Diagnosis and treatment of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequently-occurring type of malignant lymphoma in the Western world. It has an aggressive natural history, with a median survival of less than one year if left untreated. Immunochemotherapy regimens, consisting of the anti-CD20 antibody rituximab typically in combination with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), are currently the treatment backbone. Despite remarkable progress in improving patient survival, clinical outcomes are still unsatisfactory for certain subsets of patients, including the elderly and very elderly and those with highly aggressive disease. This review outlines some of the current treatment strategies for DLBCL and discusses the main issues that affect clinical practice.

Diagnosis and treatment of follicular lymphoma.

Follicular lymphoma is a slow-growing disease exhibiting a heterogeneous clinical course, with a subset of patients experiencing a rapid disease course in the first two years and some developing disease transformation to a more aggressive phenotype. The advent of highly effective therapies has resulted in an increasing number of patients who achieve long-term progression-free survival alongside a good quality of life. Monoclonal antibodies, such as rituximab, either alone or in combination with chemotherapy regimens or radioimmunotherapy have been used with significant improvements in outcome. New treatment strategies such as new antibodies, biologic agents or vaccination therapy are also under investigation for the treatment of relapsed or refractory disease, further expanding the available options for patients and physicians alike. This article presents an overview of the current therapeutic strategies for the management of follicular lymphoma, focusing on the issues encountered in clinical practice.

A cellular assay for measuring the modulation of glucose production in H4IIE cells.

Type II diabetes and its associated complications are a major health concern of the developed world. One of the hallmarks of diabetes is insulin resistance, where secreted insulin no longer has any effect on its target tissues, namely, liver, muscle, and fat. An important therapeutic strategy is to modulate blood glucose levels using pharmacological agents. Glycogen synthase kinase-3 (GSK3) is a serine-threonine protein kinase that plays important roles in regulating glucose metabolism. It is a key negative regulator of insulin action and is an important contributing factor to insulin resistance in liver, muscle, and adipose tissue. We describe the development of a cell-based assay designed to measure glucose production in rat hepatoma cell line H4IIE liver cells in response to treatment with small molecule inhibitors, including GSK3 inhibitors. The assay is set up in a 96-well format, and glucose production is assessed using a convenient fluorescence-based readout. This disease-relevant cellular assay is a valuable tool for the progression of small molecules that modulate glucose production.

A cellular assay for measuring the inhibition of glycogen synthase kinase-3 via the accumulation of beta-catenin in Chinese hamster ovary clone K1 cells.

Glycogen synthase kinase-3 (GSK3) is a serine-threonine protein kinase that exists as two isozymes, GSK3alpha and GSK3beta. It plays important roles in regulating cell structure, function, and survival, and dysregulation of its function is linked to disorders such as Alzheimer's disease and type II diabetes. In resting cells, GSK3 is active and regulates the function of many downstream targets, including beta-catenin. We describe the development of a cell-based assay designed to measure the activity of GSK3 by directly measuring the accumulation of beta-catenin in Chinese hamster ovary clone K1 (CHOK1) cells. Beta-catenin levels were assessed using an antibody-based staining protocol with a luminometric readout. The assay is set up in a 96-well format. The use of GSK3 inhibitors demonstrated that this assay could be used to compare the effects of various small molecules on GSK3 inhibition in CHOK1 cells.

Cellular imaging in drug discovery.

Traditional screening paradigms often focus on single targets. To facilitate drug discovery in the more complex physiological environment of a cell or organism, powerful cellular imaging systems have been developed. The emergence of these detection technologies allows the quantitative analysis of cellular events and visualization of relevant cellular phenotypes. Cellular imaging facilitates the integration of complex biology into the screening process, and addresses both high-content and high-throughput needs. This review describes how cellular imaging technologies contribute to the drug discovery process.

The p38 pathway regulates Akt both at the protein and transcriptional activation levels during myogenesis.

The molecular signalling pathways governing skeletal muscle differentiation remain unclear. Recent work has demonstrated that both the phosphatidylinositol 3-kinase (PI3K)/Akt and p38 pathways play important roles in myogenesis. Here, we describe the interactions between these pathways in C2C12 cells. Overall, our results suggest that Akt acts downstream of p38 in myogenic cell differentiation. Activating the p38 pathway results in the concurrent activation of Akt; conversely, activating Akt does not affect p38. We have analysed Akt messenger RNA and protein levels in a C2C12 cell line stably expressing a dominant negative (DN) form of the p38 activator MKK3. Compared to control cells, this cell line exhibits reduced levels of Akt messenger RNA and total protein. In addition, blocking the p38 pathway during differentiation inhibits Akt activation. Our results show for the first time that p38 can directly affect Akt at the transcriptional level as well as at the protein activation level during myogenic differentiation.

Regulation of C2C12 myogenic terminal differentiation by MKK3/p38alpha pathway.

The signal transduction pathways connecting cell surface receptors to the activation of muscle-specific promoters and leading to myogenesis are still largely unknown. Recently, a contribution of the p38 mitogen-activated protein kinase (MAPK) pathway to this process was evoked through the use of pharmacological inhibitors. We used several mutants of the kinases composing this pathway to modulate the activity of the muscle-specific myosin light chain and myogenin promoters in C2C12 cells by transient transfections. In addition, we show for the first time, using a stable C2C12 cell line expressing a dominant-negative form of the p38 activator MAPK kinase (MKK)3, that a functional p38 MAPK pathway is indeed required for terminal muscle cell differentiation. The most obvious phenotype of this cell line, besides the inhibition of the activation of p38, is its inability to undergo terminal differentiation. This phenotype is accompanied by a drastic inhibition of cell cycle and myogenesis markers such as p21, p27, MyoD, and troponin T, as well as a profound disorganization of the cytoskeleton.

Dynamic characterization of the molecular events during in vitro epidermal wound healing.

The aim of this study was to characterize some of the molecular events stimulated in vitro in response to injury within a confluent culture of normal epidermal keratinocytes as a model to understand the mechanisms of wound healing. To this end, an original device was developed specifically designed to perform calibrated injuries of great lengths within mono-stratified or pluri-stratified keratinocyte cultures. The experiments performed in this study validate this device as an appropriate tool for studying epidermal wound healing; this is because it performs mechanical injuries that stimulate the expression of multiple healing markers also known to be upregulated during wound healing in vivo (growth factors, cytokines, proteinases, extracellular matrix proteins). Using this device, it was demonstrated in human keratinocytes: mechanical injuries (i) immediately stimulate the tyrosine phosphorylation of numerous cellular proteins; (ii) induce molecular cascades leading to the activation of p21ras, mitogen-activated protein kinases, extracellular signal-regulated kinases 1/2, c-Jun NH2 terminal kinase, and p38 mitogen-activated protein kinase; and (iii) increase the phosphorylation of their respective substrates, c-jun and activator transcription factor 1. Wounding of these cells also results in increases in the DNA binding activities of several jun/fos activator protein-1 transcription factor complexes. It is important to note that the development of an appropriate wounding system was essential for performing this study, as use of a classical wounding procedure did not enable the detection of the biologic parameters reported above. In conclusion, these data indicate that using the appropriate system, it is possible to identify the signaling pathways activated in normal human keratinocyte cells after injury. In this study, it was shown that the mitogen-activated protein kinase pathways and activator protein-1 are stimulated in response to physical injury, and may be involved in regulating the expression of healing markers.

Sorsby's fundus dystrophy tissue inhibitor of metalloproteinases-3 (TIMP-3) mutants have unimpaired matrix metalloproteinase inhibitory activities, but affect cell adhesion to the extracellular matrix.

The TIMP family of matrix metalloproteinase inhibitors consists of four members, of which TIMP-1, -2 and -4 are secreted, freely diffusible proteins, whereas TIMP-3 is ECM-associated. Mutations in the TIMP3 gene have been linked to Sorsby's fundus dystrophy (SFD), an autosomal dominant inherited retinal degenerative disease that leads to blindness. The SFD mutations characterized result in introduction of an unpaired cysteine residue in the C-terminal domain of TIMP-3. We have expressed four SFD mutant TIMP-3 proteins in baby hamster kidney (BHK) cells and evaluated their characteristics alongside wild-type TIMP-3. Analysis of the mutant proteins (Ser156Cys, Gly167Cys, Tyr168Cys and Ser181Cys) by SDS-PAGE and reverse zymography revealed that each of the mutants retained gelatinase A and gelatinase B inhibitory activity, and were localized to the ECM. Association rate constants for Ser156Cys TIMP-3 with gelatinase-A, gelatinase-B, stromelysin-1 and collagenase-3 were only moderately reduced compared to wild-type TIMP-3. However, all of the mutants displayed aberrant protein-protein interactions, resulting in the presence of additional proteins or complexes in ECM preparations. Two of the mutants (Ser156Cys and Ser181Cys) showed a marked propensity to form multiple higher molecular-weight complexes that retained TIMP activity on reverse zymography. Expression of the SFD mutant TIMP-3 (and to a lesser extent, wild-type TIMP-3) proteins in BHK cells conferred increased cell adhesiveness to the ECM. Our findings indicate that the pathogenesis of Sorsby's fundus dystrophy cannot be attributed to a failure to localize SFD TIMP-3 proteins to the ECM or defects in MMP inhibition, but may involve the formation of aberrant TIMP-3-containing protein complexes and altered cell adhesion.

Expression of Sorsby's fundus dystrophy mutations in human retinal pigment epithelial cells reduces matrix metalloproteinase inhibition and may promote angiogenesis.

Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the macula caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. Choroidal neovascularization is a hallmark of this disease, which closely resembles the exudative form of age-related macular degeneration. However, the mechanism by which TIMP-3 mutations induce the disease phenotype in SFD remains unknown. To address this question we established human retinal pigment epithelial cell lines expressing wild type or S156C (Ser(156) changed to cysteine) mutant TIMP-3. S156C TIMP-3 had reduced matrix metalloproteinase (MMP) inhibitory activity in retinal pigment epithelial cells and resulted in increased secretion and activation of gelatinase A and B. The conditioned medium from these cells induced angiogenesis in "in vivo" chick chorioallantoic membrane assays that could be reversed with recombinant wild type TIMP-3. Our data indicate that the choroidal neovascularization in SFD may be a result of increased MMP activity, which could lead to the stimulation of angiogenesis. These results also suggest the potential therapeutic use of TIMP-3 or synthetic MMP inhibitors in this disease.