Elucidation of the mechanism of homozygous deletion of 3p12-13 in the U2020 cell line reveals the unexpected involvement of other chromosomes.

Homozygous deletions in tumor cells have been useful in the localization and validation of tumor suppressor genes. We have described a homozygous deletion in a lung cancer cell line (U2020) which is located within the most proximal of the three regions on the short arm of chromosome 3 believed to be lost in lung cancer development. Construction of a YAC contig map indicates that the deletion spans around 8 Mb, but no large deletion was apparent on conventional cytogenetic analysis of the cell line. To investigate this paradox, whole chromosome, arm-specific, and regional paints have been used. This analysis has revealed that genetic loss has occurred by complex rearrangements of chromosomes 3, rather than simple interstitial deletion. These studies emphasize the power of molecular cytogenetics to disclose unsuspected tumor-specific translocations within the extremely complex karyotypes characteristic of solid tumors.

A combined approach of conventional and molecular cytogenetics for detailed karyotypic analysis of the small cell lung carcinoma cell line U2020.

Until recently the ability to analyze complex karyotypic rearrangements was totally dependent upon light microscopy of G-banded chromosomes. Developments in the area of molecular cytogenetics have revolutionized such analysis, making it possible to determine the nature of complex rearrangements. An extensive analysis has been made of the small cell lung carcinoma (SCLC) cell line U2020, using a combined approach of conventional and molecular cytogenetics, enabling a highly detailed karyotype to be constructed revealing rearrangements previously undetected by G-banding alone. This approach offers the opportunity to reassess other tumor karyotypes, particularly those of high complexity found in solid tumors, for tumor-specific consistent rearrangements indecipherable by conventional karyotyping.

Recovery of Cryptosporidium oocysts from small and large volume water samples using a compressed foam filter system.

A novel filter system comprising open cell reticulated foam rings compressed between retaining plates and fitted into a filtration housing was evaluated for the recovery of oocysts of Cryptosporidium from water. Mean recoveries of 90.2% from seeded small and large volume (100-2000 l) tap water samples, and 88.8% from 10-20 l river water samples, were achieved. Following a simple potassium citrate flotation concentrate clean-up procedure, mean recoveries were 56.7% for the tap water samples and 60.9% for river water samples. This represents a marked improvement in capture and recovery of Cryptosporidium oocysts from water compared with conventional polypropylene wound cartridge filters and membrane filters.

Multicolour fluorescence in situ hybridisation to order small, single-copy probes on metaphase chromosomes.

In constructing complete human chromosome maps, the relative order of markers and their precise chromosomal location will be combined. Multicolour in situ hybridisation, in which two probes are simultaneously hybridised to chromosomes and subsequently distinguished, potentially will provide both types of information. Using this technique, we have produced an ordered map of eight human chromosome 3 DNA markers, using small, single-copy probes that can detect target sequences ranging in size from 4 kb to as little as 500 bp.

A 190-kilodalton protein overexpressed in non-P-glycoprotein-containing multidrug-resistant cells and its relationship to the MRP gene.

BACKGROUND: A 190k (190-kilodalton) membrane protein has been identified in several multidrug-resistant (MDR) cell lines that show decreased drug accumulation without expression of P-glycoprotein. It is not clear whether this 190k protein is involved directly in drug efflux. Recently, a gene for a putative transporter protein, MRP (multidrug resistance-associated protein) has been sequenced and localized to chromosome 16. The protein encoded by this gene contains a 7-amino-acid sequence present in the synthetic peptide used to generate the antiserum recognizing the 190k protein. PURPOSE: The study was undertaken to clarify the relationship of the 190k protein to MRP gene expression in non-P-glycoprotein-containing MDR cells of the large-cell and adenocarcinoma lung cancer lines, COR-L23 and MOR. METHODS: Expression of the 190k protein was determined by Western blot analysis and that of the MRP gene by polymerase chain reaction amplification of complementary DNA reverse transcribed from RNA. Abnormalities of chromosome 16 were investigated in chromosome spreads by fluorescence in situ hybridization. RESULTS: The amount of detectable 190k protein is closely associated with degree of drug resistance. Cell lines surviving in higher drug concentrations have greater amounts of protein, and revertant lines grown without drug for up to 28 weeks show reduced expression of the protein together with enhanced drug sensitivity. The 190k protein appears to be one of the major proteins differentially expressed in membranes of drug-resistant cells. The amount of MRP messenger RNA correlates closely with that of the 190k protein. The MDR cells contain amplified chromosome 16 material with many double minutes in the large-cell lung tumor lines and an enlarged chromosome 16 in the adenocarcinoma lines. CONCLUSION: The 190k protein detected immunologically is likely to be the protein, encoded by the MRP gene, which becomes overexpressed in these cells as a consequence of chromosomal amplification and fragmentation. IMPLICATION: Though associated with drug resistance, enhanced drug efflux, and decreased drug accumulation in cell lines, the role of this protein in clinical resistance has yet to be determined.

The probe BMS1271 identifies a new polymorphic locus (D3S1207) and maps to 3p26-pter.

We report here the characterization of a new polymorphic locus (D3S1207), shown by fluorescence in situ hybridization to map to 3p26-pter. The distal region of chromosome 3 has been implicated in both von Hippel-Lindau and 3p deletion syndrome, lung, uterine, breast, testicular, and ovarian cancers, renal cell and nasopharyngeal carcinomas, mesotheliomas, and various haematological malignancies, yet relatively few polymorphic marker probes are well mapped within this region. The identification of this new probe will therefore be particularly useful for the study of these diseases.

Molecular characterization of a large homozygous deletion in the small cell lung cancer cell line U2020: a strategy for cloning the putative tumor suppressor gene.

Homozygous deletions are instrumental in the detection and cloning of tumor suppressor genes. We report the isolation and characterization of 39 new single-copy probes saturating a submicroscopic homozygous deletion detected in the DNA of the small cell lung cancer (SCLC) cell line U2020. The probes were selected from a large collection, covering the entire length of chromosome 3 with an estimated average spacing of 100-150 kb. Based on the number of probes in the deletion and the probe density, the size of the U2020 submicroscopic deletion was estimated to be in the range of 4-7 megabases. Among the deleted loci, 17 showed conservation across species, probably representing potential coding gene sequences. By genetic and physical mapping of a large randomly chosen fraction of the deleted probes, we defined the location of the U2020 deletion within chromosome band 3p12. Our cloning strategy is based on narrowing the region of interest by eliminating probes that retain heterozygosity in SCLC samples, thus selecting for probes in the region of common loss.

Ordering of six polymorphic DNA markers important in the delineation of 3p deletions in neoplasia.

Using fluorescence in situ hybridisation (FISH) the chromosomal location and relative order of six human chromosome 3 probes has been determined. The sensitivity of the technique has enabled the relative mapping of probes carrying inserts as small as 500 basepairs (bp), thus allowing the following proximal-distal probe order to be proposed: D3S30 (3p13-14), D3S4 (3p13-14), D3S2 (distal 3p14), D3S32 (3p21), D3S48E (3p21-23), and D3S11 (3p22-23). These data combined with the deletion mapping data of other researchers raise the possibility that the loss of more than one region of the short arm of chromosome 3 may be important in the development of small cell lung cancer.

Robertsonian translocation and an extra microchromosome: independent origin identified by in situ hybridization.

Robertsonian translocation may result in either a monocentric or a dicentric product involving the long arms of the participating chromosomes. In the former case a microchromosome involving the short arms of both acrocentrics would be expected to arise, but in practice is rarely observed. Where such small bisatellited markers are seen in association with Robertsonian translocations, they are assumed to represent such reciprocal products. We present a case, involving a t(14q21q) and a DA/DAPI positive microchromosome, where using in situ hybridization we show that this assumption is incorrect and that the microchromosome has arisen independently.