Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases.

Decreased affinity of the retinoblastoma protein (RB) for the nuclear compartment has been correlated with cell cycle-dependent phosphorylation of the RB protein during the G1/S phase of the cell cycle. We examined the effects of microinjected protein-serine/threonine phosphatases types 1 (PP1) and 2A (PP2A) on nuclear association of RB monitored as the resistance of RB to extraction at the G1/S transition. Microinjection of PP1 into either the nucleus or the cytoplasm of cells synchronized in G1 increased the amount of RB that was resistant to extraction from the nucleus. Microinjection of PP2A, however, required direct injection into the nucleus to generate this effect. In addition, we found that nuclear injection of only the PP2A catalytic subunit (PP2AC) and not the complex containing the A and C subunits inhibited RB extraction. Microinjection of either PP1 or PP2A and the resultant increased affinity of RB for the nucleus corresponded with the inhibition of cell cycle progression into S phase. Injection of either phosphatase into cells that had entered S phase did not block DNA synthesis, suggesting that the effect of the injected phosphatases on cell cycle arrest was specific. In vitro biochemical studies with purified PP1 and PP2A showed that intact RB protein phosphorylated by cdc2 kinase served as a substrate for both protein phosphatases. Our results suggest that protein phosphatases may be important regulators of RB function and support the idea that cell cycle progression is regulated by the phosphorylation state of the RB protein.     

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

Elevated dihydrofolate reductase and impaired methotrexate transport as elements in methotrexate resistance in childhood acute lymphoblastic leukemia

A retrospective study of clinical resistance to methotrexate (MTX) was performed on 29 archival specimens of frozen lymphoblasts obtained from children with acute lymphoblastic leukemia (ALL), including 19 at initial presentation and 10 at first relapse. Blasts were assayed for dihydrofolate reductase and MTX transport by flow cytometry using the fluorescent methotrexate analog, PT430 (Rosowsky et al, J Biol Chem 257:14162, 1982). In contrast to tissue culture cells, patient blasts were often heterogeneous for dihydrofolate reductase content. Of the 19 specimens at initial diagnosis, 7 exhibited dual blast populations, characterized by threefold to 10-fold differences in relative dihydrofolate reductase; the dihydrofolate reductase-overproducing populations comprised 12% to 68% of the total blasts for these specimens. Remission duration intervals for patients exhibiting dual blast populations were notably shorter than for patients expressing a single blast population with lower dihydrofolate reductase ( < or = 9 months v > or = 15 months, respectively), a difference that was statistically significant (P = .045). There was no apparent correlation between expression of increased dihydrofolate reductase at diagnosis and known patient and disease prognostic features (immunophenotype, age, sex, and white blood count). For the relapsed patients, 4 of 10 exhibited dual lymphoblast populations with elevated dihydrofolate reductase. The majority of the patient lymphoblast specimens were entirely competent for MTX transport and, likewise, expressed immunoreactive reduced folate carriers by indirect immunofluorescence staining with specific antiserum to the transporter. Three patients (2 at relapse and 1 at diagnosis) exhibited heterogeneous expression of imparied MTX transport (14% to 73% of blasts). In only 1 of these patients did the majority of the lymphoblasts (73%) show impaired MTX transport and for this specimen, immunoreactive carrier proteins were virtually undetectable. These results suggest that heterogeneous expression of elevated dihydrofolate reductase and impaired MTX transport are important modes of resistance in childhood ALL patients undergoing chemotherapy with MTX and that these parameters may serve as predictive indices of clinical response to MTX.     

7q32-q36 translocations in childhood T cell leukemia: cytogenetic evidence for involvement of the T cell receptor beta-chain gene

Blast cell chromosomal rearrangements involving the long arm of chromosome 7 were identified in eight of 197 cases of childhood acute lymphoblastic leukemia (ALL). Breakpoints were variable but tended to cluster in either the proximal or the terminal 7q region, depending on the immunophenotype of the cells. The 7q32-q36 region, the locus of the T cell receptor beta-chain gene, was the site of breakpoints in four of 31 cases of T cell ALL but was not involved in any of the 166 cases originating from B cell precursors (P less than .0004). In three of the four T cell cases it was possible to identify the chromosomal segment that had been translocated to the 7q32-q36 region: 1p32, 2p21, and 6p21. The 1p32 and 6p21 bands are particularly interesting, as they contain the sites of two known protooncogenes, c-L-myc and hpim, respectively. Our findings suggest that the locus of the beta-chain gene of the T cell receptor is a preferential site for certain chromosomal rearrangements in leukemic T lymphoblasts, analogous to the T cell receptor alpha-chain gene on human chromosome 14. Translocation of proto-oncogenes to a site near the beta-chain regulatory sequences provides a potential mechanism for oncogene activation.     

Ki-ras proto-oncogene mutations in sporadic colorectal adenomas: relationship to histologic and clinical characteristics

BACXKGROUND & AIMS: The goal of this study was to examine the relationship between Ki-ras mutations in colorectal adenomas and characteristics of both the subject (age, gender, and family/personal history of colonic neoplasia) and the adenoma (multiplicity, size, location, and histologic features). METHODS: Ki-ras mutations were detected by direct sequencing in 738 adenomatous polyps removed at baseline from 639 participants in a nutritional trial of adenoma recurrence. RESULTS: Ki-ras mutations were detected in 17.2% of the adenomas. Ki-ras mutations were unrelated to gender, family, or personal history of colonic neoplasia, location within the colorectum, or adenoma multiplicity, but were more common in older subjects (P = 0.01 for trend), in larger adenomas (P < 0.0001 for trend), in adenomas with villous histology (odds ratio [OR], 3.2; 95% confidence interval [CI], 2.1-4.9 vs. tubular), and in adenomas with high-grade dysplasia (32.0% vs. 13.6%; OR, 3.0; 95% CI, 1.9-4.6 vs. low-grade dysplasia). Multivariate analysis showed Ki-ras mutations to be independently associated with subject age (P = 0.01 for trend), tubulovillous/villous histology (OR, 2.3; 95% CI, 1.5-3.7), and high-grade dysplasia (OR, 1.9; 95% CI, 1.2-3.1). Adenoma size was not independently related to Ki-ras mutation. CONCLUSIONS: Ki-ras mutations are associated with the histologic features of adenoma progression (villous histology and high-grade dysplasia) rather than with adenoma growth.     

Prolonged survival of B-lineage acute lymphoblastic leukemia cells is accompanied by overexpression of bcl-2 protein

Overexpression of bcl-2 delays the onset of apoptosis in lymphohematopoietic cells. We measured levels of bcl-2 protein in normal and leukemic human B-cell progenitors with a specific monoclonal antibody and flow cytometry. Normal immature B cells had low levels of bcl-2 protein; the intensity of fluorescence, expressed as molecules of soluble fluorochrome per cell, within CD10+ cells was 3,460 +/- 1,050 (mean +/- SD; 5 samples). In 16 cases of B-lineage acute lymphoblastic leukemia (ALL), cells had levels of bcl-2 that were strikingly higher than those of their normal counterparts (33,560 +/- 14,570; P < .001 by t-test analysis). We next investigated whether the intensity of bcl-2 expression correlated with the resistance of immature B cells to in vitro culture. In 12 cases of B-lineage ALL, the cells recovered after 7 days of culture on allogeneic bone marrow stromal layers were 69% to 178% (median, 95.5%) of those originally seeded. Prolonged survival of leukemic cells in vitro was observed even in the absence of stromal layers in 6 of these 12 cases; the intensity of bcl-2 protein expression in these cases was 45,000 +/- 13,270, compared with 21,500 +/- 7,260 in the 6 cases in which greater than 99.5% of cells rapidly died by apoptosis under the same culture conditions (P = .003). Five immature B-cell lines, continuously growing in the absence of stroma, had the highest bcl-2 expression (79,400 +/- 20,330). By contrast, most normal CD19+, sIg-immature B cells died despite the presence of bone marrow stromal layers; 9.7% to 28.2% were recovered after 7 days of culture in three experiments. We conclude that abnormal bcl-2 gene expression influences the survival ability of B-cell progenitors. This may contribute to leukemogenesis and explain the aptitude of leukemic lymphoblasts to expand outside the bone marrow microenvironment.     

Human interleukin-6 supports granulocytic differentiation of hematopoietic progenitor cells and acts synergistically with GM-CSF

Recombinant human (rh) interleukin-6 (IL-6), in a dose range of 1 to 10 U/mL, was able to induce a low number of neutrophilic-granulocytic colonies in a CFU-GM clonogenic assay, using T cells and adherent cells, depleted low density marrow cells. A synergistic increase in the number of granulocytic colonies was observed when rhGM-CSF at suboptimal doses and IL-6 at effective doses were both present in the assay; the increase was only additive when either rhIL-1 alpha or rhIL- 3 was used together with IL-6. To determine whether the increase in colony number reflects the interactions of these factors on the same hematopoietic progenitor target cells or, instead, represents activation of accessory cells, we analyzed the effect of IL-6 on the proliferation and differentiation of three growth factor-dependent leukemic cell lines that respond with continuous proliferation to the presence of GM-CSF and IL-3 in culture. One of the three cell lines (AML-193) showed limited proliferation in the presence of IL-6 followed by terminal differentiation after 14 days into basophilic-granulocytic- like cells. A synergistic proliferative response was observed on the same cells treated with both GM-CSF and IL-6. These data support the hypothesis that IL-6 may have a direct effect on myeloid hematopoietic progenitor cells, and that GM-CSF interacts synergistically with IL-6 by acting on the same target cells.     

Susceptibility to Atrial Fibrillation:

Propensity for Extrastimulus to Induce AF. Introduction : The development of susceptibility to atrial fibrillation (AF) is a common consequence of many forms of cardiovascular disease, especially heart failure. In this study we used a sheep model of pacing-induced stable early heart failure to describe, quantify, and relate the level of susceptibility to AF to changes in structural and electrophysiologic parameters.
Methods and Results : Epicardial electrodes were implanted on the atria and right ventricles of nine sheep. The AF thresbold, atrial vulnerability period, atrial effective refractory period (ERP), and interatrial conduction time were examined during control and over a 6-week period of ventricular pacing at 190 beats/min. Left atrial (LA) area and left ventricular (LV) fractional shortening were monitored using echocardiography. There were significant increases in LA susceptibility to AF (P < 0.0003), LA area (P < 0.0002), and LA FRP400 (P < 0.0002). Rate of increase in LA area was related positively to AF susceptibility (P = 0.02) and inversely to LA ERP400 (P = 0.002). LV fractional shortening decreased to approximately 50% of control value (P < 0.00001). No changes were observed in right atrial electrophysiology.
Conclusion : In this study, susceptibility (the ability of an extrastimulus to induce AF) was rigorously measured within a predetermined format. Significant relationships were found to exist between susceptibility, certain of the observed changes in atrial electrophysiology and structure.     

The rising rate of admissions for tonsillitis and neck space abscesses in England, 1991–2011

Introduction

Sore throats and tonsillitis represent a considerable health burden as well as a significant source of expenditure for the National Health Service (NHS). As part of the recent NHS savings drive, the introduction of ‘procedures of low clinical effectiveness’ (PoLCE) lists has reinforced a large reduction in the number of tonsillectomies performed. We carried out a cross-sectional study of trends in emergency sore throat admissions in the context of the number of tonsillectomies performed.

Methods

Hospital Episode Statistics (HES) data were extracted. Office for National Statistics data were also used.

Results

Between 1991 and 2011, the overall tonsillectomy rate fell by 44%. In the same time, the admission rate for tonsillitis rose by 310% (Pearson’s r=–0.67, p=0.01). The peritonsillar abscess admission rate rose by 31% (r=–0.79, p<0.01). Between 1996 and 2011, the overall tonsillectomy rate fell by 41% and the retro and parapharyngeal abscess admission rate rose by 39% (r=–0.55, p=0.026). There was a 14% overall increase in tonsillectomy and sore throat associated bed days. This was despite the large fall in tonsillectomy numbers and the reduction in length of hospital stay.

Conclusions

Efforts to reduce the tonsillectomy rate are correlated with a significant rise in emergency admissions. The rise in the retro and parapharyngeal abscess rate is perhaps most alarming given the very high mortality of these conditions. Bed day data suggest that no net saving has been made despite the new measures.