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Biomedical subjects

F M Richards

Publications and source records attributed to F M Richards.

At least 19 recordsLinked to original sources
PubMed2006

Identification of novel VHL targets that are associated with the development of renal cell carcinoma.

von Hippel-Lindau (VHL) disease is a dominantly inherited family cancer syndrome characterized by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC) and phaeochromocytoma. Specific germline VHL mutations may predispose to haemangioblastomas, RCC and phaeochromocytoma to a varying extent. Although dysregulation of the hypoxia-inducible transcription factor-2 and JunB have been linked to the development of RCC and phaeochromocytoma, respectively, the precise basis for genotype-phenotype correlations in VHL disease have not been defined. To gain insights into the pathogenesis of RCC in VHL disease we compared gene expression microarray profiles in a RCC cell line expressing a Type 1 or Type 2B mutant pVHL (RCC-associated) to those of a Type 2A or 2C mutant (not associated with RCC). We identified 19 differentially expressed novel VHL target genes linked to RCC development. Eight targets were studied in detail by quantitative real-time polymerase chain reaction (three downregulated and five upregulated by wild-type VHL) and for six genes the effect of VHL inactivation was mimicked by hypoxia (but hypoxic-induction of smooth muscle alpha-actin 2 was specific for a RCC cell line). The potential role of four RCC-associated VHL target genes was assessed in vitro. NB thymosin beta (TMSNB) and proteinase-activated receptor 2 (PAR2) (both downregulated by wt pVHL) increased cell growth and motility in a RCC cell line, but aldehyde dehydrogenase (ALDH)1 and ALDH7 had no effect. These findings implicate TMSNB and PAR2 candidate oncogenes in the pathogenesis of VHL-associated RCC.

Base Sequence↗
PubMed2006

The elusive multiple self-healing squamous epithelioma (MSSE) gene: further mapping, analysis of candidates, and loss of heterozygosity.

The MSSE gene predisposes to multiple invasive but self-healing skin tumours (multiple self-healing epitheliomata). MSSE was previously mapped to chromosome 9q22-q31 and a shared haplotype in affected families suggested a founder mutation. We have refined the MSSE critical region (<1 cM, <1 Mb) between the zinc-finger gene ZNF169 and the Fanconi anaemia gene FANCC. By genetic mapping we have excluded ZNF169 and FANCC as well as PTCH (PATCHED) and TGFBR1 (transforming growth factor beta receptor type-1) genes. The CDC14B cell cycle phosphatase gene also lies in the region but screening of the complete coding region revealed no mutation in MSSE patients. Somatic cell hybrids created by haploid conversion of an MSSE patient's cells enabled screening of the MSSE chromosome 9 and showed no CDC14B deletion or mutation that abrogates CDC14B mRNA expression. Thus, CDC14B is unlikely to be the MSSE gene. We also report the first molecular analysis of MSSE tumours showing loss of heterozygosity of the MSSE region, with loss of the normal allele, providing the first evidence that MSSE is a tumour suppressor gene.

Base Sequence↗
PubMed2004

Molecular genetic analysis of FIH-1, FH, and SDHB candidate tumour suppressor genes in renal cell carcinoma.

BACKGROUND: Overexpression of the hypoxia inducible factor 1 (HIF-1) and HIF-2 transcription factors and the consequent upregulation of hypoxia inducible mRNAs is a feature of many human cancers and may be unrelated to tissue hypoxia. Thus, the VHL (von Hippel-Lindau) tumour suppressor gene (TSG) regulates HIF-1 and HIF-2 expression in normoxia by targeting the alpha subunits for ubiquitination and proteolysis. Inactivation of the VHL TSG in VHL tumours and in sporadic clear cell renal cell carcinoma (RCC) results in overexpression of HIF-1 and HIF-2. However, RCC without VHL inactivation may demonstrate HIF upregulation, suggesting that VHL independent pathways for HIF activation also exist. In RCC, three candidate HIF activating genes exist-FIH-1 (factor inhibiting HIF), SDHB, and FH-which may be dependent or independent of VHL inactivation. AIMS: To investigate FIH-1, SDHB, and FH for somatic mutations in sporadic RCC. METHODS: Gene mutation was analysed in primary RCCs (clear cell RCCs, papillary RCCs, and oncocytomas) and RCC cell lines. SDHB mutation analysis was performed by denaturing high performance liquid chromatography followed by direct sequencing of aberrant PCR products. FH and FIH-1 mutation analysis were performed by single stranded conformational polymorphism and direct sequencing of PCR products. RESULTS: No mutations were identified in the three genes investigated. CONCLUSIONS: There was no evidence to suggest that somatic mutations occur in the FH, FIH-1, or SDHB TSGs in sporadic RCCs.

Base Sequence↗
PubMed2002

Genetic and functional analysis of the von Hippel-Lindau (VHL) tumour suppressor gene promoter.

The VHL gatekeeper tumour suppressor gene is inactivated in the familial cancer syndrome von Hippel-Lindau disease and in most sporadic clear cell renal cell carcinomas. Recently the VHL gene product has been identified as a specific component of a SCF-like complex, which regulates proteolytic degradation of the hypoxia inducible transcription factors HIF-1 and HIF-2. pVHL is critical for normal development and mRNA expression studies suggest a role in nephrogenesis. Despite the importance of VHL in oncogenesis and development, little is known about the regulation of VHL expression. To investigate VHL promoter activity, we performed comparative sequence analysis of human, primate, and rodent 5' VHL sequences. We then proceeded to deletion analysis of regions showing significant evolutionary conservation between human and rat promoter sequences, and defined two positive and one negative regulatory regions. Analysis of specific putative transcription factor binding sites identified a functional Sp1 site, which was shown to be a regulatory element. Overlapping Sp1/AP2 sites were also identified and candidate E2F1 binding sites evaluated. Three binding sites for as yet unidentified transcription factors were mapped also. These investigations provide a basis for elucidating the regulation of VHL expression in development, the molecular pathology of epigenetic silencing of VHL in tumourigenesis, and suggest a possible link between Sp1, VHL, and nephrogenesis.

Animals↗
PubMed2001

Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma.

Renal cell carcinoma (RCC), the most common adult kidney neoplasm, is histopathologically heterogeneous, with most sporadic RCCs ( approximately 80%) classified as clear cell (CC) tumors. Chromosome 3p allele loss is the most frequent genetic alteration in RCC but is associated specifically with sporadic and hereditary forms of clear cell RCC (CC-RCC) and is not a feature of non-CC-RCC, such as papillary (chromophilic) RCC. The VHL tumor suppressor gene (TSG) maps to chromosome 3p25, and somatic inactivation of the VHL gene occurs in up to 70% of CC-RCC tumors and cell lines. However, VHL inactivation is not sufficient for CC-RCC tumorigenesis, and inactivation of 3p12-p21 TSG(s) appears to be necessary in CC-RCC irrespective of VHL gene inactivation status. Recently, we demonstrated that the candidate 3p21 TSG, RASSF1A, is hypermethylated in most small cell lung cancers. We have now investigated the role of RASSF1A inactivation in primary RCC tumors. RASSF1A promoter methylation was detected in 23% (32 of 138) of primary CC-RCC tumors. In CC-RCC cell lines, RASSF1A methylation was associated with silencing of RASSF1A expression and restoration of expression after treatment with 5'-azacytidine. The frequency of RASSF1A methylation was similar in CC-RCC with and without VHL gene inactivation (24% versus 21%), and there was no association between epigenetic silencing of the RASSF1A and VHL TSGs, because 0 of 6 tumors with VHL hypermethylation had RASSF1A methylation, and VHL was not methylated in 26 CC-RCCs with RASSF1A methylation. Although 3p allele loss has been reported rarely in papillary RCC, we identified RASSF1A methylation in 44% (12 of 27) of papillary RCCs analyzed. Thus: (a) inactivation of RASSF1A is a frequent event in both CC-RCC and papillary RCC tumors; (b) there is no relationship between epigenetic silencing of RASSF1A and VHL inactivation status in CC-RCC. Fifty-four CC-RCCs analyzed for RASSF1A methylation were informative for 3p21 allele loss, and 20% (7 of 35) with 3p21 allele loss demonstrated RASSF1A methylation. All informative CC-RCCs with 3p21 allele loss and no RASSF1A methylation also demonstrated allele losses at other regions of 3p so that tumorigenesis in these cases may result from: (a) haploinsufficiency of RASSF1A; (b) inactivation of other 3p21 TSGs; or (c) inactivation of 3p TSGs from outside of 3p21. RASSF1A is the first TSG to be inactivated frequently in both papillary and CC-RCCs. The finding of frequent epigenetic inactivation of RASSF1A in papillary RCCs despite previous studies reporting infrequent 3p21 allele loss in this tumor type illustrates how the systematic identification of all major human cancer genes will require detailed analysis of the cancer genome and epigenome.

Adenocarcinoma, Clear Cell↗
PubMed2000

Protein packing: dependence on protein size, secondary structure and amino acid composition.

We have used the occluded surface algorithm to estimate the packing of both buried and exposed amino acid residues in protein structures. This method works equally well for buried residues and solvent-exposed residues in contrast to the commonly used Voronoi method that works directly only on buried residues. The atomic packing of individual globular proteins may vary significantly from the average packing of a large data set of globular proteins. Here, we demonstrate that these variations in protein packing are due to a complex combination of protein size, secondary structure composition and amino acid composition. Differences in protein packing are conserved in protein families of similar structure despite significant sequence differences. This conclusion indicates that quality assessments of packing in protein structures should include a consideration of various parameters including the packing of known homologous proteins. Also, modeling of protein structures based on homologous templates should take into account the packing of the template protein structure.

Alcohol Dehydrogenase↗
PubMed2000

Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: overview and feasibility: properties of diazirine as a precursor.

Methylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C-H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra :CH2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 Kd protein and a noninteracting 1.7 Kd peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13CH2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble.

Animals↗
PubMed1999

Germline E-cadherin gene (CDH1) mutations predispose to familial gastric cancer and colorectal cancer.

Inherited mutations in the E-cadherin gene ( CDH1 ) were described recently in three Maori kindreds with familial gastric cancer. Familial gastric cancer is genetically heterogeneous and it is not clear what proportion of gastric cancer susceptibility in non-Maori populations is due to germline CDH1 mutations. Therefore, we screened eight familial gastric cancer kindreds of British and Irish origin for germline CDH1 mutations, by SSCP analysis of all 16 exons and flanking sequences. Each family contained: (i) two cases of gastric cancer in first degree relatives with one affected before age 50 years; or (ii) three or more cases of gastric cancer. Novel germline CDH1 mutations (a nonsense and a splice site) were detected in two families (25%). Both mutations were predicted to truncate the E-cadherin protein in the signal peptide domain. In one family there was evidence of non-penetrance and susceptibility to both gastric and colorectal cancer; thus, in addition to six cases of gastric cancer, a CDH1 mutation carrier developed colorectal cancer at age 30 years. We have confirmed that germline mutations in the CDH1 gene cause familial gastric cancer in non-Maori populations. However, only a minority of familial gastric cancers can be accounted for by CDH1 mutations. Loss of E-cadherin function has been implicated in the pathogenesis of sporadic colorectal and other cancers, and our findings provide evidence that germline CDH1 mutations predispose to early onset colorectal cancer. Thus, CDH1 should be investigated as a cause of inherited susceptibility to both gastric and colorectal cancers.

Adult↗
PubMed1999

Familial gastric cancer: overview and guidelines for management.

Families with autosomal dominant inherited predisposition to gastric cancer have been described. More recently, germline E-cadherin/CDH1 mutations have been identified in hereditary diffuse gastric cancer kindred. The need to have protocols to manage and counsel these families in the clinic led a group of geneticists, gastroenterologists, surgeons, oncologists, pathologists, and molecular biologists to convene a workshop to produce consensus statements and guidelines for familial gastric cancer. Review of the available cancer pathology from people belonging to families with documented germline E-cadherin/CDH1 mutations confirmed that the gastric cancers were all of the diffuse type. Criteria to define the different types of familial gastric cancer syndromes were agreed. Foremost among these criteria was that review of histopathology should be part of the evaluation of any family with aggregation of gastric cancer cases. Guidelines for genetic testing and counselling in hereditary diffuse gastric cancer were produced. Finally, a proposed strategy for clinical management in families with high penetrance autosomal dominant predisposition to gastric cancer was defined.

Cadherins↗
PubMed1998

Hepatocyte growth factor-stimulated renal tubular mitogenesis: effects on expression of c-myc, c-fos, c-met, VEGF and the VHL tumour-suppressor and related genes.

Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway.

Cells, Cultured↗
PubMed1998

Molecular genetic analysis of von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is a dominantly inherited multisystem family cancer syndrome predisposing to retinal and central nervous system haemangioblastomas, renal carcinoma, phaeochromocytoma, pancreatic islet cell tumours and endolymphatic sac tumours. In addition, renal, pancreatic and epididymal cysts occur. Morbidity and mortality from VHL disease can be reduced by the identification and surveillance of affected individuals and at-risk relatives so that complications are diagnosed at an early presymptomatic stage. The detailed mapping and subsequent isolation of the VHL tumour suppressor gene has enabled molecular genetic analysis in families and patients with definite or possible VHL disease. Initially, linked DNA markers were used in informative families to modify individual risks and then to make appropriate alterations in surveillance programs. However, currently most DNA analysis involves the characterisation of germline mutations. World-wide, mutations have been identified in almost 500 families (including 132 in our laboratory). These studies have revealed considerable heterogeneity both in the type and in the location of mutations within the VHL gene. In our experience, most recurrent mutations result from de novo mutations at hypermutable sequences, although a founder effect for the Tyr98His ('Black Forest') mutation has been reported in German and American families. Although many mutations are predicted to impair the ability of pVHL to combine with the elongin regulatory subunits, analysis of genotype-phenotype relationships suggests that the VHL protein has multiple and tissue specific functions. Calculation of tumour risks for different classes of VHL mutations has provided important prognostic information especially with respect to the likelihood of phaeochromocytoma. However, there is evidence that retinal involvement does not correlate with allelic heterogeneity, but that the variability in retinal angiomatosis is influenced by modifier gene effects. VHL gene mutation analysis also provides a basis for investigating the genetic basis of familial phaeochromocytoma and renal cell carcinoma, and apparently isolated retinal angiomas. Results to date suggest that a substantial proportion of patients with familial pheochromocytoma have VHL gene mutations but in contrast, most familial clusters of clear cell renal cell carcinoma (RCC) without evidence of VHL do not have germline VHL mutations.

Age of Onset↗
PubMed1998

An analysis of phenotypic variation in the familial cancer syndrome von Hippel-Lindau disease: evidence for modifier effects.

von Hippel-Lindau disease (VHL) is a dominantly inherited familial cancer syndrome predisposing to ocular and CNS hemangioblastomas, renal-cell carcinoma (RCC), and pheochromocytoma. Both interfamilial and intrafamilial variability in expression is well recognized. Interfamilial differences in pheochromocytoma susceptibility have been attributed to allelic heterogeneity such that specific missense germ-line mutations confer a high risk for this complication. However, in most cases, tumor susceptibility does not appear to be influenced by the type of underlying VHL mutation. To probe the causes of phenotypic variation, we examined 183 individuals with germ-line VHL gene mutations, for the presence and number of ocular tumors. The prevalence of ocular angiomatosis did not increase with age, and the distribution of these tumors in gene carriers was significantly different than the expected stochastic distributions. Individuals with ocular hemangioblastomas had a significantly increased incidence of cerebellar hemangioblastoma and RCC (hazard ratios 2.3 and 4.0, respectively). The number of ocular tumors was significantly correlated in individuals of 12 degree relatedness but not in more distantly related individuals. These findings suggest that the development of VHL ocular tumors is determined at an early age and is influenced by genetic and/or environmental modifier effects that act at multiple sites. Functional polymorphisms in the glutathione-S-transferase M1 gene (GSTM1) or the cytochrome P450 2D6 gene (CYP2D6) did not show a significant association with the severity of ocular or renal involvement.

Adolescent↗
PubMed1997

Comparison of straight chain and cyclic unnatural amino acids embedded in the core of staphylococcal nuclease.

We have determined by X-ray crystallography the structures of several variants of staphylococcal nuclease with long flexible straight chain and equivalent length cyclic unnatural amino acid side chains embedded in the protein core. The terminal atoms in the straight side chains are not well defined by the observed electron density even though they remain buried within the protein interior. We have previously observed this behavior and have suggested that it may arise from the addition of side-chain vibrational and oscillational motions with each bond as a side chain grows away from the relatively rigid protein main chain and/or the population of multiple rotamers (Wynn R, Harkins P, Richards FM. Fox RO. 1996. Mobile unnatural amino acid side chains in the core of staphylococcal nuclease. Protein Sci 5:1026-1031). Reduction of the number of degrees of freedom by cyclization of a side chain would be expected to constrain these motions. These side chains are in fact well defined in the structures described here. Over-packing of the protein core results in a 1.0 A shift of helix 1 away from the site of mutation. Additionally, we have determined the structure of a side chain containing a single hydrogen to fluorine atom replacement on a methyl group. A fluorine atom is intermediate in size between methyl group and a hydrogen atom. The fluorine atom is observed in a single position indicating it does not rotate like methyl hydrogen atoms. This change also causes subtle differences in the packing interactions.

Amino Acids↗
PubMed1997

Protein stability: still an unsolved problem.

This brief review suggests that molecular packing, the efficient filling of space, may be the most generally applicable factor that leads to the unique structures of most globular proteins. While simple in concept, the details of packing can lead to very subtle effects. The mechanical properties of a protein, dynamics and deformations under stress, tend to be asymmetric. In terms of structural alterations and thermostability, responses to genetic mutations are context dependent and remain difficult to predict with any confidence. Through small shifts proteins can frequently accommodate major changes in composition of the core region without substantial alteration in the basic chain conformation. Extending a jigsaw puzzle analogy, all of the pieces (side chains) are convex, varying flexible, and cannot be packed together without leaving cavities. Although large cavities do occasionally occur, a relatively even distribution of empty space is more common, and the overall packing does seem to specify the unique native structure. While it might appear that the translation machinery of the cell could have been designed with any set of alpha amino acids, the packing requirements, while strong, must be flexible enough to permit nondestructive single site mutations. This flexibility, combined with the need to produce a unique structure, may limit the average number of allowed side chain rotamers per residue. This in turn will reduce the allowable asymmetry of the side chains in order to maintain the largest number of structural motifs. It may be hard to improve on current set of amino acids.

Amino Acids↗
PubMed1997

Mapping the multiple self-healing squamous epithelioma (MSSE) gene and investigation of xeroderma pigmentosum group A (XPA) and PATCHED (PTCH) as candidate genes.

The MSSE gene predisposes to the development of multiple invasive but self-healing skin tumours (multiple self-healing squamous epitheliomata, MSSE). MSSE (previously named ESS1) was mapped to chromosome 9q by linkage analysis; haplotype analysis in families then suggested a common founder mutation and indicated that the gene lies in the interval D9S1-D9S29 (9q22-q31). Squamous cell carcinomata also develop as one of the complications of xeroderma pigmentosum, and one of the xeroderma pigmentosum genes (XPA) maps within the MSSE interval. We have investigated the hypothesis that a novel dominant mutation in XPA is responsible for MSSE. We screened the entire coding region, 3' untranslated region (UTR) and 5'UTR of XPA for germline mutations in MSSE families by single-stranded conformation polymorphism analysis and by direct DNA sequencing. No mutations were detected but a novel intragenic polymorphism was identified in the 5'UTR of XPA, in both MSSE-affected and unrelated normal individuals. This XPA polymorphism and nine new polymorphic markers that map in the MSSE region were typed in eleven MSSE families; XPA was excluded as the MSSE gene and the most likely location of MSSE was reduced to the interval between D9S197 and (D9S287, D9S1809). The Patched (PTCH) gene, which is mutated in naevoid basal cell carcinoma syndrome (NBCCS or Gorlin syndrome) lies in this interval and all MSSE families have been shown to share a common haplotype at three novel intragenic PTCH polymorphisms. Although no mutation has been detected in MSSE families, PTCH has not been excluded as the MSSE gene.

Basal Cell Nevus Syndrome↗