Sequence-dependent variations associated with H2A/H2B depletion of nucleosomes.

Mechanisms that can alter nucleosome structure to enhance DNA accessibility are of great interest because of their potential involvement in genomic processes. One such mechanism is H2A/H2B release from nucleosomes; it occurs in vivo and is involved in the in vitro activities of several transcription-associated complexes. Using fluorescence approaches based on Förster resonance energy transfer, we previously detected sequence-dependent structure/stability variations between 5S and two types of promoter nucleosomes (from yeast GAL10 or mouse mammary tumor virus promoters). Those variations included differing responses when nucleosomes were diluted to concentrations (sub-nM) known to produce H2A/H2B loss. Here, we show that treatment of these same three types of nucleosomes with the histone chaperone yNAP-1, which causes H2A/H2B release from nucleosomes in vitro, produces the same differential Förster resonance energy transfer responses, again demonstrating sequence-dependent variations associated with conditions that produce H2A/H2B loss. Single-molecule population data indicate that DNA dynamics on the particles produced by diluting nucleosomes to sub-nM concentrations follow two-state behavior. Rate information (determined by fluorescence correlation spectroscopy) suggests that these dynamics are enhanced in MMTV-B or GAL10 compared to 5S particles. Taken together, the results indicate that H2A/H2B loss has differing effects on 5S compared to these two promoter nucleosomes and the differences reflect sequence-dependent structure/stability variations in the depleted particles.

AFM imaging of protein movements: histone H2A-H2B release during nucleosome remodeling.

Being able to follow assembly/disassembly reactions of biomolecular complexes directly at the single molecule level would be very useful. Here, we use an AFM technique that can simultaneously obtain topographic images and identify the locations of a specific type of protein within those images to monitor the histone H2A component of nucleosomes acted on by human Swi-Snf, an ATP-dependent nucleosome remodeling complex. Activation of remodeling results in significant H2A release from nucleosomes, based on recognition imaging and nucleosome height changes, and changes in the recognition patterns of H2A associated directly with hSwi-Snf complexes.

Conformational transition in DNA on a cold surface.

The contour length of DNA fragments, deposited and imaged on mica under buffer, was measured as a function of deposition temperature. Extended DNA molecules (on Ni- and silane-treated surfaces) contract rapidly with falling temperature, approaching the contour length of A-DNA at 2 degrees C. The contraction is not unique to a specific sequence and does not occur in solution at 2 degrees C or on a surface at 25 degrees C, indicating that it arises from a combination of low temperature and surface contact. It is probably a consequence of reduced water activity at a cold surface.

Yeast chromatin structure and regulation of GAL gene expression.

Yeast genomic DNA is covered by nucleosome cores spaced by short, discrete length linkers. The short linkers, reinforced by novel histone properties, create a number of unique and dynamic nucleosome structural features in vivo: permanent unpeeling of DNA from the ends of the core, an inability to bind even full 147 bp core DNA lengths, and facility to undergo a conformational transition that resembles the changes found in active chromatin. These features probably explain how yeast can maintain most of its genome in a transcribable state and avoid large-scale packaging away of inactive genes. The GAL genes provide a closely regulated system in which to study gene-specific chromatin structure. GAL structural genes are inactive without galactose but are highly transcribed in its presence; the expression patterns of the regulatory genes can account for many of the features of GAL structural gene control. In the inactive state, GAL genes demonstrate a characteristic promoter chromosomal organization; the major upstream activation sequence (UASG) elements lie in open, hypersensitive regions, whereas the TATA and transcription start sites are in nucleosomes. This organization helps implement gene regulation in this state and may benefit the organism. Induction of GAL expression triggers Gal4p-dependent upstream nucleosome disruption. Disruption is transient and can readily be reversed by a Gal80p-dependent nucleosome deposition process. Both are sensitive to the metabolic state of the cell. Induction triggers different kinds of nucleosome changes on the coding sequences, perhaps reflecting the differing roles of nucleosomes on coding versus promoter regions. GAL gene activation is a complex process involving multiple Gal4p activities, numerous positive and negative cofactors, and the histone tails. DNA bending and chromosomal architecture of the promoter regions may also play a role in GAL regulation. Regulator-mediated competition between nucleosomes and the TATA binding protein complex for the TATA region is probably a central aspect of GAL regulation and a focal point for the numerous factors and processes that contribute to it.

Fractionated cyclophosphamide and etoposide for children with advanced or refractory solid tumors: a phase II window study.

PURPOSE: Cyclophosphamide (CPA) has a broad spectrum of activity against solid tumors. Hepatic self-induction of the active metabolite 4-hydroxycyclophosphamide occurs after repeated administration. We evaluated the clinical efficacy of a window regimen that administers fractionated CPA in conjunction with etoposide (VP16) in children with advanced or refractory solid tumors. PATIENTS AND METHODS: Seventeen children with advanced (n = 12) or refractory (n = 5) solid tumors were entered onto this phase II window study. The treatment regimen consisted of intravenous (IV) CPA 500 mg/m(2)/d and IV VP16 100 mg/m(2)/d. Both drugs were administered daily by short infusions for 5 consecutive days. RESULTS: A total of 34 courses were administered, with a median of two courses per patient. The median interval between chemotherapy courses was 21 days (range, 17 to 35 days). Thirty-three courses were assessable for toxicity, and all patients were assessable for response. No life-threatening toxicities were observed. The incidence of grade 3 or 4 neutropenia was 94% and of fever and neutropenia 38%. Fever and neutropenia occurred after 12 of 26 courses without recombinant human granulocyte colony-stimulating factor (rhG-CSF) and after one of eight courses with rhG-CSF (P =. 09). Grade 3 or 4 thrombocytopenia occurred after 10 courses (29%). There were no positive blood cultures. One heavily pretreated patient developed a localized perirectal abscess that required drainage. There were 10 patients (59%) with partial responses, four (23.5%) with stable disease, and three with progressive disease. CONCLUSION: Fractionated IV CPA and VP16 over 5 days can be safely administered in children with advanced or refractory solid tumors and has notable antineoplastic activity.

Kinetics of early therapeutic response as measured by quantitative PCR predicts survival in a murine xenograft model of human T cell acute lymphoblastic leukemia.

The identification of prognostic parameters and surrogate markers for defining patient risk has been beneficial in effectively guiding therapy and increasing the survival of leukemia patients. It has been hypothesized that the therapeutic response, as measured by a change in tumor burden during therapy, might serve as a new surrogate marker of survival. Here we describe the development of a murine SCID xenograft model of human T cell acute lymphoblastic leukemia (T-ALL), and the use of a sensitive, quantitative PCR assay for the measurement of tumor levels to investigate the relationships between tumor burden quantification, therapeutic response and survival. Animals engrafted with the CCRF-CEM (CEM) human T-ALL cell line develop leukemia that closely resembles the human disease. Quantitative PCR detects the expanding tumor mass in the peripheral blood of the animals several weeks before death. In response to induction therapy with chemotherapeutic agents, both the level of minimal residual disease (MRD) in peripheral blood at the end of therapy and the rate of tumor reduction in peripheral blood during therapy strongly correlated with animal survival. Thus, these surrogate markers, which can be measured during the early stages of therapy, may help improve patient survival through dynamic risk stratification.

Glutathione S-transferase genotypes in children who develop treatment-related acute myeloid malignancies.

Epipodophyllotoxin-associated secondary myeloid leukemia is a devastating complication of acute lymphoblastic leukemia (ALL) therapy. The risk factors for treatment-related myeloid leukemia remain incompletely defined. Genetic deficiencies in glutathione S-transferase (GST) activities have been linked to higher frequencies of a number of human malignancies. Our objective was to determine whether the null genotype for GSTM1, GSTT1, or both, was more frequent in children with ALL who developed treatment-related myeloid malignancies as compared to those who did not. A PCR technique was used to assay for the null genotype for GSTM1 and GSTT1 in 302 children with ALL, 57 of whom also subsequently developed treatment-related acute myeloid leukemia or myelodysplastic syndrome. Among children with ALL who did not develop treatment-related myeloid malignancies, the frequencies of GSTM1 and GSTT1 wild-type, GSTM1 null-GSTT1 wild-type, GSTM1 wild-type-GSTT1 null, and GSTM1 and GSTT1 null genotypes were 40%, 42%, 9% and 9%, respectively. The corresponding frequencies for patients who developed acute myeloid malignancies were 42%, 32%, 11% and 16%, respectively (P = 0.26). A statistically significant increase in the frequency of the GST null genotype was observed in male patients who developed myeloid malignancies as compared to male ALL control patients (P = 0.036), but was not observed in female patients (P = 0.51). Moreover, a logistic regression analysis of possible predictors for myeloid malignancies, controlling for gender and race, did not reveal an association of GSTM1 or GSTT1 null genotypes (P = 0.62 and 0.11, respectively) with treatment-related malignancies. Our data suggest that GSTM1 and GSTT1 null genotypes may not predispose to epipodophyllotoxin-associated myeloid malignancies.

Topographic mapping of electrophysiologic measures in patients with homonymous hemianopia.

We analyzed electroencephalographic (EEG) activity and spatial distribution of the pattern-reversal visual evoked potential (PVEP) in 20 patients with unilateral lesions in the retrochiasmal visual pathways. Focal abnormalities that were consistent with lesion location were present in the topographically analyzed EEG or VEP of 85% of the patients, compared with a 70% detection rate for conventional analysis techniques. Quantitative analysis of spectrally analyzed EEG revealed focal abnormality in 7 patients whose conventional EEG was interpreted as either normal or diffusely slow. However, focal paroxysmal spikes present in the EEG of 1 patient were missed by EEG mapping, and a false localization of quantitative EEG abnormality contralateral to the lesion occurred in 1 patient. Topographic analysis of the PVEP was no more sensitive to retrochiasmatic lesions than conventional analysis of 2 lateral occipital electrodes. We conclude that topographic mapping is a valid technique in the detection of localized cerebral lesions.

Value of C-reactive protein determination in the initial diagnostic evaluation of the febrile, neutropenic child with cancer.

We studied prospectively the value of administration C-reactive protein (CRP) in the diagnostic evaluation of the child with cancer hospitalized for fever and neutropenia. During a 7-month period 74 patients with malignant disease had 122 hospital admissions because of fever and neutropenia. All patients had a serum CRP obtained 8 to 24 hours after the onset of fever as part of their initial evaluation. There was a borderline correlation between serum CRP concentration and temperature at admission (P = 0.06). Patients with fever without an identifiable source had significantly lower CRP concentrations compared with those having focal or microbiologically documented infection (34.9 +/- 6 vs. 70.2 +/- 12 mg/liter; P = 0.0005). Twelve patients had positive blood cultures, 5 of which were coagulase-negative staphylococci considered to be central venous catheter-related infection or colonization. CRP concentrations were significantly lower in these 5 patients compared with the 7 patients with septicemia caused by other organisms (21 +/- 9 vs. 113 +/- 23 mg/liter; P = 0.01). In distinguishing between septicemic and nonsepticemic children, serum CRP was found to have excellent sensitivity and negative predictive value at concentration limits of 20, 50 and 100 mg/liter. However, both specificity and positive predictive value were low at these respective levels, thus limiting the overall utility of serum CRP in the initial empiric management of the febrile, neutropenic child with cancer.

Two-component atomic force microscopy recognition imaging of complex samples.

Biological complexes are typically multisubunit in nature and the processes in which they participate often involve protein compositional changes in themselves and/or their target substrates. Being able to identify more than one type of protein in complex samples and to track compositional changes during processes would thus be very useful. Toward this goal, we describe here a single-molecule technique that can simultaneously identify two types of proteins in compositionally complex samples. It is an adaptation of the recently developed atomic force microscopy (AFM) recognition imaging technique but involves the tethering of two different types of antibodies to the AFM tip and sequential blocking with appropriate antigenic peptides to distinguish the recognition from each antibody. The approach is shown to be capable of simultaneously identifying in a single AFM image two specific components, BRG1 and beta-actin, of the human Swi-Snf ATP-dependent nucleosome remodeling complex and two types of histones, H2A and H3, in chromatin samples.

Using atomic force microscopy to study chromatin structure and nucleosome remodeling.

Atomic force microscopy (AFM) is a technique that can directly image single molecules in solution and it therefore provides a powerful tool for obtaining unique insights into the basic properties of biological materials and the functional processes in which they are involved. We have used AFM to analyze basic features of nucleosomes in arrays, such as DNA-histone binding strength, cooperativity in template occupation, nucleosome stabilities, nucleosome locations and the effects of acetylation, to compare these features in different types of arrays and to track the response of array nucleosomes to the action of the human Swi-Snf ATP-dependent nucleosome remodeling complex. These experiments required several specific adaptations of basic AFM methods, such as repetitive imaging of the same fields of molecules in liquid, the ability to change the environmental conditions of the sample being imaged and detection of specific types of molecules within compositionally complex samples. Here, we describe the techniques that allowed such analyses to be carried out.

Sequence-dependent nucleosome structure and stability variations detected by Förster resonance energy transfer.

Nucleosomes, the basic unit of eukaryotic chromosome structure, cover most of the DNA in eukaryotes, including regulatory sequences. Here, a recently developed Förster resonance energy transfer approach is used to compare structure and stability features of sea urchin 5S nucleosomes and nucleosomes reconstituted on two promoter sequences that are nucleosomal in vivo, containing the yeast GAL10 TATA or the major transcription response elements from the mouse mammary tumor virus promoter. All three sequences form mononucleosomes with similar gel mobilities and similar stabilities at moderate salt concentrations. However, the two promoter nucleosomes differ from 5S nucleosomes in (1) diffusion coefficient values, which suggest differences in nucleosome compaction, (2) intrinsic FRET efficiencies (in solution or in gels), and (3) the response of FRET efficiency to high (>or=600 mM) NaCl concentrations, subnanomolar nucleosome concentrations, and elevated temperatures (to 42 degrees C). These results indicate that nucleosome features can vary depending on the DNA sequence they contain and show that this fluorescence approach is sufficiently sensitive to detect such differences. Sequence-dependent variations in nucleosome structure or stability could facilitate specific nucleosome recognition, working together with other known genomic regulatory mechanisms. The variations in salt-, concentration-, and temperature-dependent responses all occur under conditions that have been shown previously to produce release of H2A-H2B dimers or terminal DNA from nucleosomes and could thus involve differences in those processes, as well as in other features.

Properties of nucleosomes in acetylated mouse mammary tumor virus versus 5S arrays.

Acetylation is one of the most abundant histone modifications found in nucleosomes. Although such modifications are thought to function mainly in recognition, acetylation is known to produce nucleosome structural alterations. These could be of functional significance in vivo. Here, the basic features of mouse mammary tumor virus (MMTV) promoter nucleosomal arrays reconstituted with highly acetylated histones prepared from butyrate-treated HeLa cells are characterized by atomic force microscopy. Results are compared to previous results obtained with hypoacetylated MMTV and hyper- or hypoacetylated 5S rDNA arrays. MMTV arrays containing highly acetylated histones show diminished intramolecular compaction compared to hypoacetylated MMTV arrays and no tendency for cooperativity in nucleosome occupation. Both features have been suggested to reflect histone tail-mediated internucleosomal interactions; these observations are consistent with that suggestion. 5S arrays show qualitatively similar behavior. Two other effects of acetylation show stronger DNA template dependence. Nucleosome salt stability is diminished in highly acetylated compared to hypoacetylated MMTV arrays, but nucleosome (histone) loading tendencies are unaffected by acetylation. However, highly acetylated histones show reduced loading tendencies on 5S templates (vs hypoacetylated), but 5S nucleosome salt stabilities are unaffected by acetylation. ATP-dependent nucleosome remodeling by human Swi-Snf is similar on hyper- and hypoacetylated MMTV arrays.

Solution AFM studies of human Swi-Snf and its interactions with MMTV DNA and chromatin.

ATP-dependent nucleosome remodeling complexes are crucial for relieving nucleosome repression during transcription, DNA replication, recombination, and repair. Remodeling complexes can carry out a variety of reactions on chromatin substrates but precisely how they do so remains a topic of active inquiry. Here, a novel recognition atomic force microscopy (AFM) approach is used to characterize human Swi-Snf (hSwi-Snf) nucleosome remodeling complexes in solution. This information is then used to locate hSwi-Snf complexes bound to mouse mammary tumor virus promoter nucleosomal arrays, a natural target of hSwi-Snf action, in solution topographic AFM images of surface-tethered arrays. By comparing the same individual chromatin arrays before and after hSwi-Snf activation, remodeling events on these arrays can be monitored in relation to the complexes bound to them. Remodeling is observed to be: inherently heterogeneous; nonprocessive; able to occur near and far from bound complexes; often associated with nucleosome height decreases. These height decreases frequently occur near sites of DNA release from chromatin. hSwi-Snf is usually incorporated into nucleosomal arrays, with multiple DNA strands entering into it from various directions, + or - ATP; these DNA paths can change after hSwi-Snf activation. hSwi-Snf appears to interact with naked mouse mammary tumor virus DNA somewhat differently than with chromatin and ATP activation of surface-bound DNA/hSwi-Snf produces no changes detectable by AFM.

Deciphering cancer complexities in genetically engineered mice.

Because the pRb pathway is disrupted in most solid human cancers, we have generated genetically engineered mouse cancer models by inactivating pRb function in several cell types, including astrocytes and mammary, prostate, ovarian, and brain choroid plexus epithelia. In every case, proliferation and apoptosis are acutely induced, predisposing to malignancy. Cell type dictates the pathways involved in tumor progression. In the astrocytoma model, we developed strategies to induce events in the adult brain, either throughout the tissue or focally. Both K-Ras activation and Pten inactivation play significant roles in progression. In the prostate model, adenocarcinoma progression depends on Pten inactivation. However, nonautonomous induction of p53 in the mesenchyme leads to evolution of both compartments, with p53 loss occurring in the mesenchyme. Thus, studies in these models continue to identify key tumorigenesis mechanisms. Furthermore, we are hopeful that the models will provide useful preclinical systems for diagnostic and therapeutic development.

Intrinsically bent DNA in the promoter regions of the yeast GAAL1-10 and GAL80 genes.

Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1-10 and GAL80 genes. These bends lie in functionally suggestive locations. On the promoter of the GAL1-10 structural genes, strong bends bracket nucleosome B, which lies between the UAS(G) and the GAL1 TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1-10 expression is induced. The strongest intrinsic bend ( approximately 70 degrees ) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it to be involved in GAL1 activation in several ways, including the recruitment of a yeast HMG protein that is required for the normally robust level of GAL1 expression in the induced state (Paull, T., Carey, M., and Johnson, R. (1996) Genes Dev. 10, 2769-2781). On the regulatory gene GAL80, the single bend lies in the non-nucleosomal hypersensitive region, between a GAL80-specific far upstream promoter element and the more gene-proximal promoter elements. GAL80 promoter region nucleosomes contain no intrinsically bent DNA.

Safety and cost effectiveness of early hospital discharge of lower risk children with cancer admitted for fever and neutropenia.

BACKGROUND: Standard treatment for fever during periods of chemotherapy-induced neutropenia includes hospitalization and administration of intravenous antibiotics until the patient is afebrile and no longer neutropenic. This study prospectively evaluates the safety and cost-effectiveness of early discharge of selected low risk children before recovery from neutropenia. METHODS: We studied 74 children with cancer during 131 consecutive admissions for fever during a period of neutropenia. All patients initially were hospitalized and received broad-spectrum antibiotics. Intravenous antibiotic therapy was discontinued, and the patients promptly were discharged even if they had an absolute neutrophil count (ANC) of less than 500 cells/mm3 as long as they were afebrile, appeared clinically well, had negative cultures, exhibited control of local infection, and showed hematologic evidence of bone marrow recovery. RESULTS: Intravenous antibiotics were discontinued in 82 cases (63%) before recovery of the ANC to more than 500 cells/mm3, and 78 patients were discharged immediately. None of 70 patients discharged while neutropenic but exhibiting a rising ANC at the time of discharge developed recurrent fever and required readmission. Thirty of these children had an improving localized infection when intravenous antibiotics were discontinued and completed a course of oral antibiotics at home. The estimated mean savings in hospital charges due to early discharge was $5058 per patient. CONCLUSIONS: Low risk children with cancer who are hospitalized and treated for fever and neutropenia but appear clinically well may have intravenous antibiotics discontinued and be discharged safely irrespective of the ANC, as long as their granulocyte count is rising. This approach shortens hospital stays and results in considerable cost savings.

The yield of routine chest radiography in children with cancer hospitalized for fever and neutropenia.

A routine admission chest radiograph (CXR) in pediatric patients with cancer who are admitted to the hospital for fever and neutropenia has been advised because the signs and symptoms of pneumonia may be absent. The authors studied 131 consecutive patient admissions for fever and neutropenia to evaluate the diagnostic yield of routine CXR. All patients had a complete history, physical examination, complete blood count, blood culture, urinalysis, urine culture, and CXR. Patients routinely started ceftazidime monotherapy. Results of the CXR were correlated with the presence or absence of signs and symptoms of respiratory disease. Of 128 CXR results, 26 (20%) were abnormal (13 with known malignant disease, 2 with atelectasis, 3 with peribronchial cuffing, and 8 with pneumonia [6%]). Three patients with pneumonia were asymptomatic. Therefore, only 3 of 128 patients (2.3%) had pneumonia on CXR not suspected by physical examination. None would have had initial therapy modified based on the CXR finding alone. The authors concluded that the incidence of pneumonia in a child with fever and neutropenia is low and that routine CXR at diagnostic evaluation is unnecessary in the asymptomatic ambulatory patient.