Biomedical subjects
Publications and source records attributed to P H Rabbitts.
Recent developments in the detection of pre-invasive lesions of the large airways by fluorescence bronchoscopy are reviewed and the clinical and biological implications discussed.
Chromosome 3 allele loss in preinvasive bronchial abnormalities and carcinogen-exposed, histologically normal bronchial epithelium indicates that it is an early, possibly the first, somatic genetic change in lung tumor development. Candidate tumor suppressor genes have been isolated from within distinct 3p regions implicated by heterozygous and homozygous allele loss. We have proposed that DUTT1, nested within homozygously deleted regions at 3p12-13, is the tumor suppressor gene that deletion-mapping and tumor suppression assays indicate is located in proximal 3p. The same gene, ROBO1 (accession number ), was independently isolated as the human homologue of the Drosophila gene, Roundabout. The gene, coding for a receptor with a domain structure of the neural-cell adhesion molecule family, is widely expressed and has been implicated in the guidance and migration of axons, myoblasts, and leukocytes in vertebrates. A deleted form of the gene, which mimics a naturally occurring, tumor-associated human homozygous deletion of exon 2 of DUTT1/ROBO1, was introduced into the mouse germ line. Mice homozygous for this targeted mutation, which eliminates the first Ig domain of Dutt1/Robo1, frequently die at birth of respiratory failure because of delayed lung maturation. Lungs from these mice have reduced air spaces and increased mesenchyme, features that are present some days before birth. Survivors acquire extensive bronchial epithelial abnormalities including hyperplasia, providing evidence of a functional relationship between a 3p gene and the development of bronchial abnormalities associated with early lung cancer.
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.
In an attempt to improve the poor survival rates for lung cancer, therapeutic strategies require a deeper understanding of the biological events contributing to the formation and progression of the disease. In particular, the importance of studying the different stages of lung cancer including early pre-neoplasia is being recognised and studies examining genetic changes in pre-invasive and invasive lesions are being used to identify key events in tumorigenesis.
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.
RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFR alpha-1). Activating RET mutations are found in the inherited cancer syndrome multiple endocrine neoplasia type 2 and in a subset of the related sporadic tumors, medullary thyroid carcinoma and pheochromocytoma, both being derived from neuroendocrine tissues. In one small study, mutations were identified in another tumor with neuroendocrine features, small cell lung carcinoma (SCLC). To determine whether RET mutations contribute to the pathogenesis of SCLC, we examined a panel of 54 SCLC cell lines. No mutations were identified in RET exons 10, 11, and 13-16, regions previously implicated in SCLC or other neuroendocrine tumors. We further examined the expression pattern of RET and the genes encoding the components of its ligand complex GDNF and GFR alpha-1, in 21 SCLC lines by using RT-PCR. Although we found no consistent pattern of expression for these three genes, RET was expressed in 57% of SCLC lines. Thus, although RET mutations appear unlikely to be an important step in the tumorigenesis of SCLC, the frequent expression of this gene suggests that RET may have a mitogenic role in a subset of SCLC cell lines.
Lung tumors, particularly squamous cell carcinomas, are believed to develop through a series of morphological abnormalities, driven by underlying somatic genetic changes. One way of studying this process is to analyze candidate somatic genetic changes in samples of squamous metaplasia and bronchial dysplasia of varying degrees of severity as well as tumor from the same patient. This assumes a clonal relationship between these lesions. In this article, we provide evidence that adjacent, physically distinct bronchial abnormalities are clonally related. This has been achieved using a plaque assay technique to detect the same p53 mutation, present throughout a tumor specimen, in a small proportion of cells in an adjacent squamous metaplasia. In addition, we have obtained two dysplasia samples from a tumor-free patient over a 9-month interval. The earlier sample had one p53 mutation, whereas the later sample has to p53 mutations on different alleles. Thus, the pattern of clonal evolution detected in the parallel samples mimics the pattern seen in longitudinal samples and supports the analysis of synchronously collected samples for the study of tumor progression.
Little is known about the biology of Merkel cell carcinoma (MCC), also called small cell carcinoma of the skin. MCC has similarities with small cell lung cancer (SCLC): both are neuroendocrine malignancies with early metastasis to distant sites and a poor prognosis. Small cell lung cancer biopsies are known to have frequent losses on chromosome 3 in the region 3p21, yet MCCs have not been reported to have 3p deletions by karyotypic analysis. Considering the similarities between SCLC and MCC, we investigated 26 MCC tumours for loss of heterozygosity (LOH) on 3p. First, RFLP analysis was performed using PCR with nine primer sets from six loci. Second, 25 tumours were examined by microsatellite analysis for 3p markers D3S1289 and D3S1285 and SST on 3q. All 26 tumours were informative at one or more loci; of these, 18 (69%) demonstrated LOH for at least one marker on the short arm. For all informative loci the frequency of LOH was greater than 30% (range 33-75%). In a cell line derived from one tumour, it was possible to demonstrate rearrangement of chromosome 3 by in situ hybridisation. No LOH was seen in 15 informative cases for the 3q locus SST. A region 3p13-p21.1, centered on the marker D3S2, was deleted in all tumours demonstrating LOH, with a secondary deletion involving D3S30 detected in some tumours at 3p13. Our results indicate that LOH on 3p is a common occurrence in MCC; however, three tumours for which DNA was also available from a corresponding cell line suggest there may be a subset of MCC whose genesis is independent of deletions of 3p.
Epithelial tumours develop through a sequence of pre-invasive lesions of increasing disarray driven by underlying somatic genetic changes. We have studied the occurrence of the two most common somatic genetic changes associated with lung cancer in a series of premalignant bronchial lesions representing different stages in lung tumorigenesis. We present evidence that allele loss on chromosome 3 precedes damage to the p53 gene. Damage to chromosome 3 itself appears to be sequential in that the pattern of allele loss seen in dysplasia is often much more discrete than in invasive tumours. This implies that preneoplastic lesions may be a useful source of material for deletion mapping studies aimed at localising the position of tumour suppressor genes. We illustrate this by the comparison of an interstitial deletion described in this study with a homozygous deletion we have described previously, which has resulted in a better definition of the localisation of a tumour suppressor gene believed to be involved in lung cancer development.
Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p21-22. We examined 31 paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p21. Allele loss was found in 19 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p21-22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehyde-fixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues.
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.
In a number of cell lines with a multidrug resistant phenotype, there is no overexpression of the putative efflux pump, P-glycoprotein. Some such lines do, however, overexpress the MRP gene which encodes a protein bearing considerable amino acid homology to P-glycoprotein. We have used in situ hybridisation to study expression of the MRP gene in human cell lines, lung tumours (representing all the major histologies) and normal lung tissue. Considerable heterogeneity of expression was seen in parental cell line COR-L23/P whereas relatively uniform high-level expression was seen in the resistant line COR-L23/R. Normal bronchial epithelium was strongly positive, but the major epithelial component of all eight lung tumours studied showed only a negative to weak signal. However, the leading edge of the tumours consistently produced a more intense signal similar to that in normal epithelium. Areas of lymphocytic infiltrate were more strongly positive than the tumour epithelium. These results suggest that expression of the MRP gene may be a significant factor determining response of lung tumours to chemotherapy, but that considerable caution is needed in the interpretation of expression studies carried out on homogenised tissue biopsies.
Both inherited predisposition and acquired somatic genetic changes contribute to lung tumour development but the former is likely to be the minor component. The relative ease with which lung tumours can be established as cell lines has resulted in extensive cytogenetic analysis, and, this together with molecular techniques which assess loss of genetic material has revealed consistent patterns of genetic damage. Some of these characteristic somatic genetic changes have been traced back to the pre-invasive stage of tumour development and this information may contribute to the future management of this currently intractable disease.
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.
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.
A combination of cytogenetic and molecular studies has implicated the p21 region of human chromosome 3 as the probable site of a gene the loss of which contributes to the development of small cell lung cancer. We report here the isolation of a gene from this region which is expressed in normal lung tissue and in cell lines derived from a number of different types of tumor, but the expression of which in small cell lung cancer cell lines is undetectable by RNA blot analysis. Although the more sensitive polymerase chain reaction did detect transcripts, a novel quantitative polymerase chain reaction assay showed that their concentration in small cell lung cancer cell lines is less than 3% of that in normal lung.
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.
The small-cell lung carcinoma cell line U2020 contains a submicroscopic, homozygous deletion that removes a chromosomal segment within 3p13-p14, including the locus D3S3. We have sublocalized 49 additional probes to the 3p13-p14.2 region and have identified 7 new DNA markers that arise from within the U2020 deletion. The estimated size of the deletion, based on marker density, is approximately 4-5 megabases (Mb). Including D3S3, 7 of the 8 markers have been linked by pulsed-field gel (PFG) electrophoresis over an area of approximately 2 Mb. Including the one unlinked marker, PFG analysis accounts for about 3 Mb of the region. The U2020 deletion appears confined to the 3p13-p14.2 region and does not include the candidate tumor suppressor gene, protein-tyrosine phosphatase gamma (PTPG).