Evaluation of the fibroblast growth factor system as a potential target for therapy in human prostate cancer.

Overexpression of fibroblast growth factors (FGFs) has been implicated in prostate carcinogenesis. FGFs function via their high-affinity interactions with receptor tyrosine kinases, FGFR1-4. Expression of FGFR1 and FGFR2 in prostate cancer (CaP) was not found to be associated with clinical parameters. In this report, we further investigated for abnormal FGFR expression in prostate cancer and explore their significance as a potential target for therapy. The expression levels of FGFR3 and FGFR4 in CaP were examined and corroborated to clinical parameters. FGFR3 immunoreactivity in benign prostatic hyperplasia (BPH) and CaP (n=26 and 57, respectively) had similar intensity and pattern. Overall, FGFR4 expression was significantly upregulated in CaP when compared to BPH. A significant positive correlation between FGFR4 expression and Gleason score was noted: Gleason score 7-10 tumours compared to BPH (P<0.0001, Fisher's exact test), Gleason score 4-6 tumours compared to BPH (P<0.0004), and Gleason 7-10 compared to Gleason 4-6 tumours (P<0.005). FGFR4 overexpression was associated with an unfavourable outcome with decreased disease-specific survival (P<0.04, log rank test). FGF-induced signalling is targeted using soluble FGF receptor (sFGFR), potent inhibitor of FGFR function. We have previously shown that sFGFR expression via a replication-deficient adenoviral vector (AdlllcRl) suppresses in vitro FGF-induced signalling and function in human CaP DU145 cells. We tested the significance of inhibiting FGF function along with conventional therapeutic modalities in CaP, and confirmed synergistic effects on in vitro cell growth (proliferation and colony formation) by combining sFGFR expression and treatment with either Paclitaxel (Taxol) or gamma-irradiation. In summary, our data support the model of FGF system as valid target for therapy in CaP.

Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13).

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of visual fields. With an incidence of approximately 1 in 4000, RP can be inherited in X-linked, autosomal dominant or autosomal recessive modes. The RP13 locus for autosomal dominant RP (adRP) was placed on chromosome 17p13.3 by linkage mapping in a large South African adRP family. Using a positional cloning and candidate gene strategy, we have identified seven different missense mutations in the splicing factor gene PRPC8 in adRP families. Three of the mutations cosegregate within three RP13 linked families including the original large South African pedigree, and four additional mutations have been identified in other unrelated adRP families. The seven mutations are clustered within a 14 codon stretch within the last exon of this large 7 kb transcript. The altered amino acid residues at the C-terminus exhibit a high degree of conservation across species as diverse as humans, Arabidopsis and trypanosome, suggesting that some functional significance is associated with this part of the protein. These mutations in this ubiquitous and highly conserved splicing factor offer compelling evidence for a novel pathway to retinal degeneration.

Expression map of human chromosome region 17p13.3, spanning the RP13 dominant retinitis pigmentosa locus, the Miller-Dieker lissencephaly syndrome (MDLS) region, and a putative tumour suppressor locus.

Chromosome region 17p13.3 is rich in genes, with 223 expressed sequence tags (ESTs) within the last 15 cM (7 Mb) of chromosome 17p in the GeneMap database. Loci for dominant retinitis pigmentosa (RP13), central areolar choroidal dystrophy (CACD), anterior polar cataract (CTAA2), Miller-Dieker lissencephaly syndrome (MDLS), and a region of tumour loss of heterozygosity (LOH) distinct from TP53 all map into the region adjacent to the 17p telomere. To date, however, there is no physical map of the region, which has resisted the efforts of the CEPH and Whitehead physical mapping programmes to generate contiguous clones across it. We have created a physical map covering approximately 3.5 Mb (6 cM)in this region, spanning the RP13 interval and extending distally to the gene MDCR (formerly, LIS1), which, when deleted, leads to the MDLS phenotype. The region covered is also the point of maximum LOH in lung cancer and has been implicated in the pathogenesis of many other human cancers. The map orders 47 sequence tagged sites, including 32 genes or ESTs, nine genetic markers, four anonymous sequences, and two YAC end clones, and highlights new candidate ESTs for involvement in RP13, MDLS, CTAA2, and a tumour-susceptibility gene.

Mutations in the RP1 gene causing autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration that affects approximately 1 in 3500 people worldwide. Recently we identified the gene responsible for the RP1 form of autosomal dominant retinitis pigmentosa (adRP) at 8q11-12 and found two different nonsense mutations in three families previously mapped to 8q. The RP1 gene is an unusually large protein, 2156 amino acids in length, but is comprised of four exons only. To determine the frequency and range of mutations in RP1 we screened probands from 56 large adRP families for mutations in the entire gene. After preliminary results indicated that mutations seem to cluster in a 442 nucleotide segment of exon 4, an additional 194 probands with adRP and 409 probands with other degenerative retinal diseases were tested for mutations in this region alone. We identified eight different disease-causing mutations in 17 of the 250 adRP probands tested. All of these mutations are either nonsense or frameshift mutations and lead to a severely truncated protein. Two of the eight different mutations, Arg677X and a 5 bp deletion of nucleotides 2280-2284, were reported previously, while the remaining six mutations are novel. We also identified two rare missense changes in two other families, one new polymorphic amino acid substitution, one silent substitution and a rare variant in the 5'-untranslated region that is not associated with disease. Based on this study, mutations in RP1 appear to cause at least 7% (17/250) of adRP. The 5 bp deletion of nucleotides 2280-2284 and the Arg677X nonsense mutation account for 59% (10/17) of these mutations. Further studies will determine whether missense changes in the RP1 gene are associated with disease, whether mutations in other regions of RP1 can cause forms of retinal disease other than adRP and whether the background variation in either the mutated or wild-type RP1 allele plays a role in the disease phenotype.

p53 and K-ras status in duodenal adenomas in familial adenomatous polyposis.

BACKGROUND: The genetic alterations in patients with familial adenomatous polyposis (FAP) and duodenal adenomas are poorly characterized when compared with data relating to colorectal tumorigenesis in the same patients. METHODS: Point mutation of the K-ras oncogene and point mutation and overexpression of the TP53 tumour suppressor gene were investigated in 32 duodenal polyps (seven without mucosal pathology, 23 with mildly dysplastic adenomas and two with moderately dysplastic adenomas) from 21 patients with FAP. RESULTS: K-ras mutation, TP53 mutation or positive p53 staining were not found in duodenal polyps without histological abnormality. Of 25 duodenal adenomas, K-ras mutation was found in three (two mildly dysplastic, one moderately dysplastic), 20 showed positive p53 immunostaining, and mutation of the TP53 gene was found in one moderately dysplastic adenoma. p53 protein overexpression in duodenal adenomas was significantly more frequent than mutation of either K-ras or TP53 (P < 0.01). CONCLUSION: p53 dysfunction is a hallmark of duodenal adenomas in patients with FAP. Overexpression may indicate DNA damage and thus an early step in tumorigenesis.

Alu-polymerase chain reaction genomic fingerprinting technique identifies multiple genetic loci associated with pancreatic tumourigenesis.

DNA fingerprinting can be used to detect genetic rearrangements in cancer that may be associated with activation of oncogenes and inactivation of tumour suppressor genes. We have developed a fingerprinting strategy based on polymerase chain reaction (PCR) amplification of genomic DNA with primers specific for the Alu repeat sequences, which are highly abundant in the human genome. This has been applied to DNA from pancreatic cancer and paired normal samples to isolate and identify fragments of genomic DNA rearranged in the malignant cells. These fragments have been sequenced and used as probes to isolate hybridising clones from gridded bacteriophage P1, phage artificial chromosome, and cosmid libraries for fluorescent in situ hybridisation mapping and the identification of expressed sequences. Further characterisation has identified a putative novel gene (ART1) that is up-regulated specifically in pancreatic cancer as well as another sequence with similarity to genes involved in differentiation (POU domains). In conclusion, we suggest that Alu-PCR fingerprinting may be a useful technique for the identification of genes involved in tumourigenesis.

Abnormalities of the RB1 and DCC tumor suppressor genes: uncommon in human pancreatic adenocarcinoma.

Evidence of RB1 allele loss was found in only 6% of pancreatic cancers, and we found no significant sequence abnormalities nor loss of RB protein expression in a panel of tumors and cell lines. Using reverse transcription-polymerase chain reaction and Southern blot analysis, we found no evidence for loss of DCC expression in pancreatic cancer cell lines, and allele loss only rarely in tumor biopsies. These findings suggest that abnormalities of RB1 and DCC are unlikely to play a major role in pancreatic carcinogenesis.

Abnormalities affecting the APC and MCC tumour suppressor gene loci on chromosome 5q occur frequently in gastric cancer but not in pancreatic cancer.

Abnormalities affecting tumour suppressor genes on chromosome 5q21 are increasingly recognised as important in the pathogenesis of a variety of human cancers, particularly of the gastrointestinal tract. We have examined a series of gastric and pancreatic cancers from European patients for loss of heterozygosity (LOH) of markers within and around the APC and MCC genes on chromosome 5q21 using restriction fragment length polymorphism and polymerase chain reaction techniques. We find that LOH of the APC and MCC genes is particularly frequent in gastric cancers of diffuse type, but very infrequent in pancreatic cancers. We have also used single-strand conformational polymorphism to screen for abnormalities of the sequence of the APC and MCC genes in a panel of pancreatic cancer cell lines. Our results suggest that there are distinct differences in the molecular pathogenesis of gastric and pancreatic cancer and that abnormalities of APC and MCC may be involved particularly in the diffuse type of gastric cancer.

Expression of the mouse HPRT gene: deletional analysis of the promoter region of an X-chromosome linked housekeeping gene.

The mouse hypoxanthine phosphoribosyltransferase gene, like several other housekeeping genes, lacks many of the features associated with promoters of RNA polymerase II-transcribed genes. HPRT transcripts have multiple initiation sites and an HPRT minigene was used to show that only 49 bases of 5' flanking sequence was necessary for normal expression in cultured cells. The essential region, which occurs within a complex series of direct repeats, is homologous to sequences upstream of other housekeeping genes. When this sequence was deleted, cryptic upstream initiation sites were revealed. Similar aberrant patterns of initiation were seen with all minigenes assayed in Xenopus oocytes. We speculate that this region of the HPRT promoter is involved in a different interaction with the transcriptional machinery to that occurring at more conventional promoters.