Topo IIIalpha and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents.

The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.

Loss of homologous recombination or non-homologous end-joining leads to radial formation following DNA interstrand crosslink damage.

High levels of interstrand cross-link damage in mammalian cells cause chromatid breaks and radial formations recognizable by cytogenetic examination. The mechanism of radial formation observed following DNA damage has yet to be determined. Due to recent findings linking homologous recombination and non-homologous end-joining to the action of the Fanconi anemia pathway, we speculated that radials might be the result of defects in either of the pathways of DNA repair. To test this hypothesis, we have investigated the role of homologous recombination proteins RAD51 and RAD52, non-homologous end-joining proteins Ku70 and LIG4, and protein MRE11 in radial formation and cell survival following interstrand crosslink damage with mitomycin C. For the studies we used small inhibitory RNA to deplete the proteins from cells, allowing for evaluation of radial formation and cell survival. In transformed normal human fibroblasts, depletion of these proteins increased interstrand crosslink sensitivity as manifested by decreased cell survival and increased radial formation. These results demonstrate that inactivation of proteins from either of the two separate DNA repair pathways increases cellular sensitivity to interstrand crosslinks, indicating each pathway plays a role in the normal response to interstrand crosslink damage. We can also conclude that homologous recombination or non-homologous end-joining are not required for radial formation, since radials occur with depletion of these pathways.

Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression.

Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis.

Mammalian SNM1 is required for genome stability.

The protein encoded by SNM1 in Saccharomyces cerevisiae has been shown to act specifically in DNA interstrand crosslinks (ICL) repair. There are five mammalian homologs of SNM1, including Artemis, which is involved in V(D)J recombination. Cells from mice constructed with a disruption in the Snm1 gene are sensitive to the DNA interstrand crosslinker, mitomycin (MMC), as indicated by increased radial formation following exposure. The mice reproduce normally and have normal life spans. However, a partial perinatal lethality, not seen in either homozygous mutant alone, can be noted when the Snm1 disruption is combined with a Fancd2 disruption. To explore the role of hSNM1 and its homologs in ICL repair in human cells, we used siRNA depletion in human fibroblasts, with cell survival and chromosome radials as the end points for sensitivity following treatment with MMC. Depletion of hSNM1 increases sensitivity to ICLs as detected by both end points, while depletion of Artemis does not. Thus hSNM1 is active in maintenance of genome stability following ICL formation. To evaluate the epistatic relationship between hSNM1 and other ICL repair pathways, we depleted hSNM1 in Fanconi anemia (FA) cells, which are inherently sensitive to ICLs. Depletion of hSNM1 in an FA cell line produces additive sensitivity for MMC. Further, mono-ubiquitination of FANCD2, an endpoint of the FA pathway, is not disturbed by depletion of hSNM1 in normal cells. Thus, hSNM1 appears to represent a second pathway for genome stability, distinct from the FA pathway.

Demonstration of Philadelphia chromosome negative abnormal clones in patients with chronic myelogenous leukemia during major cytogenetic responses induced by imatinib mesylate.

Imatinib mesylate, an Abl-specific kinase inhibitor, produces sustained complete hematologic responses (CHR) and major cytogenetic responses (MCR) in chronic myeloid leukemia (CML) patients, but long-term outcomes in these patients are not yet known. This article reports the identification of clonal abnormalities in cells lacking detectable Philadelphia (Ph) chromosome/BCR-ABL rearrangements from seven patients with chronic- or accelerated-phase CML, who were treated with imatinib. All seven patients were refractory or intolerant to interferon therapy. Six of seven patients demonstrated MCR and one patient, who had a cryptic translocation, achieved low-level positivity (2.5%) for BCR-ABL by fluorescence in situ hybridization. The median duration of imatinib treatment before the identification of cytogenetic abnormalities in BCR-ABL-negative cells was 13 months. The most common cytogenetic abnormality was trisomy 8, documented in three patients. All patients had varying degrees of dysplastic morphologic abnormalities. One patient exhibited increased numbers of marrow blasts, yet consistently demonstrated no Ph-positive metaphases and the absence of morphologic features of CML. The presence of clonal abnormalities in Ph-negative cells of imatinib-treated CML patients with MCR and CHR highlights the importance of routine metaphase cytogenetic testing and long-term follow-up of all imatinib-treated patients.

The 4N cell cycle delay in Fanconi anemia reflects growth arrest in late S phase.

Fanconi anemia (FA) is a human genetic disorder characterized by hypersensitivity to DNA crosslinking agents. Its cellular phenotypes include increased chromosome breakage and a marked cell-cycle delay with 4N DNA content after introduction of interstrand DNA crosslinks (ICL). To further understand the nature of this delay previously described as a G2/M arrest, we introduced ICL specifically during G2 and monitored the cells for passage into mitosis. Our results showed that, even at the highest doses, postreplication ICL produced neither G2/M arrest nor chromosome breakage in FA-A or FA-C cells. This suggests that, similar to wild-type cells, DNA replication is required to trigger both responses. Therefore, the 4N cell DNA content observed in FA cells after ICL treatment also represents incomplete DNA replication and arrest in late S phase. FA fibroblasts from complementation groups A and C were able to recover from the ICL-induced cell-cycle arrest, but took approximately 3 times longer than controls. These results indicate that the FA pathway is required for the efficient resolution of ICL-induced S-phase arrest.

SNRPN methylation patterns in germ cell tumors as a reflection of primordial germ cell development.

Studies examining altered imprinted gene expression in cancer compare the observed expression pattern to the normal expression pattern for a given tissue of origin, usually the somatic expression pattern for the imprinted gene. Germ cell tumors (GCTs), however, require a developmental stage-dependent comparison. To explore using methylation as an indicator of germ cell development, we determined the pattern of methylation at the 5' untranslated region of SNRPN in 89 GCTs from both children and adults. Fifty-one of 84 tumors (60.7%) (12/30 (40%) of cultured pediatric GCTs, 23/36 (63.9%) of frozen adult GCTs, and 16/23 (69.5%) of frozen pediatric GCTs, with five samples having results from both cultured and uncultured material) demonstrated a nonsomatic methylation pattern after dual digestion with XbaI, NotI, and Southern blot analysis. In contrast, only 2 of 18 (11%) control samples (16 non-GCTs and 2 normal ovaries) exhibited a nonsomatic pattern. In both cases, the result was shown to be due to copy number differences between maternal and paternal homologs, unlike the GCTs in which there was no evidence of an uneven homolog number. A comparison of the data for only the gonadal GCTs and the control data showed a highly significant difference in the proportion of tumors with methylation alterations at this locus (P = 0.0000539). Since there is no published evidence of the involvement of SNRPN methylation changes in the development of malignancy, the data suggest that the methylation pattern of SNRPN in GCTs reflects that of the primordial germ cell giving rise to the tumor.

Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor.

The trace amine para-tyramine is structurally and functionally related to the amphetamines and the biogenic amine neurotransmitters. It is currently thought that the biological activities elicited by trace amines such as p-tyramine and the psychostimulant amphetamines are manifestations of their ability to inhibit the clearance of extracellular transmitter and/or stimulate the efflux of transmitter from intracellular stores. Here we report the discovery and pharmacological characterization of a rat G protein-coupled receptor that stimulates the production of cAMP when exposed to the trace amines p-tyramine, beta-phenethylamine, tryptamine, and octopamine. An extensive pharmacological survey revealed that psychostimulant and hallucinogenic amphetamines, numerous ergoline derivatives, adrenergic ligands, and 3-methylated metabolites of the catecholamine neurotransmitters are also good agonists at the rat trace amine receptor 1 (rTAR1). These results suggest that the trace amines and catecholamine metabolites may serve as the endogenous ligands of a novel intercellular signaling system found widely throughout the vertebrate brain and periphery. Furthermore, the discovery that amphetamines, including 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy"), are potent rTAR1 agonists suggests that the effects of these widely used drugs may be mediated in part by this receptor as well as their previously characterized targets, the neurotransmitter transporter proteins.

Chromosomes 1 and 12 abnormalities in pediatric germ cell tumors by interphase fluorescence in situ hybridization.

Chromosome studies of pediatric germ cell tumors (GCTs) show differences in abnormalities dependent on age, sex, tumor location, and histology. Previous studies suggest that loss of 1p is associated with a malignant phenotype, while amplification of 12p, a common finding in adult testicular GCTs, is uncommon in pediatric GCTs. Fifty-three pediatric GCTs were analyzed for 1p36 loss and 12p amplification by G-banding and dual-color interphase FISH with probes for the centromere and short arm of chromosomes 1 or 12. Twelve tumors with loss of 1p36 were identified. No deletion was detected in tumors with nonmalignant histology, such that there was a significant association of 1p loss with malignancy in these tumors (P = 0.00115). Five of 18 tumors from male patients had amplification of 12p, consistent with G-band results. Combined analysis of our data with those in the literature revealed a significant correlation of 12p amplification with patient age (P = 0.000196). Amplification of 12p was only seen in one of 35 tumors from female patients. Five female GCTs had numerical abnormalities of chromosome 12, and two tumors showed complete lack of 12p. This spectrum of abnormalities differs from what is seen in the male tumors, providing further evidence for different etiologies of GCTs between the sexes.

Trisomy 20 mosaicism in two unrelated girls with skin hypopigmentation and normal intellectual development.

The prenatal diagnosis of trisomy 20 mosaicism presents a challenge for practitioners and parents. The diagnosis implies an uncertain risk for an inconsistent set of physical and developmental findings, as well as a substantial chance for a child that is normal physically and developmentally. We report two girls (ages nine years one month and eight years one month) with normal intelligence and hypopigmented skin areas. Both girls were born after a prenatal diagnosis of trisomy 20 mosaicism in amniocytes. Case 1 had 83% and 57% trisomy 20 cells from two separate amniocenteses and Case 2 had 90% trisomy 20 cells from an amniocentesis. Trisomy 20 was confirmed after birth in urinary sediment (25%) and chorionic villus cells (15%) in Case 1, while cord blood lymphocytes (30 cells) and skin fibroblasts (50 cells) had only 46,XX cells. Trisomy 20 was confirmed after birth in urinary sediment (100%), placenta (100%), cord (10%), amniotic membrane (50%), and skin fibroblasts (30%) in Case 2, while cord blood lymphocytes (100 cells) had only 46,XX cells. This is the first report of a hypopigmented pigmentary dysplasia associated with isolated trisomy 20 mosaicism. Our patients are the oldest reported children with trisomy 20 mosaicism confirmed after birth.

DNA replication is required To elicit cellular responses to psoralen-induced DNA interstrand cross-links.

Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.

Localization of the Fanconi anemia complementation group D gene to a 200-kb region on chromosome 3p25.3.

Fanconi anemia (FA) is a rare autosomal recessive disease manifested by bone-marrow failure and an elevated incidence of cancer. Cells taken from patients exhibit spontaneous chromosomal breaks and rearrangements. These breaks and rearrangements are greatly elevated by treatment of FA cells with the use of DNA cross-linking agents. The FA complementation group D gene (FANCD) has previously been localized to chromosome 3p22-26, by use of microcell-mediated chromosome transfer. Here we describe the use of noncomplemented microcell hybrids to identify small overlapping deletions that narrow the FANCD critical region. A 1.2-Mb bacterial-artificial-chromosome (BAC)/P1 contig was constructed, bounded by the marker D3S3691 distally and by the gene ATP2B2 proximally. The contig contains at least 36 genes, including the oxytocin receptor (OXTR), hOGG1, the von Hippel-Lindau tumor-suppressor gene (VHL), and IRAK-2. Both hOGG1 and IRAK-2 were excluded as candidates for FANCD. BACs were then used as probes for FISH analyses, to map the extent of the deletions in four of the noncomplemented microcell hybrid cell lines. A narrow region of common overlapping deletions limits the FANCD critical region to approximately 200 kb. The three candidate genes in this region are TIGR-A004X28, SGC34603, and AA609512.

Selective pressure as an essential force in molecular evolution of myeloid leukemic clones: a view from the window of Fanconi anemia.

Specific chromosomal deletions are commonly found in bone marrow cells of children with Fanconi anemia (FA) whose disease has evolved to myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Identical deletions are found in adults with MDS/AML with a history of exposure to alkylating agents (secondary MDS/AML). While deleted chromosomal regions likely harbor genes encoding proteins with tumor suppressor (TS) function, such genes have not been identified and the environmental forces by which these mutant clones are selected remain unclear. A consistent signaling abnormality in cells bearing mutations of the Fanconi anemia complementation group C (FA-C) gene (FANCC) has revealed a potential selective force. Hematopoietic progenitor cells from patients and mice with FANCC mutations are hypersensitive to the inhibitory effects of IFNgamma and TNFalpha. Consequently, clonal outgrowths in FA likely result from strong selective pressure for stem and/or progenitor cells resistant to these inhibitory cytokines. Additional mutations that inactivate signaling pathways for these inhibitors would create a cell with a profound proliferative advantage over its apoptosis-prone counterparts. Here, we present preliminary evidence supporting a selection-based model of leukemic evolution and argue that MDS in FA patients is a de facto model of secondary MDS in non-FA adults.

Chromosome abnormalities of eighty-one pediatric germ cell tumors: sex-, age-, site-, and histopathology-related differences--a Children's Cancer Group study.

The chromosomes of 81 pediatric germ cell tumors (GCTs) were analyzed as part of two clinical treatment trials, INT-0098 and INT-0097, conducted by the Children's Cancer Group. The analysis of chromosome results showed differences with respect to sex, age, tumor location, and histology. Sixteen of 17 benign teratomas of infants and children less than 4 years old and from gonadal and extragonadal locations were chromosomally normal. Twenty-three malignant GCTs from gonadal and extragonadal locations of the same age group were endodermal sinus tumors and varied in their karyotypic findings. The most common abnormalities were gains of 1q and chromosome 3. Of eight benign ovarian teratomas from older girls, five with normal G-banded karyotypes were determined to be homozygous for Q-band heteromorphisms, suggesting a meiosis II error. Among the 12 malignant ovarian GCTs from older girls, the common abnormalities were loss of 1p/gain of 1q, +3, +8, +14, and +21. Four of eight extragonadal tumors from older boys demonstrated +21; one had +X. Five of the eight had associated constitutional chromosome abnormalities, including one trisomy 21 and three with Klinefelter syndrome. The testicular GCTs of adolescents had abnormalities resembling those found in adult testicular GCT, including near-triploidy, loss of chromosomes 11, 13, and 18, and gain of chromosomes 7, 8, the X chromosome, and an isochromosome 12p. The gain of an isochromosome 12p was only frequent in the tumors from adolescent boys. Deletion of 1p/gain of 1q and +3 were the most common abnormalities among the malignant tumors from both sexes.

Environmental contamination with vancomycin-resistant enterococci in an outpatient setting.

Eleven cancer patients colonized with vancomycin-resistant enterococci (VRE) were followed as outpatients. Environmental cultures were obtained from clinic rooms before and after patients care. Environmental contamination occurred in 29% of encounters. Superficial disinfection did not eradicate contamination, although more thorough cleaning did. We conclude that environmental VRE contamination occurs in the outpatient setting. Infection control practices, similar to those used in the inpatient setting, may be necessary for outpatient clinics.

Clinical outcomes of four patients with microdeletion in the long arm of chromosome 2.

We present clinical outcome, through several years of follow-up, of 4 mentally retarded patients, each with a small interstitial deletion in the long arm of chromosome 2, within a region on which clinical reports are infrequent. Our patient 1 was found to have del(2)(q22.3q23.3); patients 2 and 3, del(2)(q23.3q24.2); and patient 4, del(2) (q24.2q31). By comparison of our cases with each other and with those previously published with comparable interstitial deletion, we attempted to identify characteristic clinical findings. Short neck with excessive cervical skin was seen with monosomy of chromosome 2 bands q22.3-q23.3, while hypertrichosis and a peculiar high pitched cry were seen with monosomy of chromosome 2 bands q23.3-q24.2. As suggested by Moller et al. [1984: Hum Genet 68:77-86], a cleft between the first and second toes was seen with monosomy of chromosome 2 bands q24.2-q31. In addition, seizure disorder was present in patients 1 and 4 (with the more proximal and distal deletions, respectively).

Functional complementation by electroporation of human BACs into mammalian fibroblast cells.

Bacterial Artificial Chromosomes (BACs) have been used to complement a metabolic defect and to transfer a drug resistance marker into mammalian cells by electroporation. The selectable markers are stable and the recipient cells have BAC DNA integrated into the chromosomes as shown by fluorescent in situ hybridization, PCR and Southern hybridization.

DNA cross-linker-induced G2/M arrest in group C Fanconi anemia lymphoblasts reflects normal checkpoint function.

Cells from individuals with Fanconi anemia (FA) arrest excessively in the G2/M cell cycle compartment after exposure to low doses of DNA cross-linking agents. The relationship of this abnormality to the fundamental genetic defect in such cells is unknown, but many investigators have speculated that the various FA genes directly regulate cell cycle checkpoints. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FAC) functions to control a cell cycle checkpoint and that cells from group C patients (FA[C]) have abnormalities of cell cycle regulation directly related to the genetic mutation. We found that retroviral transduction of FA(C) lymphoblasts with wild-type FAC cDNA resulted in normalization of the cell cycle response to low-dose mitomycin C (MMC). However, when DNA damage was quantified in terms of cytogenetic damage or cellular cytotoxicity, we found similar degrees of G2/M arrest in response to equitoxic amounts of MMC in FA(C) cells as well as in normal lymphoblasts. Similar results were obtained using isogenic pairs of uncorrected, FAC- or mock-corrected (neo only) FA(C) cell lines. To test the function of other checkpoints we examined the effects of hydroxyurea (HU) and ionizing radiation on cell cycle kinetics of FA(C) and normal lymphoblasts as well as with isogenic pairs of uncorrected, FAC-corrected, or mock-corrected FA(C) cell lines. In all cases the cell cycle response of FA(C) and normal lymphoblasts to these two agents were identical. Based on these studies we conclude that the aberrant G2/M arrest that typifies the response of FA(C) cells to low doses of cross-linking agents does not represent an abnormal cell cycle response but instead represents a normal cellular response to the excessive DNA damage that results in FA(C) cells following exposure to low doses of cross-linking agents.

Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism.

About 2 per cent of specimens from chorionic villus sampling (CVS) analysed either on direct preparation of cytotrophoblast cells or after culture of mesenchymal stroma reveal confined placental mosaicism (CPM), most commonly involving chromosomal trisomy. A significantly higher rate of prenatal loss (22 per cent) as well as the presence of intrauterine growth retardation (IUGR) has been reported among pregnancies with CPM. To evaluate more precisely the effect of these aneuploid cell lines confined to the placenta on intrauterine fetal growth and fetal survival, we have studied 34 term placentae from pregnancies with CPM diagnosed on CVS and confirmed identical mosaicism in 17 of these placentae. There was a direct correlation between a high number of aneuploid cells present at CVS and a high likelihood of their detection in term placenta. Also, the proportion of aneuploid cells in the mosaic term placentae correlated with that observed in CVS specimens. Among 17 gestations with confirmed CPM at delivery, there were six cases of IUGR identified, five in liveborns and one associated with intrauterine death.