Optimisation of DNA and RNA extraction from archival formalin-fixed tissue.

Archival, formalin-fixed, paraffin-embedded tissue is an invaluable resource for molecular genetic studies but the extraction of high quality nucleic acid may be problematic. We have optimised DNA extraction by comparing 10 protocols, including a commercially available kit and a novel method that utilises a thermal cycler. The thermal cycler and Chelex-100 extraction method yielded DNA capable of amplification by PCR from every block and 61% of sections versus 54% using microwave and Chelex-100, 15% with classical xylene-based extraction and 60% of sections using the kit. Successful RNA extraction was observed, by beta-actin amplification, in 83.7% sections for samples treated by the thermal cycler and Chelex-100 method. Thermal cycler and Chelex-100 extraction of nucleic acid is reliable, quick and inexpensive.

Molecular genetic analysis of the cytochrome P450-debrisoquine hydroxylase locus and association with cancer susceptibility.

The cytochrome P450-dependent monooxygenases play a central role in the metabolism of chemical carcinogens. The action of these enzymes can lead to either carcinogen detoxication or activation. Differences in P450 expression in animal models give rise to large differences in susceptibility to chemical carcinogens, so genetic polymorphisms in P450 expression may be expected to be an important factor in individual human susceptibility to cancer. Of particular interest is the genetic polymorphism at the cytochrome P450-debrisoquine/sparteine hydroxylase locus (CYP2D6). Although this is a minor liver P450, its polymorphic expression is associated with the abnormal metabolism of at least 30 therapeutic drugs, including beta-blockers and tricyclic antidepressants. Conflicting reports have been made on the association of this polymorphism with cancer susceptibility. This disagreement may be attributable to limitations of the phenotyping assay used to identify affected individuals (poor metabolizers, PMs). In order to clarify these anomalies, we have developed a simple DNA-based assay with which we can identify the majority of PMs. The assay is centered around the primary gene defect responsible for the polymorphism, a G to A transition at the junction of intron 3/exon 4 which results in a frame-shift in the resultant mRNA. The frequency of this mutation is 70-80% in PMs. We have studied the frequency of mutated alleles in a control population and in a wide range of cancer patients. No association between this polymorphism and lung cancer susceptibility was observed; however, in other populations of cancer patients some very interesting shifts were found in the proportion of PMs and heterozygotes from that in the normal population.

Assignment of an anonymous DNA sequence IB/D22 (DXS371) to XP11.1-11.4.

In densely mapped and characterized chromosomes such as the X chromosome there are still regions which have a paucity of markers. One of these areas lies in the Xp11 region. Using human-rodent somatic cell hybrids we have assigned a newly isolated clone IB/D22 to the Xp11.1-p11.4 region.

Cloning of the mouse homologue of the yeast cell cycle control gene cdc2.

The cdc2 cell cycle control genes of yeast and man encode 34 kDa protein kinases. Using the human CDC2 gene as a DNA probe we have isolated cDNA clones corresponding to the mouse cdc2 gene. Sequencing of the mouse clones show 96% identity at the amino acid level to the human CDC2 gene and detects a 2 kb RNA product indistinguishable to that seen in human cells. The mouse gene should be useful in studying the functional control of the cell cycle in mouse cells.

The human cytochrome P450 CYP3 locus: assignment to chromosome 7q22-qter.

The cytochrome P-450s are a large multigene family of enzymes involved in the metabolism and detoxification of drugs and chemicals. Using a full length cDNA clone for the human nifedipine oxidase gene (CYP3) and a panel of human-rodent somatic cell hybrids, we have assigned this gene family to chromosome 7q22-qter. A search for RFLPs using this probe yielded no results.

Bombesin family receptor and ligand gene expression in human colorectal cancer and normal mucosa.

Bombesin-like peptides and their receptors are widely distributed throughout the gut and are potential mitogens for a number of gastrointestinal (GI) cancers. We have analysed the expression of bombesin-like peptides and their receptor subtypes in normal and neoplastic colorectal tissue. Expression was analysed by reverse transcription polymerase chain reaction (RT-PCR) using receptor and ligand subtype-specific primers and then expression localized by in situ hybridization (ISH) with riboprobes synthesized by in vitro transcription of cloned PCR product. Colorectal cancer tissue and matched normal mucosa from 23 patients were studied. Two of these patients had synchronous adenomatous polyps and two had synchronous hepatic metastases which were also studied. An additional two patients with adenomatous polyps were studied along with matched normal mucosa. Gastrin releasing peptide (GRP) receptor and ligand expression was present in all samples but with overall greater expression in the tumour samples. Neuromedin B (NMB) receptor expression was not detectable. NMB ligand was detected in all but one mucosal sample with overall overexpression in the tumour samples. Bombesin receptor subtype 3 (BRS-3) receptor expression was not detectable. These data support the possibility that GRP may be an autocrine growth factor in colorectal cancer.

Chromosome assignment of the human glutathione S-transferase mu 3 gene (GSTM3) to chromosome 1 by gene specific polymerase chain reaction.

Oligonucleotide primers specific for exons 4 and 5 sequences were used to amplify a unique 199-bp fragment in the human GSTM3 gene. Using DNA from a panel of somatic cell hybrids we assigned the GSTM3 locus to chromosome 1p.

Polymorphisms in drug-metabolizing enzymes as modifiers of cancer risk.

The identification of low-penetrance genes, the polymorphisms of which increase an individual's risk of developing cancer, are likely to be extremely important in the general population. In this report we analyzed two genes involved in detoxification. In a number of loci, we identified polymorphic variation correlating with the expression of the gene product. We analyzed two such loci, the cytochrome P-450 gene CYP2D6 and the N-acetyltransferase 2 (NAT2) genes, in patients with bladder and colon cancer, respectively. We observed no statistically significant associations between the control and cancer populations; however, there was a small increase in heterozygote number in bladder cancer.

Localization of the gene for human simple epithelial keratin 18 to chromosome 12 using polymerase chain reaction.

Many human genes encoding keratin intermediate filament proteins are clustered on chromosomes 17 (the type I genes) and 12 (the type II genes). Some have not yet been localized, notably the genes for the primary embryonic keratins 8 and 18, normally expressed in simple epithelia: this is because the numerous pseudogenes for these keratins have made it difficult to identify the true functional gene in each case. Through the use of human-specific primers from within introns of the published gene sequence for human type I keratin 18, human genomic DNA has been specifically amplified using the polymerase chain reaction. A single reaction product was obtained. DNA from a characterized series of mouse-human somatic cell hybrid lines was tested for the presence of sequences able to initiate the chain reaction from these primers, and the presence or absence of this genomic DNA PCR product allowed us to assign a gene for human keratin 18 to chromosome 12 unambiguously. This differs from the location of other human type I keratins on chromosome 17 and may indicate the early divergence of the genes for stratifying cell keratins from that of simple, or embryonic, keratin 18.

Analysis of somatostatin receptor subtype mRNA expression in human breast cancer.

Somatostatin is a widely distributed inhibitory peptide with growth-inhibitory effects in several human tumours, including breast cancer, raising the possibility that it may have therapeutic potential. The effects of somatostatin are mediated via a family of cell-surface receptors that differ in their tissue distribution, pharmacological properties and intracellular response mediators, suggesting that they mediate different functions of the peptide. We have analysed the expression of somatostatin receptor subtype (SSTR1-5) mRNA in normal and malignant breast tissue. Receptor expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR) using receptor subtype-specific primers and by in situ hybridization (ISH) with riboprobes synthesized by in vitro transcription of cloned PCR products. A total of 51 breast carcinomas, 36 samples of matched normal tissue, two axillary node metastases and eight normal/benign breast tissue samples were analysed. SSTR2 expression was ubiquitous in both normal and malignant breast tissue. Expression of SSTR5 was detected in approximately one-third of tumour and normal tissue, but fewer than 13% of all tissues expressed SSTR1, 3 and 4. These data suggest that SSTR2 gene expression is ubiquitous in breast cancer. Although this is unlikely to have diagnostic or prognostic significance, SSTR2-specific somatostatin analogues may have therapeutic potential in breast cancer.

Somatostatin receptor subtype mRNA expression in human colorectal cancer and normal colonic mucosae.

Somatostatin analogues may be useful novel agents in the systemic treatment of advanced colorectal cancer, as somatostatin inhibits proliferation in a wide variety of cell types. Here, we report the expression profiles of somatostatin receptor mRNAs in 32 pairs of malignant and normal colonic epithelia. Receptor subtype 2 (hSSTR2) mRNA was detected throughout nearly 90% of both malignant and normal tissue by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Subtype 5 (hSSTR5) mRNA was detected in 46% and 45% of tumour and mucosal samples respectively, but in 75% (9/12) of early-stage tumours (tubulovillous adenomas, Dukes' A and B) compared with 31% (5/16) of late-stage tumours (Dukes' C and 'D' tumours), 0.05>P>0.025 (chi2 with Yates' correction). There was also reduced expression of hSSTR5 in samples of metastatic tumour (11%, 1/9) compared with all tumour samples (56%, 18/32) 0.025>P>0.01 (chi2 with Yates' correction). Other hSSTRs (1, 3 and 4) were expressed infrequently. Thus, hSSTR2 expression is retained after malignant transformation in colonic epithelium and, although it may potentially be a target for antiproliferative therapy, its ubiquitous expression militates against this. hSSTR5 warrants investigation as a tumour suppressor.

A simplified assay for the arylamine N-acetyltransferase 2 polymorphism validated by phenotyping with isoniazid.

Human arylamine N-acetyltransferase (NAT) activity is determined by two distinct genes, NAT1 and NAT2, and the classical acetylation polymorphism in NAT2 has been associated with a variety of disorders, including lupus erythematosus and arylamine induced cancers. Over 50% of the white population exhibit a slow acetylator phenotype. The genetic basis of the defect has been identified and several DNA based assays are available for genotyping studies. We present here a simplified, rapid PCR based assay for the identification of the major slow acetylator genotypes and validate it using isoniazid as probe drug. This assay was 100% predictive of phenotype. The three genotypes (homozygous mutated, heterozygous, and homozygous rapid) corresponded to a trimodal distribution of Ac-INH/INH metabolic ratios (slow, intermediate, and rapid) without overlapping.

Minisatellite instability is found in colorectal tumours with mismatch repair deficiency.

Microsatellite instability (MSI) in colorectal tumours is demonstrated by PCR amplification of several different microsatellite loci. Minisatellites, which are repeats of longer sequences also found throughout the genome, may also be affected by tumorigenesis. Certain minisatellite alleles contain 2 types of similar repeat unit that are randomly interspersed. The interspersion pattern can be analysed by mapping variant repeat units along an amplified allele, minisatellite variant repeat unit mapping PCR (MVR-PCR). We have applied microsatellite analysis with 10 markers and MVR-PCR for locus D7S21 to 33 cases of colorectal neoplasia, 27 sporadic and 6 from patients suspected of having hereditary non-polyposis colorectal cancer (HNPCC). Of the 27 sporadic cases, 3 were MSI-high on microsatellite analysis and one MSI-low. Instability with MVR-PCR was observed, but only in the MSI-high cases. Four of the HNPCC patients had mismatch repair (MMR) gene mutations in either hMLH1 or hMSH2. All 4 had DNA instability by MVR-PCR, but only two of these had MSI (one high, one low). The other 2 of the 6 patients with suspected HNPCC were negative to mutation analysis. One had features strongly suggestive of HNPCC and was unstable by both microsatellite analysis (MSI-high) and by MVR-PCR. The other tumour, from an Amsterdam criteria positive kindred, did not demonstrate instability by any technique. Thus MVR-PCR detects DNA instability in MSI-high sporadic tumours and in those associated with HNPCC where MSI is observed. Further, in some MMR mutation positive cases MSI was not seen but instability was observed by MVR-PCR. MVR-PCR may be a valuable adjunct to the detection of MMR deficiency in colorectal tumours and it may allow new insights into the nature of DNA instability in this condition.

Localization of the CYP2D gene locus to human chromosome 22q13.1 by polymerase chain reaction, in situ hybridization, and linkage analysis.

Using a combination of somatic cell hybrids, in situ hybridization, and linkage mapping, we have been able to localize the cytochrome P450 CYP2D6 gene to chromosome 22 in the region q13.1. Linkage analysis, using locus-specific primers, showed a maximum sex-average lod score of 8.12 (theta = 0.00) between the marker pH130 (D22S64) and CYP2D6, of 6.92 (theta = 0.00) between the marker KI839 (D22S95) and CYP2D6, and of 4.80 (theta = 0.036) between the platelet-derived growth factor beta subunit gene (PDGFB) and CYP2D6.

A novel human cytochrome P450 gene (P450IIB): chromosomal localization and evidence for alternative splicing.

We have isolated from a single human liver cDNA library two clones which are highly homologous (78% over the coding region) to the major phenobarbital-inducible P450 from rat (P450IIB1). This is the first direct demonstration of the presence of the P450IIB gene subfamily in humans. This subfamily is much less extensive than the rodent homologues, but does appear to contain at least two genes. Of the cDNA clones isolated one is apparently normally spliced, whereas the other lacks exon 8 and retains all or part of intron 5. Both clones contain transcribed Alu sequences. The human P450IIB gene has been located to chromosome 19q12----19q13.2 using a probe derived from intron 5, and is close to the CYP 2A locus encoding cytochrome P450IIA2. Restriction fragment length polymorphisms have been found with the enzymes BamHI and MspI which will enable linkage to be determined between these two loci.

Restriction fragment length polymorphism analysis and assignment of the metalloproteinases stromelysin and collagenase to the long arm of chromosome 11.

Collagenase and stromelysin are two metalloproteinases produced mainly by connective tissue cells and involved in the breakdown of the extracellular matrix. cDNA clones for both of these genes have been isolated and sequencing has shown them to be closely related. The collagenase and stromeylsin cDNA clones have been used to assign these genes to the long arm of chromosome 11 in the regions 11q21-22.1 and 11q22.2-22.3, respectively. This has been achieved using somatic cell hybrids and in situ hybridization. In addition a Taq1 restriction fragment length polymorphism has been demonstrated using the stromelysin cDNA.

Molecular genetics of the human cytochrome P450-dependent monooxygenases.

In this work, the role of genetic as well as environmental factors in determining cytochrome P450 isozyme levels in man have been studied. Simple DNA based assays for the identification of individuals nulled at the CYP2D6 locus are described and have been applied to investigate whether this gene defect is associated with altered cancer susceptibility. In contrast to literature reports, in no cancer type were poor metabolizers underrepresented, indeed in several cancers the mutant allele frequency was increased. A model using human tumours grown as xenografts is described that should help elucidate the factors which regulate P450 levels in man.

Identification of the primary gene defect at the cytochrome P450 CYP2D locus.

The mammalian cytochrome P450-dependent monooxygenase system is involved in the metabolism of drugs and chemical carcinogens. The role of these enzymes in toxicological response is exemplified by an autosomal recessive polymorphism at the cytochrome P450 CYP2D6 debrisoquine hydroxylase locus which results in the severely compromised metabolism of at least 25 drugs, and which in some cases can lead to life-threatening side-effects. In addition, this polymorphism, which affects 8-10% of the caucasian population, has been associated with altered susceptibility to lung and bladder cancer. Here we report the identification of the primary mutation responsible for this metabolic defect and the development of a simple DNA-based genetic assay to allow both the identification of most individuals at risk of drug side-effects and clarification of the conflicting reports on the association of this polymorphism with cancer susceptibility.