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M Buchwald

Publications and source records attributed to M Buchwald.

At least 19 recordsLinked to original sources
PubMed1995

Classification of Fanconi anemia patients by complementation analysis: evidence for a fifth genetic subtype.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms, life-threatening progressive panmyelopathy, and cellular hypersensitivity to cross-linking agents. Currently, 4 genetic subtypes or complementation groups (FA-A through FA-D) have been distinguished among 7 unrelated FA patients. We report the use of genetically marked FA lymphoblastoid cell lines representing each of the 4 presently known complementation groups to classify 13 unrelated FA patients through cell fusion and complementation analysis. Twelve cell lines failed to complement cross-linker sensitivity in fusion hybrids with only 1 of the 4 reference cell lines and could thus be unambiguously classified as FA-A (7 patients), FA-C (4 patients), or FA-D (1 patient). One cell line complemented all 4 reference cell lines and therefore represents a new complementation group, designated FA-E. These results imply that at least 5 genes appear to be involved in a pathway that, when defective, causes bone marrow failure in FA patients.

Cell Fusion↗
PubMed1995

CFTR expression and organ damage in cystic fibrosis.

The cloning of the defective gene in cystic fibrosis (CFTR) is the most important step to date toward understanding the pathogenesis of the disease and developing novel therapeutic strategies. Although many studies have provided insights into the molecular defects and knowledge of the expression and role of the gene, the basic defect and its pathogenesis are still unclear. We hypothesize that organ damage in cystic fibrosis is the result of a combination of at least three main factors: the genotype (the type of mutation that alters the function of the cystic fibrosis transmembrane regulator [CFTR]), the rate of CFTR-mediated chloride secretion in the epithelium of each organ (inferred from the level of expression of the gene), and the anatomical and physiologic characteristics of the affected organs (the size and contents of the ducts). Confirmation of this hypothesis should allow a better understanding of the pathogenesis of the disease and help prevent organ damage.

Chloride Channels↗
PubMed1995

Human cDNA clones that modify radiomimetic sensitivity of ataxia-telangiectasia (group A) cells.

Genes responsible for genetic diseases with increased sensitivity to DNA-damaging agents can be identified using complementation cloning. This strategy is based on in vitro complementation of the cellular sensitivity by gene transfer. Ataxia-telangiectasia (A-T) is a multisystem autosomal recessive disorder involving cellular sensitivity to ionizing radiation and radiomimetic drugs. A-T is genetically heterogeneous, with four complementation groups. We attempted to identify cDNA clones that modify the radiomimetic sensitivity of A-T cells assigned to complementation group [A-T(A)]. The cells were transfected with human cDNA libraries cloned in episomal vectors, and various protocols of radiomimetic selection were applied. Thirteen cDNAs rescued from survivor cells were found to confer various degrees of radiomimetic resistance to A-T(A) cells upon repeated introduction, and one of them also partially influenced another feature of the A-T phenotype, radioresistant DNA synthesis. None of the clones mapped to the A-T locus on chromosome 11q22-23. Nine of the clones were derived from known genes, some of which are involved in cellular stress responses. We concluded that a number of different genes, not necessarily associated with A-T, can influence the response of A-T cells to radiomimetic drugs, and hence the complementation cloning approach may be less applicable to A-T than to other diseases involving abnormal processing of DNA damage.

Antibiotics, Antineoplastic↗
PubMed1995

Characterization of the 5' region of the Fanconi anaemia group C (FACC) gene.

Fanconi anaemia (FA) is an autosomal recessive disease characterised by progressive pancytopenia, chromosome instability and an increased risk of cancer. The Fanconi Anaemia Complementation Group C (FACC) gene is mutated in patients of complementation group C. Several different forms of FACC mRNA that share the same coding region have been isolated. At least two species result from the use of alternative exons at the 5' end and three result from the use of distinct polyadenylation signals. As a first step toward the characterization of this gene we have isolated the genomic clones corresponding to the 5' region, including a putative promoter and two alternate 5' exons. These exons, named -1 and -1a, were found to be separated by a small intron, with exon -1 located 5' to exon -1a. Further, these exons are flanked by consensus sequences of donor sites at the 5' ends of introns. An acceptor splice site was not evident 5' of exon -1a, suggesting that exon -1 is not spliced onto exon -1a. The sequences upstream of exons -1 and -1a have no obvious TATA or CAAT boxes but include CG-rich sequences. Functional analysis of the sequence upstream of the putative transcription start site of both alternative exons indicates that the region upstream exon -1 is sufficient to drive the expression of the luciferase reporter gene in CaCo-2 cells and that the transcriptional regulation of this gene is complex.

Base Sequence↗
PubMed1994

Phosphatase inhibitors activate normal and defective CFTR chloride channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis.

1-Methyl-3-isobutylxanthine↗
PubMed1994

A functionally active retrovirus vector for gene therapy in Fanconi anemia group C.

Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of < or = 5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.

3T3 Cells↗
PubMed1994

Fanconi anemia revisited: old ideas and new advances.

This review summarizes both historical and more recent data on the clinical, cellular and genetic features of Fanconi anemia (FA), a rare autosomal recessive disorder. FA patients are characterized by pancytopenia, congenital malformations, growth delay and an increased susceptibility to the development of malignancies, particularly acute myelogenous leukemia. FA cells show chromosomal fragility, slow growth and increased sensitivity to DNA crosslinking agents. FA can be caused by defects in any one of at least four genes. Two general hypotheses have been proposed to explain the underlying defect: loss of a DNA repair function or of a step in the defense toward oxygen toxicity. After many attempts to clone the FA genes, the first one, that defective in group C, has been cloned by complementation of the increased sensitivity of FA(C) cells to mitomycin C and diepoxybutane. This gene (FACC) codes for a novel protein and is ubiquitously expressed. Mutations in various FA(C) patients that cause loss of function have been identified. The review concludes by suggesting directions for future research in FA.

Animals↗
PubMed1994

Screening cosmid libraries with oligonucleotides corresponding to splice-site consensus sequences.

To facilitate the identification of genes within genomic DNA, we have developed a method based on the use of short oligonucleotides designed from the consensus sequences of splice sites. We describe here the hybridization and washing conditions under which such oligonucleotides can be used to screen cosmid libraries. We confirm the presence of genes within cosmids identified by screening with one oligonucleotide by showing that DNA isolated from such cosmids will hybridize to another splice-site oligonucleotide.

Animals↗
PubMed1994

Regional expression of CFTR in developing human respiratory tissues.

Morbidity and mortality in cystic fibrosis (CF) patients is strongly related to their respiratory disease. We have analyzed, by means of in situ hybridization, the localization and levels of CFTR mRNA in fetal, newborn, and infant respiratory tissues. Measurable levels of CFTR transcript are present in the fetal primordial epithelium of the pseudoglandular stage lung. During the following stages of lung development, CFTR expression decreases in cells of the future alveolar spaces and is gradually limited to the epithelium of the small airways. After birth, expression decreases in the small airways and is not detected in alveolar epithelia. In trachea and large bronchi, a differential pattern of expression is also observed. No CFTR expression is found in fetal submucosal glands during fetal development, but appears gradually in the newborn period. Since CFTR codes for a secretory Cl- channel, these data probably reflect the changes that occur in the lung transition from a fluid-secreting to an absorbing organ. The pattern of expression seems paradoxical in view of the clinical-pathological manifestations of CF. Although CFTR is expressed in the normal fetus and lung development is influenced by the amount of fetal lung liquid, newborns affected with CF have normal lungs. In addition, the earliest pathologic change described in CF lungs in hyperplasia of the submucosal glands, yet expression in these structures is seen only after birth. An improved understanding of the factors that alter the expected relationship between CFTR expression and pathologic lesions in the fetal lung may provide important insights into the pathogenesis and potential treatment of lung disease in CF patients.

Child, Preschool↗
PubMed1994

Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line.

We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogen-coated filters was permeabilized with nystatin, a forskolin-stimulated Cl- permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogen-regulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them.

Adenoviridae↗
PubMed1994

Differential cellular expression of cystic fibrosis transmembrane regulator in human reproductive tissues. Clues for the infertility in patients with cystic fibrosis.

Cystic fibrosis (CF) is characterized by a wide spectrum of clinical manifestations, including reproductive problems. Practically all males affected by the disease are infertile due to azoospermia associated with pathology of the male ducts, whereas females with CF have reduced fertility. To study the mechanism of reproductive pathology in CF patients, we analyzed the levels and localization of expression of the cystic fibrosis transmembrane regulator (CFTR) gene in relevant postnatal tissues. Significant expression was detected in the epithelium of the epididymis and vas deferens. Minimal expression, not associated with specific cell types, was seen in the mature testis. In female genitalia, variable levels of expression were seen in the cervical epithelium and fallopian tube. The endometrial epithelium and glands expressed CFTR at high levels only after puberty. No expression was seen in ovaries. Deficient secretory function of CFTR in males but not in females may lead to organ damage probably as a consequence of excessive concentration of viscid luminal contents.

Adult↗
PubMed1993

Mapping of the murine and rat Facc genes and assessment of flexed-tail as a candidate mouse homolog of Fanconi anemia group C.

Fanconi anemia is a rare, autosomal recessive disorder characterized at the cellular level by a combination of hypersensitivity to DNA-damaging agents and chromosomal instability. Clinical features include pancytopenia, often associated with specific congenital malformations, and a predisposition to leukemia. We previously cloned the gene defective in Fanconi anemia group C by complementation of the intrinsic sensitivity of Fanconi anemia cells to DNA cross-linking agents, and we recently cloned its mouse homolog (Facc). In this report, we localized Facc to mouse Chromosome (Chr) 13 and its rat homolog to rat Chr 17. A previously described anemic mouse mutant, flexed-tail, maps to the same chromosomal region. Differences were detected between DNA of the flexed-tail and congenic mice, indicating the proximity of the Facc probe to the disease mutation. Analysis of flexed-tail RNA did not reveal detectable difference in Facc message level or size between flexed-tail and congenic mice. On this basis, we conclude that, although flexed-tail remains a candidate for Fanconi anemia in the mouse, there is no evidence currently that Facc is mutated in flexed-tail mice.

Animals↗
PubMed1993

CFTR expression is regulated during both the cycle of the seminiferous epithelium and the oestrous cycle of rodents.

Severely reduced fertility is a common finding in cystic fibrosis (CF). We used in situ hybridization to examine the cell-specific expression of CFTR in the reproductive organs of rodents. In males CFTR mRNA is found in the round spermatids (spermatogenic stages V-X) and in the principal cells that line the initial segment of the epididymis. In both the testis and the epididymis, CFTR expression is developmentally regulated suggesting that the defect in the genital tract of male CF patients is of developmental origin. CFTR expression in the luminal and glandular epithelium of the uterus is regulated during the oestrous cycle and is maximal at pro-oestrus. Our results provide a biological rationale for the reduced fertility of CF patients, and suggest a possible cause for the comparatively poorer prognosis for women with CF.

Animals↗
PubMed1993

Cell-specific localization of CFTR mRNA shows developmentally regulated expression in human fetal tissues.

An improved understanding of the expression of the cystic fibrosis gene (CFTR) will assist our approach to preventing the organ damage caused by cystic fibrosis (CF). We have studied the expression of CFTR in human fetal tissues at different gestational ages using in situ hybridization to detect CFTR mRNA. CFTR was principally expressed in less differentiated cells of endodermal origin. The highest levels were seen in specific areas of the developing pancreas, liver, gall bladder and intestine, with lower but significant levels in lung and trachea. Expression was also seen in reproductive tissues, such as epididymis and third trimester uterus and fallopian tubes, and in addition, sweat and salivary glands. No detection of CFTR mRNA was found in many other relevant tissues. The detection of CFTR transcript in these organs is consistent with the clinical manifestations of CF and the function of CFTR as a chloride channel early in development. The localization and levels of expression described have implications regarding the pathogenesis of organ damage and the potential gains that can be achieved by early therapy in the disease.

Cystic Fibrosis↗
PubMed1993

Cloning and analysis of the murine Fanconi anemia group C cDNA.

Fanconi anemia (FA) is one of a group of disorders characterized at the cellular level by a combination of hypersensitivity to DNA-damaging agents, chromosomal instability, and defective DNA repair. Clinical features of FA include pancytopenia, often accompanied by specific congenital malformations, and a predisposition to leukemia. Since the hematological manifestations are the critical defect in terms of prognosis, FA is a candidate disease for gene replacement therapy, and the development of a mouse model system is essential for the initial stages of this work. Previously, we have cloned the gene defective in FA group C by complementation of the intrinsic sensitivity of FA cells to DNA cross-linking agents. We have now cloned the murine homologue of the human FACC cDNA. The mouse cDNA (Facc) shares 79% amino acid sequence similarity with the human gene product. The expression of the mouse cDNA in human FA(C) cells restores the cellular drug sensitivity to normal levels. Thus, the function of the protein has been conserved despite the significant sequence divergence. PCR analysis of mouse tissue RNA reveals that the gene is expressed in all adult tissues, while in situ RNA hybridization experiments show tissue specific expression at late stages of fetal development. Cross-hybridizing sequences exist in DNA from other mammals, chicken and Drosophila. These results support the hypothesis that the FACC gene product has a role in a basic aspect of cellular protection against DNA damaging agents and that this function has been conserved during evolution.

Amino Acid Sequence↗
PubMed1993

A nonsense mutation and exon skipping in the Fanconi anaemia group C gene.

Fanconi anaemia (FA) is an autosomal recessive disorder associated with bone-marrow failure and hypersensitivity to DNA cross-linking agents. At least four complementation groups have been defined, and a cDNA which corrects the defect in group C cells (FACC) has recently been isolated. We have screened the FACC coding sequence for mutations in FA patients and found one patient to be homozygous for a nonsense mutation in exon 6 of the FACC coding sequence (R185X). Exon 6 was spliced out of a proportion of this patient's transcripts, providing further support for the proposal that nonsense mutations may alter splice site selection. Alternatively spliced transcripts which lacked exon 13 were detected in both patients and controls.

Base Sequence↗