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D I Smith

Publications and source records attributed to D I Smith.

At least 73 records · Page 4Linked to original sources
PubMed1997

Aphidicolin-induced FRA3B breakpoints cluster in two distinct regions.

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE and Alu repetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.

Aphidicolin↗
PubMed1997

Frequent breakpoints in the region surrounding FRA3B in sporadic renal cell carcinomas.

The constitutive fragile site at chromosomal band 3p14.2, FRA3B, is the most active common fragile site in the human genome. We have localized aphidicolin-induced breakpoints to two distinct clusters, separated by 200 Kb, in FRA3B (Paradee et al., 1996). Sequence analysis of these regions identified two polymorphic microsatellite markers immediately adjacent to each of these breakpoint clusters. In this report we have used these two new microsatellites and 14 additional 3p microsatellites to analyse chromosome 3p breakage and loss in 94 sporadic RCC samples, including nonpapillary, papillary and oncocytomas. We have found heterozygous loss of 3p14 sequences in >60% of the RCC samples, including both clear cell and papillary renal cell carcinomas. We have found frequent breakage in the region immediately surrounding FRA3B, demonstrating that FRA3B does play a role in chromosome breakage and loss in RCC. In contrast to other reports, >50% of the papillary tumors also showed LOH of 3p markers. We also observed microsatellite instability (MIN) with most of the tested markers in seven of eight oncocytomas and one of 69 clear cell carcinomas. The MIN in some oncocytomas was of the RER+ (replication error) type I phenotype. None of the five 3p14.2 markers detected any homozygous deletions in tumor samples, but 69/94 (73%) of the tumors had LOH for the region, which includes the recently identified FHIT gene.

Adult↗
PubMed1997

Maternal uniparental disomy of chromosome 2 and confined placental mosaicism for trisomy 2 in a fetus with intrauterine growth restriction, hypospadias, and oligohydramnios.

We present a case of maternal uniparental heterodisomy for chromosome 2 (UPD 2) detected after trisomy 2 mosaicism was found on placental biopsy. This case presented prenatally with severe intrauterine growth restriction (IUGR) and oligohydramnios. The diploid newborn had hypospadias and features consistent with oligohydramnios sequence. He died shortly after birth of severe pulmonary hypoplasia. The term placenta had high levels of trisomy 2 in both the trophoblast and the stroma. A comparison of this case with others reported in the literature suggests that the IUGR and oligohydramnios are likely related to placental insufficiency due to the high levels of trisomy 2 present in the trophoblast of the term placenta and the presence of UPD 2 in the diploid placental line.

Chromosomes, Human, Pair 2↗
PubMed1997

Normal FHIT transcripts in renal cell cancer- and lung cancer-derived cell lines, including a cell line with a homozygous deletion in the FRA3B region.

The recently identified FHIT gene encompasses the FRA3B region and the breakpoint of a constitutive t(3;8) occurring in a family with hereditary renal cell cancer. Occurrence of aberrant transcripts in different types of tumours has led to the suggestion that FHIT might play a critical role in the development of various types of cancer. We have analyzed the gene and its transcripts in lung cancers and renal cell cancer-derived cell lines. A lung adenocarcinoma cell line, GLC-A2, appeared to have a homozygous deletion in intron 5 of FHIT. RT-PCR analysis revealed a normal-sized PCR product in all of the cell lines: Including GLC-A2. A number of them had an additional aberrant product. Analysis of a great number of control cell lines and tissues showed that the majority of these also had aberrant PCR products in addition to a normal-sized PCR product. Different specimens of the same cell type showed variable additional RT-PCR products. Normal-sized PCR products had a sequence identical to the FHIT sequence. PCR products longer than normal had insertions of different sizes at different positions. With three exceptions, PCR products shorter than normal represented FHIT sequences missing one or more entire exons. Thus, the presence of aberrant transcripts is not cancer-specific. Conceivably, sequence responsible for the instability of the FRA3B region are being transcribed into FHIT pre-mRNA and may cause the abnormal splicing and processing of the transcripts.

Acid Anhydride Hydrolases↗
PubMed1996

Mutations in the arginine-rich protein gene, in lung, breast, and prostate cancers, and in squamous cell carcinoma of the head and neck.

Arginine-rich protein (ARP) is a highly conserved gene that maps to human chromosomal band 3p21.1. This gene contains an imperfect trinucleotide repeat which encodes a string of arginines. We previously detected a specific mutation (ATG50-->AGG) within this region of the gene in 10 of 21 sporadic renal cell carcinomas. Here, we report the detection of the same mutation in 5 of 21 squamous cell carcinomas of the head and neck, 1 of 2 small cell lung cancer cell lines, 6 of 18 non-small cell lung carcinomas, 9 of 22 breast tumors, and 5 of 13 prostate tumors. This mutation was seen in several early stage tumors and may thus be an early event in tumorigenesis. We also detected a mutation at codon 53 of this gene in both primary and metastatic tumors from one patient. Other nucleotide changes were observed in a few PCR subclones, but their frequency was the same in both tumor and control samples, suggesting that many of these changes were PCR or subcloning artifacts rather than mutations in the tumor cells themselves.

Breast Neoplasms↗
PubMed1996

Frequent breakpoints in the 3p14.2 fragile site, FRA3B, in pancreatic tumors.

FRA3B, at chromosomal band 3p14.2, is the most active common fragile site in the human genome. Homozygous deletions in the region of FRA3B have been observed in many types of solid tumors. Recently, the FHIT gene was reported to span FRA3B and was shown to be homozygously deleted in several gastric and colonic tumor cell lines. Several microsatellite markers that precisely define the 1.0-Mb region surrounding FRA3B and FHIT have been utilized, along with other 3p microsatellites, to analyze the loss of 3p sequences in 25 primary pancreatic adenocarcinomas. The high density of microsatellite markers in the 3pl4.2 region enabled us to both identify losses within and flanking FRA3B in pancreatic cancer and define the breakpoints. We observed loss of het erozygosity of 3pl4.2 markers in 16 of 25 pancreatic tumors and loss of heterozygosity of 3p markers outside of 3pl4.2 in only 2 of 25 tumors of this type. There appears to be a dramatic clustering of chromosomal breakpoints at 3pl4.2 in and immediately distal to FRA3B in pancreatic cancer. We detected no homozygous deletions in this region.

Adenocarcinoma↗
PubMed1996

A 350-kb cosmid contig in 3p14.2 that crosses the t(3;8) hereditary renal cell carcinoma translocation breakpoint and 17 aphidicolin-induced FRA3B breakpoints.

The constitutive fragile site at human chromosomal band 3p14.2, FRA3B, has been described as the most active common fragile site in the human genome. FRA3B is cytologically indistinguishable from the chromosome 3 breakpoint observed in the hereditary renal cell carcinoma (hRCC) translocation t(3;8) (p14.2;q24.13). Previous work demonstrated that a 1330-kb YAC clone, YC850A6, spans both the t(3;8) translocation and FRA3B and also encompasses FRA3B-associated breakpoints induced in hamster-human hybrids. This YAC was used to construct a multi-hit cosmid library. Screening of this library resulted in a 350-kb cosmid contig that extends distally from the t(3;8) translocation breakpoint. Seventeen aphidicolin-induced 3p14. 2 breakpoints derived from hamster-human hybrids were mapped within this cosmid contig. These breakpoints were found to localize as two distinct clusters, separated by 200 kb, which lie on either side of a region of frequent breakage within FRA3B as defined by FISH analysis using cosmids from the contigs. The most proximal of the breakpoint clusters lies approximately 100 kb distal to the hRCC t(3;8) breakpoint. The distribution of these breakpoints, together with the region of frequent chromosomal breakage mapped by FISH analysis, further confirms the position of FRA3B and helps to define the extent over which its fragility is exerted. These data indicate that FRA3B comprises several hundred kilobases of DNA sequence within 3p14.2. The 350-kb contig and the cosmid library constructed from YAC YC850A6 will be essential for further characterization of the region surrounding FRA3B and in experiments to determine the molecular basis of the fragility of FRA3B.

Animals↗
PubMed1996

A gene from human chromosomal band 3p21.1 encodes a highly conserved arginine-rich protein and is mutated in renal cell carcinomas.

We have identified a gene, called ARP for Arginine-rich protein, in human chromosomal band 3p21. It is approximately 600 Kb telomeric to the ACY1 locus (Miller et al., 1989) and encodes a previously unidentified 234 amino acid long, highly basic protein. This gene is highly conserved at the DNA and RNA level. It is found in all species including hamster, rat, mouse, bovine and yeast. We have detected a point mutation (ATG50 to AGG) or deletion of ATG50 in 10 of 21 sporadic renal cell carcinomas. The mutable region is in an imperfect trinucleotide repeat in the coding region which is non-polymorphic among 50 normal individuals examined. The point mutation (ATG50 to AGG) or deletion of codon 50 removes a methionine and increases the stretch of arginines encoded by the AGG repeats in the ARP gene.

Amino Acid Sequence↗
PubMed1996

FRA3B extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites.

The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes for the formation of gaps and breaks when DNA replication is perturbed by aphidicolin or folate stress. Although rare fragile sites are known to arise through the expansion of CCG repeats, the mechanism responsible for common fragile sites is unknown. Beyond being a basic component of chromosome structure, no biological effects of common fragile sites have been convincingly shown, although suggestions have been made that breakage and recombination at these sites may sometimes be mechanistically involved in deletions observed in many tumors and in constitutional deletions. In an observation related to the high rate of recombination at fragile sites, a number of studies have shown a statistical association between the integration of transforming DNA viruses and chromosomal fragile sites. Using FISH analysis we recently identified a 1.3 Mb YAC spanning both FRA3B and the t(3;8) translocation associated with hereditary RCC. Here we report the further localization of FRA3B within this YAC. Using lambda subclones of the YAC as FISH probes, gaps and breaks were found to occur over a broad region of at least 50 kb. Neither CCG nor CAG repeats were found in this region suggesting a different mechanism for fragility than seen with rare fragile sites. We further show that an area of frequent gaps and breaks within FRA3B, defined by a lambda contig, coincides with a previously characterized site of HPV16 integration in a primary cervical carcinoma. The HPV16 integration event gave rise to a short chromosomal deletion limited to the local FRA3B region within 3p14.2. Interestingly, 3p14.2 lies within the smallest commonly deleted region of 3p in cervical cancers, which are often HPV16 associated. To our knowledge this is the first molecular characterization of an in vivo viral integration event within a confirmed fragile site region, supporting previous cytogenetic observations linking viral integration sites and fragile sites.

Bacteriophage lambda↗
PubMed1996

Construction of a consistent YAC contig for human chromosome region 3p14.1.

Chromosomal deletions and translocations of human chromosome region 3p14 are observed in various human malignancies and suggest the existence of a tumor suppressor gene locus within this region. Tumors most frequently affected by these aberrations are small-cell lung cancer and renal-cell carcinoma. In continuation of our previously published YAC contig of chromosome region 3p14.2-p14.3, we report here on the construction of a YAC contig of at least 11 Mb that consisted of 171 YACs and covers the entire subregion 3p14.1. This contig includes the t(3;8) breakpoint of a hereditary renal-cell carcinoma localized in 3p14.2 and extends into human chromosome region 3p12-p13. It defines the order of 34 DNA probes in relation to reference markers D3S6 and D3S30 as well as the human protein tyrosine phosphatase-gamma gene. For 31 DNA probes we identified nonchimeric YACs by fluorescence in situ hybridization. The minimal tilling pathway consists of 16 yeast artificial chromosomes. As a prerequisite for identification of a putative tumor suppressor gene within this region, this contig renders human chromosome region 3p14.1 accessible to gene isolation.

Blotting, Southern↗
PubMed1996

A cloned major Schistosoma mansoni egg antigen with homologies to small heat shock proteins elicits Th1 responsiveness.

In schistosomiasis mansoni, soluble egg antigens of the worm induce chronic T-cell-mediated granulomatous tissue responses. Since the first preparation of crude soluble egg antigen extract, a dearth of highly purified antigens has hampered the identification of granuloma inducer molecules. Here we report that a cloned 38-kDa egg polypeptide (r38) with homologies to small heat shock proteins is a strong immunogen. The recombinant and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated and eluted native 38-kDa (p38) polypeptides, used in microgram amounts and unaided by adjuvant, sensitized mice for a Th1-type immune response, with strong interleukin-2 (IL-2) and gamma interferon secretion but no IL-4 and IL-10 secretion. Extensive cross-reactivity between these two polypeptides was evident. THis pattern was confirmed by reverse transcription-PCR that showed strong IL-2 and gamma interferon message expression but trace amounts of IL-4 message expression in r38-sensitized splenocytes. In mice, the polypeptide induced pulmonary mononuclear granuloma formation around antigen-coupled beads or worm eggs. We propose that the superior immunogenicity of r38 is linked to its relatedness to small heat shock proteins and that the 38-kDa polypeptide may induce the Th1 cytokine responses observed during the early development phase of the egg-induced granuloma.

Animals↗
PubMed1996

Precise localisation of 3p25 breakpoints in four patients with the 3p-syndrome.

In patients with the 3p-syndrome, hemizygous deletion of 3p25-pter is associated with profound growth failure, characteristic facial features, and mental retardation. We performed a molecular genetic analysis of 3p25 breakpoints in four patients with the 3p- syndrome, and a fifth patient with a more complex abnormality, 46,XY,der(3)t(3;?)(p25.3;?). EBV transformed lymphoblasts from each of the patients were initially characterised using fluorescent in situ hybridisation (FISH) and polymorphic microsatellite analyses. The 3p-chromosome from each patient was isolated from the normal chromosome 3 in somatic cell hybrid lines and subsequently analysed with polymorphic and monomorphic PCR amplifiable markers from 3p25. The analysis clearly shows that all five breakpoints are distinct. Furthermore, we have identified yeast artificial chromosomes that cross the 3p25 breakpoints of all four 3p-patients. Two of the patients were deleted for the von Hippel-Lindau (VHL) tumour suppressor gene, although neither has yet developed evidence of VHL disease. The patient with the most centromeric breakpoint, between D3S1585 and D3S1263, had the most severe clinical phenotype including an endocardial cushion defect that was not observed in any of the four patients who had more telomeric breakpoints. This study should provide useful insights into critical regions within 3p25 that are involved in normal human growth and development.

Abnormalities, Multiple↗
PubMed1995

Precise localization of aphidicolin-induced breakpoints on the short arm of human chromosome 3.

The common fragile site at 3p14.2 (FRA3B) has been described as the most active fragile site in the human genome. This locus may predispose chromosome 3 to specific losses due to deletions and translocations that have been associated with several malignancies, including hereditary renal cell carcinoma. We have previously described induction of breakage around FRA3B using aphidicolin in a somatic cell hybrid whose only human component was a single intact chromosome 3. That work led to the isolation of hybrids with breakpoints in the 3p13-p21.1 region with loss of all sequences distal to their respective breakpoints. In this report we describe the further characterization of the breakpoints in many of these cell lines using newly available molecular markers. We also report the identification of YAC clones that span the breakpoints present in many of these hybrids.

Aphidicolin↗
PubMed1995

Isolation and characterization of a 1 Mb region of 5q23.3-q31.2 surrounding the human lysyl oxidase gene.

Lysyl oxidase (EC 1.4.3.13) plays a pivotal role in the maintenance of tissue integrity in both the normal and pathological states. It is a member of a newly discovered gene family that exhibits a complex mode of regulation. To date the resources necessary to begin to address its regulation have not been assembled. In part, this reflects the instability of this region of the genome when cloned into cosmid vectors. The paucity of long range restriction endonuclease sites suitable for mapping this region of the genome has further hampered progress. To begin to address this issue 2 YAC clones of 920 kb and 245 kb that contain the human lysyl oxidase gene were isolated. Long range physical mapping revealed that the 245 kb clone was centrally located within the 920 kb clone. The corresponding map of this region is congruent with that observed in the human genome. Thus, these YACs faithfully represent this region of the human genome. The results of our cloning and mapping studies described in this communication should accelerate the advance of our understanding of this new connective tissue gene family.

Base Sequence↗
PubMed1995

Cloning of a putative human voltage-gated chloride channel (CIC-2) cDNA widely expressed in human tissues.

We have cloned a cDNA from the human epithelial cell line T84 whose predicted amino acid sequence shows 93.9% identity with rat CIC-2. Mapping by somatic cell hybrids and polymerase chain reaction localizes the gene corresponding to this cDNA to chromosome 3q26-qter. The major transcription start site assessed by RNA primer extension is 100 nt upstream of the putative translation initiation codon. Analysis of the 5' flanking sequence revealed a high GC content and lack of common transcriptional elements such as TATA and CCAAT boxes. Northern blot analysis indicated wide organ distribution including tissues affected in cystic fibrosis (CF) and expression in an airway epithelial cell line derived from a CF patient. The high degree of sequence similarity and similar tissue distribution to rat CIC-2 suggests that this cDNA encodes the human CIC-2 voltage-gated chloride channel. Since this chloride channel is present in epithelial tissues it may be amenable to manipulation to circumvent the chloride secretion defect observed in CF.

Amino Acid Sequence↗
PubMed1994

Genetic instability of microsatellite sequences in many non-small cell lung carcinomas.

We have analyzed DNA obtained from 38 lung tumors and normal lung or blood DNA for microsatellite instability. Instability was examined at 10 different microsatellite loci on chromosome 3p, as well as loci on 3q, 11p, 11q, and 13q, and two on Xq. We observed microsatellite instability at one or more loci in 13 of the lung tumors analyzed, and this instability ranged from tumors showing instability in only a single microsatellite to two adenocarcinomas that had alterations in all 16 tested microsatellites. Microsatellite instability could therefore play a significant role in the development of a sizable portion of lung tumors.

Alleles↗