Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

Loss of matrix adhesion triggers rapid transformation-selective apoptosis in fibroblasts.

Cell-matrix and cell-cell adhesion are recognized physiological determinants of cell growth and survival. In epithelial and endothelial cell systems, oncogenic transformation has in several cases been shown to confer resistance to apoptosis upon depriving cells of substrate adhesion. We examined the effects of oncogenic transformation in adherent versus adhesion- deprived primary embryonic fibroblasts. Whereas untransformed early passage fibroblasts undergo cell cycle arrest, their Myc/Ras- or E1A/Ras-transformed counterparts rapidly enter apoptosis when placed into suspension. This phenomenon also occurs upon incubation with a soluble, RGD-containing integrin ligand and is blocked by a peptide antagonist to ICE family proteases or by aggregation of cells plated at high density. Loss of wild-type p53 modulates the kinetics but does not abrogate this death pathway. Transformation with activated Src rather than Ras rendered fibroblasts selectively resistant to adhesion-dependent apoptosis, an effect likely related to Src's role in integrin signaling, while simultaneously sensitizing the cells to radiation-induced apoptosis. Thus cell adhesion events regulate transformation-selective apoptosis in fibroblasts and provide potentially important targets for understanding and interfering with tumor cell viability.

Ribosomal S6 kinase 1 (RSK1) activation requires signals dependent on and independent of the MAP kinase ERK.

BACKGROUND: The rsk1 gene encodes the 90 kDa ribosomal S6 kinase 1 (RSK1) protein, which contains two kinase domains. RSK1, which is involved in regulating cell survival and proliferation, lies at the end of the signaling cascade mediated by the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases. ERK activation and subsequent phosphorylation of the RSK1 carboxy-terminal catalytic loop stimulates phosphotransferase activity in the RSK1 amino-terminal kinase domain. When activated, RSK1 phosphorylates both nuclear and cytoplasmic substrates through this amino-terminal catalytic domain. It is thought that stimulation of the ERK/MAP kinase pathway is sufficient for RSK1 activation, but how ERK phosphorylation activates the RSK1 amino-terminal kinase domain is not known. RESULTS: The individual isolated RSK1 kinase domains were found to be under regulatory control. In vitro kinase assays established that ERK phosphorylates RSK1 within the carboxy-terminal kinase domain, and the phosphoinositide-dependent kinase 1 (PDK1) phosphorylates RSK1 within the amino-terminal kinase domain. In transiently transfected HEK 293E cells, PDK1 alone stimulated phosphotransferase activity of an isolated RSK1 amino-terminal kinase domain. Nevertheless, activation of full-length RSK1 in the absence of serum required activation by both PDK1 and ERK. CONCLUSIONS: RSK1 is phosphorylated by PDK1 in the amino-terminal kinase-activation loop, and by ERK in the carboxy-terminal kinase-activation loop. Activation of phosphotransferase activity of full-length RSK1 in vivo requires both PDK1 and ERK. RSK1 activation is therefore regulated by both the mitogen-stimulated ERK/MAP kinase pathway and a PDK1-dependent pathway.

Rsk1 mediates a MEK-MAP kinase cell survival signal.

BACKGROUND: Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. RESULTS: We identify here a pro-survival role for the serine/threonine kinase Rsk1, a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonized the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminated the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restored the MEK-MAP kinase survival signal. Experiments in vitro and in vivo demonstrated that Rsk1 directly phosphorylated the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bad's pro-apoptotic function. Constitutively active Rsk1 caused constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevented phosphorylation by Rsk1 also inhibited Rsk1-mediated cell survival. CONCLUSIONS: These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad.

Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

Histone H1 represses transcription from minichromosomes assembled in vitro.

We have previously shown that transcription from a Xenopus 5S rRNA gene assembled into chromatin in vitro can be repressed in the absence of histone H1 at high nucleosome densities (one nucleosome per 160 base pairs of DNA) (A. Shimamura, D. Tremethick, and A. Worcel, Mol. Cell. Biol. 8:4257-4269, 1988). We report here that transcriptional repression may also be achieved at lower nucleosome densities (one nucleosome per 215 base pairs of DNA) when histone H1 is present. Removal of histone H1 from the minichromosomes with Biorex under conditions in which no nucleosome disruption was observed led to transcriptional activation. Transcriptional repression could be restored by adding histone H1 back to the H1-depleted minichromosomes. The levels of histone H1 that repressed the H1-depleted minichromosomes failed to repress transcription from free DNA templates present in trans. The assembly of transcription complexes onto the H1-depleted minichromosomes protected the 5S RNA gene from inactivation by histone H1.

Purification and properties of Streptococcus mutans extracellular glucosyltransferase.

Extracellular glucosyltransferase (sucrose:1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) was purified about 10 000-fold from the culture supernatant of Streptococcus mutans 6715. The enzyme preparation was homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing and ultracentrifugation analyses. The specific activity of the enzyme was 34.9 I.U. per mg of protein and the carbohydrate content was less than 1% (w/w). The molecular weight was determined to be 149 000 +/- 5000 by sedimentation equilibrium experiment. The acidic and basic amino acids of the enzyme comprised 29 and 8.4% of total amino acid, respectively, and the isoelectric point was pH 4.1. The enzyme had the optimum pH of 5.5 and the Km value of 2.4 mM for sucrose. The water-soluble glucan, which was de novo-synthesized from sucrose by the purified enzyme, was analyzed by a gas-liquid chromatography-mass spectroscopy and was found to be 1,6-alpha-D-glucan with highly (35%) branched structure of 1,3,6-linked glucose residue.

Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c.

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltransferase (sucrose: 1,6-alpha-D-glucan 3-alpha and 6-alpha-glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1-8.4). The molecular weight was estimated to be 151000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had Km values of 7.1 micro M for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6-alpha-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.

Assembly and properties of chromatin containing histone H1.

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.

Three kinds of extracellular glucosyltransferases from Streptococcus mutans 6715 (serotype g).

In addition to the 1,3-alpha-D-glucan synthetase (pI 4.9) and the highly-branched 1,6-alpha-D-glucan synthetase (pI 3.9-4.1), Streptococcus mutans 6715 (serotype g) was found to secrete the third glucosyltransferase in multiple forms (pI 5.5-7.0), which exhibited 87% 1,6-alpha-bond-, 6% 1,3-alpha-bond- and 7% 1,3,6-branch-forming activities. The production of this enzyme was extremely enhanced when the organism was grown in Tween 80-supplemented medium. The 3 glucosyltransferases from the same organism were enzymatically and immunologically distinct from each other, and they were commonly found among the serotype g strains.

Impaired use of organizational strategies in free recall following frontal lobe damage.

Free recall, use of organizational strategies, and interference effects were assessed in patients with frontal lobe lesions and control subjects. In three experiments, patients with frontal lobe lesions exhibited impaired free recall and reduced use of organizational strategies in tests of memory. Reduced use of strategies was observed on tests of recall of unrelated items, as measured by subjective organization, and on tests of recall of related items, as measured by both category clustering and subjective organization. Frontal patients benefited from strategy instruction at either study or test, suggesting that both encoding and retrieval processes are impaired by frontal lobe damage. These findings indicate that the free recall impairments exhibited by patients with frontal lobe lesions may be caused at least in part by deficits in the use of organizational strategies. In addition, when first-list learning was matched for patients and control subjects, patients with frontal lobe lesions exhibited relatively increased sensitivity to proactive interference during second-list learning.

Strength and duration of priming effects in normal subjects and amnesic patients.

In three separate experiments, we assessed the strength and duration of word completion effects in amnesic patients and two control groups. In Experiment 1 subjects studied words under a semantic orienting condition and were given tests of word completion and recognition memory after an immediate, 2-hr or 4-day delay. In the word completion test for Experiment 1, we presented three-letter word stems that could be completed to form several common words, one of which had been presented previously (e.g. MOT for MOTEL), and subjects completed each stem with the first word that came to mind. Priming effects were equivalent in amnesic patients and control subjects and they reached baseline levels within 2 hr. In Experiments 2 and 3, subjects studied words under either a semantic or a nonsemantic orienting condition, and word completion was tested at the same three delays using cues that uniquely specified the study words (e.g. JUI for JUICE; or A--A--In for ASSASSIN). In these experiments, amnesic patients exhibited both smaller and shorter lasting word completion effects than control subjects. Specifically, amnesic patients exhibited word completion effects that seldom lasted as long as 2 hr, whereas control subjects usually exhibited completion effects lasting 4 days. An important additional finding was that control subjects exhibited larger and longer-lasting word completion effects when tested under the semantic orienting condition than when tested under the nonsemantic orienting condition. Amnesic patients were not affected by this manipulation. Moreover, under the nonsemantic orienting condition, control subjects and amnesic patients performed similarly. The results show that word completion performance is not always fully intact in amnesic patients. Long-lasting word completion effects found in normal subjects may be mediated by declarative or elaborative retrieval processes, which are impaired in amnesic patients. If so, priming as measured by word completion methods is shorter lasting than recent studies of normal subjects would suggest.

Impaired word-stem priming in patients with temporal-occipital lesions.

In the word-stem priming test, words are presented (e.g., MOTEL, PARADE), and later subjects are shown three-letter word stems (e.g., MOT, PAR) and asked to complete each stem with the first word that comes to mind. Word-stem priming, as well as other aspects of implicit memory, are intact in amnesic patients with medial temporal lesions. However, this form of priming has been shown to be impaired in patients with Alzheimer's disease, suggesting that damage to neocortical areas outside the medial temporal lobe contributes to impaired priming in these patients. To examine the role of posterior cortical areas on word-stem priming, we administered the test to patients with unilateral temporal-occipital lesions. Patients with temporal-occipital lesions exhibited significantly impaired priming on this test. The findings suggest a critical role of the inferior posterior neocortex in the expression of this form of implicit memory.

Memory for the temporal order of events in patients with frontal lobe lesions and amnesic patients.

Patients with frontal lobe lesions, amnesic patients with Korsakoff's syndrome, other (non-Korsakoff) amnesic patients, and control subjects were given tests of memory for temporal order. In the first experiment, subjects were presented with a list of 15 words and then asked to reproduce the list order from a random array of the words. In the second experiment, they were asked to arrange in chronological order a random display of 15 factual events that occurred between 1941 and 1985. In both experiments, patients with frontal lobe lesions were impaired in placing the items in the correct temporal order, despite normal item memory (i.e. normal recall and recognition memory for the words and facts). The two groups of amnesic patients exhibited impaired memory for temporal order as well as impaired item memory. Patients with Korsakoff's syndrome exhibited poorer temporal order memory than the other amnesic patients, despite similar levels of item memory. These findings demonstrate that patients with frontal lobe lesions have difficulty organizing information temporally. Patients with Korsakoff's syndrome, who have both diencephalic and frontal damage, have memory impairment together with a disproportionate deficit in memory for temporal order.

Source memory impairment in patients with frontal lobe lesions.

In two experiments, we investigated memory for recently learned facts and memory for the source of the facts (i.e. where and when the facts were learned) in patients with frontal lobe lesions, age-matched elderly control subjects, and younger subjects. In both experiments, patients with frontal lobe lesions recalled as many facts as their age-matched subjects and the younger subjects, but they frequently attributed facts to incorrect sources. In the second experiment, both patients with frontal lobe lesions and their age-matched subjects committed more source errors than younger subjects. These findings suggest that the frontal lobes may play a special role in associating facts to the context in which they were learned. The results are also discussed in the light of the source memory impairment that occurs in amnesic patients.

Intact implicit memory in patients with frontal lobe lesions.

Patients with frontal lobe lesions and control subjects were administered tests of word-stem completion priming. In this implicit memory test, subjects are first presented words (e.g. MOTEL, PARADE) in an incidental learning paradigm. Following word presentation, subjects are shown word stems (e.g. MOT, PAR) and asked to produce the first word that comes to mind. Patients with frontal lobe lesions exhibited normal levels of word-stem completion. These findings indicate that implicit memory can operate normally despite damage to the prefrontal cortex. The present results substantiate previous neuropsychological and positron emission tomography findings which indicate that word priming depends critically on posterior cortical areas.

Distinct mathematical behavior of apoptotic versus non-apoptotic tumor cell death.

PURPOSE: The presence or absence of a p53-dependent apoptosis response has previously been shown to greatly influence radiosensitivity in tumor cells. Here, we examine clonogenic survival curves for two genetically related oncogene transformed cell lines differing in the presence or absence of p53 and apoptosis. Solid tumor radiosensitivity patterns have been previously described for these lines. MATERIALS AND METHODS: Oncogene-transformed fibroblasts derived from E1A + Ras transfection of p53-wild-type or p53-null mouse embryonic fibroblasts were plated as single cells and irradiated at increasing radiation doses in single fractions from 1.5 to 11 Gy. Clonogenic cell survival assays were obtained. Survival data are fit to a linear-quadratic relationship: S = e(-alphaD-betaD2). Apoptosis was assessed and quantitated morphologically by staining with the fluorescent nuclear dye DAPI, by TUNEL assay for DNA fragmentation, and by measurement of apoptotic cysteine protease cleavage activity in cytosolic extracts. RESULTS: Whereas radiation triggers massive apoptosis in the presence of p53, it produces no measurable DNA fragmentation, apoptotic cysteine protease cleavage activity, or morphological changes of apoptosis in the cells lacking p53. These contrasting mechanisms of death display dramatically different quantitative behavior: log-survival of apoptotic cells is linearly proportional to dose (S = e(-alphaD)), whereas survival of non-apoptotic (p53 null) is linear-quadratic with a significant quadratic contribution. The surviving fraction at 2 Gy (SF-2) for p53-null cells was 70% verses 12% for p53-intact cells. CONCLUSIONS: In this system, apoptosis appears to exhibit a dominance of single-event which produces a very high alpha/beta ratio, and no significant shoulder; whereas non-apoptotic death in this system exhibits a comparatively small linear component, a low alpha/beta ratio, and a larger shoulder.

Small cell sweat gland carcinoma in childhood.

Sweat gland carcinomas are rare skin tumors that typically occur in older patients. The spectrum of their clinical and pathologic features is broad, and many different types of sweat gland carcinomas have been described, ranging from fairly indolent to highly aggressive neoplasms. We present two cases of sweat gland carcinoma with a predominant small cell morphology. Both tumors occurred in children. One lesion developed in an 8-year-old girl as an asymptomatic papule on her left forearm, which ultimately was evaluated using biopsy because of rapid growth and change in color. The other lesion occurred on the hand of a 12-year-old boy. Both tumors were pandermal and extended into fat. They were composed of monotonous cuboidal cells with scant cytoplasm that formed tubules and grew in anastomosing cords and trabeculae. The tumor cells were immunoreactive for cytokeratins but not for cytokeratin 20. Ultrastructural analysis (available in one case only) showed that the tumor cells lacked neurosecretory granules. This variant of sweat gland carcinoma needs to be distinguished from other small cell neoplasms of the skin, especially Merkel cell carcinoma, its closest mimic.

Paired-associate learning and priming effects in amnesia: a neuropsychological study.

Despite severe deficits of recall and recognition, amnesic patients can exhibit normal priming effects. Amnesic patients have also been reported to perform well on tests of paired-associate learning that involve related word pairs (e.g., table-chair). The present study investigated the role of priming effects in paired-associate learning. Experiment 1 illustrated the distinction between the memory impairment of amnesic patients and their intact priming ability. Amnesic patients were markedly deficient in learning unrelated word pairs, despite exhibiting normal priming as measured by a word-completion test involving the same words. In Experiment 2A, amnesic patients showed good paired-associate learning for related word pairs, though control subjects still performed significantly better. In addition, the good performance by amnesic patients was short-lived, and performance fell to baseline after a 2-hr delay. Control subjects performed well above baseline at all delay conditions. Experiment 2B showed that the forgetting of related word pairs by amnesic patients followed the same time course as the decay of word priming. Experiment 3 showed that amnesic patients were as good as control subjects at learning related word pairs when incidental learning and test procedures were used (a word-association test). The advantage of control subjects over amnesic patients in Experiments 2A and 2B could therefore be attributed to the explicit learning instructions that are standard in paired-associate tests. Finally, Experiment 4 showed that amnesic patients exhibited normal priming when they were asked to "free associate" to words (e.g., child) that were semantically related to previously presented words (e.g., baby). The results indicate that both priming effects and paired-associate learning of related word pairs depend on activation, a process that is preserved in amnesia. Activation can account for the findings of good performance by amnesic patients on tests of word priming (Experiments 1 and 2B), related paired associates (Experiments 2A and 2B), and word association (Experiments 3 and 4). Activation is a transient phenomenon presumed to operate on and facilitate access to preexisting representations. Control subjects can establish new associations and can strengthen preexisting associations by engaging processes that are impaired in amnesia. As a result, when explicit learning instructions are used to test paired-associate learning of related word pairs, control subjects can learn better and can remember longer than can amnesic patients (Experiments 2A and 2B).(ABSTRACT TRUNCATED AT 400 WORDS)