3. We have now isolated the correct 5' end of LIS1, constructed a approximately 500 kb genomic contig encompassing LIS1, and estimated its gene to be approximately 80 kg. Fluorescence in situ hybridization analysis of an ILS patient with a de novo balanced translocation, as well as analysis of several other key MDS and ILS deletion patients, localizes the lissencephaly critical region within the LIS1 gene. Therefore, LIS1 remains the strongest candidate gene for the lissencephaly phenotype in ILS and MDS. Our analyses also suggest that additional genes distal to LIS1 may be responsible for the facial dysmorphology and other abnormalities seen in MDS but not in ILS patients, supporting our original concept MDS as a contiguous gene deletion syndrome.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma.

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.