Rapamycin-induced G1 arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3, cyclin E,cyclin A,and survivin

In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G(0)/early G(1) phase of the cell cycle, and defective apoptosis might be involved in disease progression. However, increasing evidence exists that B-CLL is more than a disease consisting of slowly accumulating resting B cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells, and has shown activity against a broad range of human tumor cell lines. Therefore, we investigated the ability of rapamycin to block cell cycle progression in proliferating B-CLL cells. We have recently demonstrated that stimulation with CpG-oligonucleotides and interleukin-2 provides a valuable model for studying cell cycle regulation in malignant B cells. In our present study, we demonstrated that rapamycin induced cell cycle arrest in proliferating B-CLL cells and inhibited phosphorylation of p70s6 kinase (p70(s6k)). In contrast to previous reports on nonmalignant B cells, the expression of the cell cycle inhibitor p27 was not changed in rapamycin-treated leukemic cells. Treatment with rapamycin prevented retinoblastoma protein (RB) phosphorylation in B-CLL cells without affecting the expression of cyclin D2, but cyclin D3 was no longer detectable in rapamycin-treated B-CLL cells. In addition, rapamycin treatment inhibited cyclin-dependent kinase 2 activity by preventing up-regulation of cyclin E and cyclin A. Interestingly, survivin, which is expressed in the proliferation centers of B-CLL patients in vivo, is not up-regulated in rapamycin-treated cells. Therefore, rapamycin interferes with the expression of many critical molecules for cell cycle regulation in cycling B-CLL cells. We conclude from our study that rapamycin might be an attractive substance for therapy for B-CLL patients by inducing a G(1) arrest in proliferating tumor cells.     

A biexponential DWI study in rat brain intracellular oedema

Purpose

To examine the changes in MR parameters derived from diffusion weighted imaging (DWI) biexponential analysis in an in vivo intracellular brain oedema model, and to apply electron microscopy (EM) to shed more light on the morphological background of MR-related observations.

Materials and methods

Intracellular oedema was induced in ten male Wistar rats (380–450 g) by way of water load, using a 20% body weight intraperitoneal injection of 140 mmol/L dextrose solution. A 3T MRI instrument was used to perform serial DWI, and MR specroscopy (water signal) measurements. Following the MR examination the brains of the animals were analyzed for EM.

Results

Following the water load induction, apparent diffusion coefficient (ADC) values started declining from 724 ± 43 μm²/s to 682 ± 26 μm²/s (p < 0.0001).ADC-fast values dropped from 948 ± 122 to 840 ± 66 μm²/s (p < 0.001). ADC-slow showed a decrease from 226 ± 66 to 191 ± 74 μm²/s (p < 0.05).There was a shift from the slow to the fast component at 110 min time point. The percentage of the fast component demonstrated moderate, yet significant increase from 76.56 ± 7.79% to 81.2 ± 7.47% (p < 0.05). The water signal was increasing by 4.98 ± 3.52% compared to the base line (p < 0.01).The results of the E.M. revealed that water was detected intracellularly, within astrocytic preivascular end-feet and cell bodies.

Conclusion

The unexpected volume fraction changes (i.e. increase in fast component) detected in hypotonic oedema appear to be substantially different from those observed in stroke. It may suggest that ADC decrease in stroke, in contrast to general presumptions, cannot be explained only by water shift from extra to intracellular space (i.e. intracellular oedema).     

Molecular imaging of cancer: MR spectroscopy and beyond

Proton magnetic resonance spectroscopic imaging is a non-invasive diagnostic tool for the investigation of cancer metabolism. As an adjunct to morphologic and dynamic magnetic resonance imaging, it is routinely used for the staging, assessment of treatment response, and therapy monitoring in brain, breast, and prostate cancer. Recently, its application was extended to other cancerous diseases, such as malignant soft-tissue tumours, gastrointestinal and gynecological cancers, as well as nodal metastasis. In this review, we discuss the current and evolving clinical applications of proton magnetic resonance spectroscopic imaging. In addition, we will briefly discuss other evolving techniques, such as phosphorus magnetic resonance spectroscopic imaging, sodium imaging and diffusion-weighted imaging in cancer assessment.     

Benzimidazole -Resistance in Haemonchus contortus: New PCR-RFLP Method for the Detection of Point Mutation at Codon 167 of Isotype 1 β-Tubulin Gene

Background

Due to the lack of a suitable and economic test for the analysis of the polymorphism at codon 167, we developed a new PCR-RFLP technique, based on a modified forward primer (UT-HC167 MF-primer), to identify simultaneously the SNPs at codons 167 and 200 of isotype 1 β-tubulin gene of Haemonchus contortus.

Methods

There already are several safe and easy methods for identification of point mutations at codons 198 and 200. Due to the lack of a reliable and easy method for the detection of the single nucleotide polymorphism (SNP) at codon 167, we developed an innovative PCR-RFLP technique based on a modified forward primer (UT-HC167 MF-primer), in which the nucleotide T at the position 443 was substituted through a nucleotide A creating a restriction site for restriction endonuclease SnaB I in the nucleotide sequences including codon 167. A total of 138 adult male H. contortus were collected from three different geo-climatic areas of Iran. The isolated genomic DNA of each single worm was amplified by PCR using primers flanking codon 167. The PCR product (527 bp) was then amplified by semi-nested PCR using the UT-HC167 MF-primer and the reverse primer achieving a PCR product of 451 bp in length. This PCR product was subsequently digested with the restriction endonucleases SnaB I and TaaI for analysis of the mutations at codons 167 and 200, respectively.

Results

All worms had two alleles encoding for phenylalanine (BZ^ss homozygote) for both codons.

Conclusion

Using the UT-HC167 MF-primer and a suitable reverse primer designed upstream from codon 200, it is possible to amplify a PCR product which can be used for analysis of the SNPs at all three mentioned codons using RFLP.     

Massive fundic gland polyposis caused by chronic proton pump inhibitor therapy

We report two cases of a massive fundic gland polyposis associated with protracted proton pump inhibitor (PPI) therapy. Both patients were females aged 51. On repeated endoscopy, the number of fundic gland polyps was increasing steeply, and they resulted in a passage disorder. In the first case, the enormous number of polyps made endoscopic removal impossible, so the patient was treated by total gastrectomy. Although our case is the second one reported in the world, we would like to draw the attention to this rare complication of long lasting PPI therapy.     

Cell markers in hairy cell leukemia studied in cells from 51 patients

To determine the maturation arrest of the neoplastic cells of hairy- cell leukemia (HCL) and the spectrum of the surface markers on these cells, a series of 51 patients with this disease was studied. The cells of all but two of the patients showed monoclonal surface Ig with respect to light chains. In about one-third of the cases, only gamma heavy chain determinants were present on the cells; the majority carried multiple heavy chain determinants as documented by the application of different fluorochromes. Two patients each showed two different clones of cells, both of the same light chain type. In one of these two patients, two paraproteins were present in the serum. Intracytoplasmic Ig was found in only 4 of 39 cases, in all instances being IgM. All cases studied concerned cells with FclgG receptors; however, the density of this receptor varied. FcIgM receptors also showed a spectrum of density, with some cases showing very few FcIgM- positive cells. Receptors C3 were not observed on the hairy cells. Serum immunoglobulin levels were normal or increased. Paraproteins were found in the sera of 4 of 38 patients. These data suggest that HCL is a neoplasm of B lymphocytes. The neoplastic cells are probably arrested at a more mature stage than the cells of chronic lymphocytic leukemia. The multiple isotypes on the cells indicate a block at the "switch" phase from the small micro-carrying lymphocyte to the larger Ig- producing lymphocyte or plasma cell.     

Interaction of platelet plasma membranes with thrombin-activated platelets

This study described the binding of platelet plasma membranes to either control or thrombin-activated platelets. Glycoproteins in plasma membranes isolated from human platelets were labeled by oxidation with periodate followed by reduction with [3H]NaBH4. Labeled membranes were incubated with either control or thrombin-activated platelets. The amount of membranes bound was measured by separating platelets with bound membranes from solution by rapid centrifugation through 27% sucrose and determining the amount of radioactivity associated with platelets. Five- to sevenfold more membranes bound to thrombin- activated platelets than to control platelets. This enhanced binding of labeled membranes was completely inhibited by an excess of unlabeled platelet membranes. Human erythrocyte membranes had little affinity for either washed or thrombin-activated platelets and therefore did not compete for platelet-membrane binding. Binding of platelet membranes to thrombin-treated platelets was inhibited by prior incubation of the platelets with PGI2 suggesting that the enhanced binding of membranes was to activated platelets. This study demonstrates that the purified platelet membranes have functional sites that can mediate membrane binding to platelets and that quantitation of membrane binding appears to reflect the increased aggregation capability of activated platelets.