The genetics of bladder cancer: a cytogeneticist's perspective.

Cytogenetic studies of bladder cancer have helped to define two clinically distinct subtypes: benign tumors with few genetic mutations and a stable karyotype and aggressive cancers with chromosomal instability and many non-random cytogenetic aberrations. While the cytogenetic data does not provide complete information, these studies have been important for suggesting pathways for bladder carcinoma initiation and its progression. In addition, molecular cytogenetic studies have proven useful for diagnosing bladder cancer and for monitoring patients for cancer recurrence. More detailed molecular genetic studies and expression array analyses are needed to fully comprehend the biologic processes associated with urothelial cancers, but cytogenetics studies have laid the foundation for further investigation.

A non-sex chromosome marker in a patient with an atypical Ullrich-Turner phenotype and mosaicism of 46,X,mar/46,XX.

The absence of a sex chromosome in conjunction with the presence of a marker chromosome generally implicates a sex chromosome origin for such marker chromosomes. These types of findings are frequently associated with Ullrich-Turner syndrome. We report a patient that presented with an atypical Ullrich-Turner phenotype and a cytogenetic mosaicism of 46,X,mar/46,XX. The marker chromosome was derived from chromosome 20, not from the X or Y chromosome. The patient's clinical features are described and discussed relative to the cytogenetic findings. This case further demonstrates the necessity of marker chromosome identification for accurate phenotype-karyotype correlation.

C60-Fullerene monomalonate adducts selectively inactivate neuronal nitric oxide synthase by uncoupling the formation of reactive oxygen intermediates from nitric oxide production.

C(60)-Fullerene monomalonate adducts inactivate selectively the neuronal nitric oxide synthase isoform in a manner completely preventable by the concurrent presence of superoxide dismutase and catalase. This inactivation is time-, fullerene concentration-, and turnover-dependent and is not reversible by dilution. The di(carboxypropan-3-ol)methano-[60]-fullerene (diol adduct) has no effect on NADPH consumption by nNOS as measured in the absence of arginine substrate, but dramatically increases NADPH consumption in the presence of arginine. This fullerene-enhanced NADPH consumption is linked to oxygen as electron acceptor and is accompanied by the increased production of hydrogen peroxide. These effects of fullerene monomalonate adducts are unique to the nNOS isoform and are not observed using either the iNOS or the eNOS isoform. The inhibitory effects of fullerene monomalonate adducts are unaltered and insurmountable by increased concentrations of arginine, tetrahydrobiopterin, or calmodulin. These observations indicate that fullerene monomalonate adducts uncouple in the presence of arginine the formation of reactive oxygen intermediates from NO production by nNOS. These reactive oxygen intermediates dissociate from the enzyme and, acting from solution, inactivate NOS NO forming activity.

Inhibition of nitric oxide synthase isoforms by tris-malonyl-C(60)-fullerene adducts.

C(3)-tris-malonyl-C(60)-fullerene and D(3)-tris-malonyl-C(60)-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citrulline formation by C(3)-tris-malonyl-C(60)-fullerene occurs with IC(50) values of 24, 17, and 123 microM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 microM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 microM C(3)-tris-malonyl-C(60)-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C(3)-tris-malonyl-C(60)-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC(50) value of 22 microM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 microM. The inhibition of nNOS activity by C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of product formation but did not alter the EC(50) value for activation by calmodulin. C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K(m) for l-arginine substrate or the EC(50) value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C(3)-tris-malonyl-C(60)-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C(3)-tris-malonyl-C(60)-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface.

Cellular and enzymatic studies of N(omega)-propyl-l-arginine and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea as reversible, slowly dissociating inhibitors selective for the neuronal nitric oxide synthase isoform.

N(omega)propyl-l-arginine (NPA) and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea (TFMPITU) inhibit selectively the neuronal nitric oxide (NO) synthase (nNOS) isoform. In the presence of Ca(2+) and calmodulin (CaM), NPA and TFMPITU produce a time- and concentration-dependent suppression of nNOS catalyzed NO formation. This suppression of activity occurs by a first order kinetic process as revealed from linear Kitz-Wilson plots but does not depend on catalytic turnover since it occurs in the absence of NADPH. Following full suppression of NO synthetic activity by either NPA or TFMPITU, NO synthesis can be restored slowly by excess arginine or by dilution, indicating that the effects of these agents are reversible. This behavior is consistent with a dissociation of NPA and TFMPITU from nNOS slowed by a conformational transition produced by Ca(2+) CaM-binding. NPA and TFMPITU bind to nNOS rapidly producing a heme-substrate interaction as revealed by difference spectrophotometry. At physiological conditions (100 microM extracellular arginine), NPA and TFMPITU inhibit Ca(2+)-dependent NO formation by GH(3) pituitary cells with IC(50) values of 19 and 47 microM, respectively, but require millimolar concentrations to inhibit NO formation by cytokine-induced RAW 264.7 murine macrophages. The inhibition of NO formation by these agents in GH(3) cells is rapidly reversible and not due to suppression of cellular arginine uptake.

Advances in laboratory evaluation of Turner syndrome and its variants: beyond cytogenetics studies.

Turner syndrome is a clinically defined phenotype that is characterized by partial or complete X chromosome monosomy. A host of cytogenetic aberrations and mosaicism have been associated with this syndrome. Some individuals, Turner syndrome variants, have cytogenetic findings consistent with Turner syndrome, but exhibit atypical clinical phenotypes. Recently, several molecular tests have been presented to allow for the refined clinical study of Turner syndrome and its variants.

Molecular determination of X inactivation pattern correlates with phenotype in women with a structurally abnormal X chromosome.

PURPOSE: To correlate the X inactivation pattern, as determined by one or more molecular assays, with phenotype in individuals with structurally abnormal X chromosomes. METHODS: We utilized methylation analysis of androgen receptor (AR) and Fragile X (FMR1) genes and expression studies of an XIST polymorphism to assess X inactivation patterns of 28 females with structurally abnormal X chromosomes. Individuals were placed in one of three categories: (1) completely nonrandom inactivation of one X chromosome, (2) preferential or skewed inactivation of one X chromosome, or (3) random inactivation of either X chromosome. RESULTS: In 19 of 21 cases with complete (>97%) skewing of X inactivation, the phenotype was either normal, consistent with a single gene disorder, or consistent with classical Turner syndrome; two cases with completely nonrandom X inactivation had unexplained mental retardation phenotypes. In contrast, six of seven cases that did not exhibit completely nonrandom X inactivation were phenotypically abnormal. Carriers of two balanced translocations, two duplicated Xs, one deleted X, and one 45,X/46,X,r(X) presented with mental retardation and/or multiple congenital anomalies. CONCLUSION: In patients with random or skewed X inactivation, the abnormal phenotype was hypothesized to be due to functional nullisomy or disomy of X-linked genes. Based on these results, we propose that X inactivation studies should be performed on all women with structurally abnormal X chromosomes. This should aid in the understanding of abnormal phenotypes in liveborn individuals with abnormal X chromosomes and may help to predict phenotypes for prenatally detected cases in the future.

Pharmacological modulation of nitric oxide synthesis by mechanism-based inactivators and related inhibitors.

Nitric oxide synthase (NOS) (EC 1.14.13.39) is a homodimeric cytochrome P450 monooxygenase analog that generates nitric oxide (NO) from the amino acid L-arginine. Enzymatically produced NO acts as an intracellular messenger in neuronal networks, blood pressure regulatory mechanisms, and immune responses. Isoform-selective pharmacological modulation of NO synthesis has emerged as a new therapeutic strategy for the treatment of diverse clinical conditions associated with NO overproduction. Mechanism-based inactivators (MBIs) represent a class of NOS mechanistic inhibitors that require catalytic turnover to produce irreversible inactivation of the ability of NOS to generate NO. Diverse isoform-selective NOS MBIs have been characterized with respect to their kinetic parameters and chemical mechanisms of inactivation. In studies with isolated and purified NOS isoforms, MBIs produce irreversible inactivation of NOS enzymatic activities. The inactivation process is associated with covalent modification of the NOS active site and proceeds either through heme destruction, its structural alteration, or covalent modification of the NOS protein chain. The behavior of NOS MBIs in intact cells is different from their behavior observed with the isolated NOS isoforms. In cytokine-induced RAW 264.7 macrophages, treatment with MBIs produces a complete loss of cellular NOS synthetic competence and inducible NOS activity. However, following drug removal, cells can recover at least partially in the absence of protein synthesis. In GH3 cells containing the neuronal NOS isoform, calcium transients are too low and abbreviated to allow significant NOS inactivation; hence, the cellular effects of MBIs on the neuronal isoform are almost completely and immediately reversible.

Studies of neuronal nitric oxide synthase inactivation by diverse suicide inhibitors.

N(G)-Amino-l-arginine, N(5)-(1-iminoethyl)-l-ornithine, N(6)-(1-iminoethyl)-l-lysine, and aminoguanidine were studied for the mechanisms by which they produce suicidal inactivation of the neuronal nitric oxide synthase isoform (nNOS). All of the inactivators that were amino acid structural analogs targeted the heme residue at the nNOS active site and led to its destruction as evidenced by the time- and concentration-dependent loss of the nNOS heme fluorescence, which reflects the disruption of the protoporphyrin-conjugated structure. The loss of heme was exclusively associated with the dimeric population of the nNOS. This inactivator-mediated loss of the nNOS heme never reached more than 60%, suggesting that only half of the dimeric heme is involved in catalytic activation of mechanism-based inactivators studied. Aminoguanidine-induced nNOS inactivation produced covalent modification of the nNOS protein chain with a stoichiometry of 0.8 mol of aminoguanidine per mole of the nNOS monomer. Specific covalent modification by aminoguanidine was exclusively associated with the oxygenase domain of the nNOS. The mechanisms by which N(6)-(1-iminoethyl)-l-lysine and aminoguanidine inactivate the nNOS and iNOS do not differ between the isoforms. The selectivity of these inactivators toward the iNOS isoform is a reflection of their much lower partition ratios, which were determined to be 0.16 +/- 0. 1 for N(6)-(1-iminoethyl)-l-lysine and 12 +/- 1.5 for aminoguanidine in case of the iNOS isoform while the same inactivators produced the partition ratios of 17 +/- 2 and 206 +/- 4, respectively, for the nNOS isoform.

Prenatal evaluation of a de novo X;9 translocation.

A case of X-autosome translocation was diagnosed prenatally [46,X, t(X;9)(p21.3 approximately 22.1;q22]. We describe the use of fluorescence in situ hybridization (FISH) to estimate the integrity of the Duchenne muscular dystrophy (DMD) gene. X-inactivation studies were used as well to assess the probability of phenotypic abnormalities associated with functional partial disomy X and monosomy 9.

Molecular characterization and delineation of subtle deletions in de novo "balanced" chromosomal rearrangements.

To test the hypothesis that the phenotypic abnormalities seen in cases with apparently balanced chromosomal rearrangements are the result of the presence of cryptic deletions or duplications of chromosomal material near the breakpoints, we analyzed three cases with apparently balanced chromosomal rearrangements and phenotypic abnormalities. We characterized the breakpoints in these cases by using microsatellite analysis by polymerase chain reaction and fluorescence in situ hybridization analysis of yeast artificial chromosome clones selected from the breakpoint regions. Molecular characterization of the translocation breakpoint in patient 1 [46,XY,t(2;6)(p22.2;q23.1)] showed the presence of a 4- to 6-Mb cryptic deletion between markers D6S412 and D6S1705 near the 6q23.1 breakpoint. Molecular characterization of the proximal inversion 7q22.1 breakpoint in patient 2 [46,XY,inv(7)(q22.1q32.1)] revealed the presence of a 4-Mb cryptic deletion between D7S651 and D7S515 markers. No deletion or duplication of chromosomal material was found near the breakpoints in patient 3 [46,XX,t(2;6)(q33.1;p12.2)]. Our study suggests that a systematic molecular study of breakpoints should be carried out in cases with apparently balanced chromosomal rearrangements and phenotypic abnormalities, because cryptic deletions near the breakpoints may explain the phenotypic abnormalities in these cases.

Neuronal nitric oxide synthase is refractory to mechanism-based inactivation in GH3 pituitary cells.

Nitric oxide formation by GH3 pituitary cells is stimulated by depolarizing concentrations of K+ and by the L-channel Ca2+ agonist Bay kappa 8644 in an additive manner that depends on extracellular Ca2+. Ca(2+)-dependent NO formation at 100 microM arginine was inhibited 50% over a 30-min period by 5 microM NG-amino-L-arginine, 30 microM N6-iminoethyl-L-ornithine (NIO) and 520 microM N5-iminoethyl-L-lysine (NIL) but required concentrations of aminoguanidine (AG) greater than 3 mM. As measured at 100 microM extracellular arginine, intracellular neuronal nitric oxide synthase (nNOS) was inactivated 50% over a 30-min period by 150 microM NG-amino-L-arginine and 1500 microM NIO, but required concentrations of NIL or AG greater than 5 mM. The inactivation of nNOS by these agents occurred only under conditions that mobilized extracellular Ca2+ and was inhibited by increased extracellular arginine. Thus these agents inhibit cellular Ca(2+)-dependent NO formation at concentrations far lower than those required to inactivate the cellular nNOS. Inhibition of NO formation by these agents was not attributable to effects on cellular arginine uptake. In contrast diphenyliodonium chloride produced a rapid and complete inactivation of cellular NO formation and nNOS activity. These inactivations produced by diphenyliodonium chloride occurred with identical kinetics but displayed no requirement for Ca2+. These data support the assertion that neuronal NO synthase is refractory to mechanism-based inactivation in GH3 pituitary cells.

Inactivation of nitric oxide synthases and cellular nitric oxide formation by N6-iminoethyl-L-lysine and N5-iminoethyl-L-ornithine.

The kinetics of inactivation of affinity-purified nitric oxide synthase isoforms by N6-iminoethyl-L-lysine (NIL) and N5-iminoethyl-L-ornithine (NIO) has been examined. Each of the agents produced a time and concentration dependent first order inactivation of the nitric oxide synthase isoforms that required exposure of the NO synthase to drug under conditions that supported catalysis, consistent with the proposal that these agents act as alternate substrate, mechanism-based inactivators. As measured at 100 microM arginine, NIL and NIO were equally efficient as inactivators of the cytokine-inducible nitric oxide synthase exhibiting apparent second order inactivation rate constants of 31.5 and 32.0 mM(-1) min(-1) respectively. By contrast, NIL and NIO were less efficient as inactivators of the constitutive neuronal nitric oxide synthase isoform exhibiting apparent second order inactivation rate constants of 0.79 and 8.4 mM(-1) min(-1) respectively. As measured at 100 microM extracellular arginine, NIL and NIO produced a time and concentration dependent inactivation of the NO synthetic capability of cytokine-induced murine macrophage RAW 264.7 cells exhibiting apparent second order inactivation rate constants of 3.1 and 1.8 mM(-1) min(-1). The inactivated RAW cell NO synthetic capability was restored to 30% of its pretreatment value over a 3-h period despite the presence of cycloheximide.

Random X inactivation in a girl with a balanced t(X;9) and an abnormal phenotype.

X inactivation is the process by which mammalian females achieve dosage compensation by transcriptionally silencing one X chromosome. In chromosomally normal females, this process is random. However, most females with one abnormal X chromosome demonstrate complete skewing of X inactivation, presumably as the result of cell selection. We present a mentally retarded girl with a 46,X,t(X;9)(q28;q12) karyotype. Analysis of this patient's lymphocytes, using late replication banding and methylation assays for the androgen receptor (AR) and fragile X mental retardation (FMR1) genes, did not show the predicted nonrandom X inactivation pattern. Thus, this patient is functionally disomic for Xq28-qter in a proportion of her cells, most likely resulting in her abnormal phenotype. This case demonstrates the utility of correlating X inactivation patterns with phenotype in females with one structurally abnormal X chromosome, and suggests that both cytogenetic and molecular X inactivation studies should be included in the routine study of these individuals.

The spreading of X inactivation into autosomal material of an x;autosome translocation: evidence for a difference between autosomal and X-chromosomal DNA.

X inactivation involves initiation, propagation, and maintenance of genetic inactivation. Studies of replication timing in X;autosome translocations have suggested that X inactivation may spread into adjacent autosomal DNA. To examine the inactivation of autosomal material at the molecular level, we assessed the transcriptional activity of X-linked and autosomal loci spanning an inactive translocation in a phenotypically normal female with a karyotype of 46,X,der(X)t(X;4)(q22;q24). Since 4q duplications usually manifest dysmorphic features and severe growth and mental retardation, the normal phenotype of this individual suggested the spreading of X inactivation throughout the autosomal material. Consistent with this model, reverse transcription-PCR analysis of 20 transcribed sequences spanning 4q24-qter revealed that three known genes and 11 expressed sequence tags (ESTs) were not expressed in a somatic-cell hybrid that carries the translocation chromosome. However, three ESTs and three known genes were expressed from the t(X;4) chromosome and thus "escaped" X inactivation. This direct assay of expression demonstrated that the spreading of inactivation from the adjoining X chromosome was incomplete and noncontiguous. These findings are broadly consistent with the existence of genes known to escape inactivation on normal inactive X chromosomes. However, the fact that a high proportion (30%) of tested autosomal genes escaped inactivation may indicate that autosomal material lacks X chromosome-specific features that are associated with the spreading and/or maintenance of inactivation.

Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine.

The inducible nitric oxide synthase (iNOS) selective inhibitors aminoguanidine (AG) and N6-(1-iminoethyl)-L-lysine (NIL), under conditions that support catalytic turnover, inactivate the enzyme by altering in different ways the functionality of the active site. NIL inactivation of the iNOS primarily targets the heme residue at the active site, as evidenced by a time- and concentration-dependent loss of heme fluorescence that accompanies the loss of NO-forming activity. The NIL-inactivated iNOS dimers that have lost their heme partially disassemble into monomers with no fluorometrically detectable heme. AG inactivation of the iNOS is not accompanied by heme destruction, as evidenced by retention of heme fluorescence and absorbance after complete loss of NO-forming activity. The AG-inactivated iNOS dimers do not disassemble into monomers as extensively as NIL-inactivated dimers. Incubation of the iNOS with 14C-labeled NIL results in no detectable protein-associated radioactivity in the NIL-inactivated iNOS, suggesting that the primary mechanism of the iNOS inactivation by NIL is heme alteration and loss. In contrast, incubations of iNOS with 14C-labeled AG result in the incorporation of radioactivity into both iNOS protein and low molecular weight structures that migrate by SDS-PAGE similarly to free heme. These observations suggest that AG inactivation proceeds through multiple pathways of covalent modification of the iNOS protein and the heme residue at the active site, but which sustain the integrity of the heme porphyrin ring.

Inactivation of nitric oxide synthase by substituted aminoguanidines and aminoisothioureas.

A series of substituted aminoguanidines and amino-substituted isothioureas have been examined as inhibitors of nitric oxide (NO) synthase (NOS) isoforms. Each of the agents produced a time- and concentration-dependent inactivation of the NO-forming activity of the affinity-purified NOS isoforms. These inactivations required exposure of NOS to the drug under conditions that supported catalysis, consistent with the proposal that they act as alternate substrate, mechanism-based inactivators. Of the aminoguanidines examined, 2-ethylaminoguanidine was the most efficient inactivator, exhibiting vs. iNOS an apparent KI value of 120 microM as measured at 100 microM arginine and a k(inact max) value of 0.48 min(-1) and thus an apparent second-order rate constant for inactivation of 4.0 mM(-1)min(-1). 2-Ethylaminoguanidine displayed a high isoform selectivity for the iNOS compared with the nNOS and eNOS isoforms. 2-Ethylaminoguanidine inactivated NO synthetic activity in cytokine-induced RAW 264.7 cells as measured at 100 microM extracellular arginine with an apparent KI value of 55 microM and a k(inact max) value of 0.09 min(-1). The inactivated RAW 264.7 cell NO synthetic capability was restored over a 3-hr period after drug removal to a value 60% of its pretreatment value. This recovery occurred despite the presence of cycloheximide sufficient to inhibit protein synthesis by >99%. 1-Amino-S-methylisothiourea by contrast with the aminoguanidines was identified as a mechanism-based inactivator selective for the nNOS isoform. In contrast to S-isopropylisothiourea, which was found to be both cell penetrant and reversible, 1-amino-S-methylisothiourea appeared cell impermeable and inhibited NOS enzyme "irreversibly."

Deletions in Xq26.3-q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern.

High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In Family 1, a 19-year-old male had facial features consistent with fragile X syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3-q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. Methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother's white blood cells and 80-85% of the sister's leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing approximately 13 Mb of DNA, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed approximately 12 Mb of DNA around the FMR1 gene. Cytogenetic, and molecular data revealed that approximately 50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3-q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3-q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients.

Duplication 14(q24.3q31) in a father and daughter: delineation of a possible imprinted region.

A number of clinical reports have described children with a variety of congenital anomalies in association with uniparental disomy (upd) of chromosome 14, suggesting that at least some genes on chromosome 14 are subject to parent of origin, or imprinting, effects. However, little else is known about this putative imprinting of chromosome 14. Both maternal and paternal upd have been observed, but a consistent phenotype has only been suggested for the former. Here we report on a child with developmental delay, microcephaly, distinct facial findings, and who has a duplication of 14q24.3q31. The same cytogenetic abnormality was found in her phenotypically normal father. We hypothesize that this segment of chromosome 14 contains maternally silenced genes, and that this duplicated segment defines an imprinted region on chromosome 14. Alternatively, this cytogenetic duplication may be unrelated to the girl's phenotypic anomalies, and this duplication may contain genes that are not subject to dosage effect.