Animal models of antimuscle-specific kinase myasthenia.

Antimuscle-specific kinase (anti-MuSK) myasthenia (AMM) differs from antiacetylcholine receptor myasthenia gravis in exhibiting more focal muscle involvement (neck, shoulder, facial, and bulbar muscles) with wasting of the involved, primarily axial, muscles. AMM is not associated with thymic hyperplasia and responds poorly to anticholinesterase treatment. Animal models of AMM have been induced in rabbits, mice, and rats by immunization with purified xenogeneic MuSK ectodomain, and by passive transfer of large quantities of purified serum IgG from AMM patients into mice. The models have confirmed the pathogenic role of the MuSK antibodies in AMM and have demonstrated the involvement of both the presynaptic and postsynaptic components of the neuromuscular junction. The observations in this human disease and its animal models demonstrate the role of MuSK not only in the formation of this synapse but also in its maintenance.

Acute severe animal model of anti-muscle-specific kinase myasthenia: combined postsynaptic and presynaptic changes.

OBJECTIVES: To determine the pathogenesis of anti-muscle-specific kinase (MuSK) myasthenia, a newly described severe form of myasthenia gravis associated with MuSK antibodies characterized by focal muscle weakness and wasting and absence of acetylcholine receptor antibodies, and to determine whether antibodies to MuSK, a crucial protein in the formation of the neuromuscular junction (NMJ) during development, can induce disease in the mature NMJ. Design, Setting, and PARTICIPANTS: Lewis rats were immunized with a single injection of a newly discovered splicing variant of MuSK, MuSK 60, which has been demonstrated to be expressed primarily in the mature NMJ. Animals were assessed clinically, serologically, and by repetitive stimulation of the median nerve. Muscle tissue was examined immunohistochemically and by electron microscopy. RESULTS: Animals immunized with 100 µg of MuSK 60 developed severe progressive weakness starting at day 16, with 100% mortality by day 27. The weakness was associated with high MuSK antibody titers, weight loss, axial muscle wasting, and decrementing compound muscle action potentials. Light and electron microscopy demonstrated fragmented NMJs with varying degrees of postsynaptic muscle end plate destruction along with abnormal nerve terminals, lack of registration between end plates and nerve terminals, local axon sprouting, and extrajunctional dispersion of cholinesterase activity. CONCLUSIONS: These findings support the role of MuSK antibodies in the human disease, demonstrate the role of MuSK not only in the development of the NMJ but also in the maintenance of the mature synapse, and demonstrate involvement of this disease in both presynaptic and postsynaptic components of the NMJ.

Cyclin D1b variant promotes cell invasiveness independent of binding to CDK4 in human bladder cancer cells.

Alternative splicing in the cyclin D1 gene produces cyclin D1b variant which lacks a C-terminal region containing the threonine-286 (T286) phosphorylation site required for nuclear export. We have shown that the expression of the cyclin D1b variant is detected in about 60% of human bladder cancer tissues (15/26) and cell lines (3/5). To examine the role of the cyclin D1b variant in bladder carcinogenesis, we introduced wild-type cyclin D1a, cyclin D1b variant or mutant cyclin D1-T286A cDNAs into a human bladder cancer cell line, SBT991, in which cyclin D1b transcript was not expressed, and compared their oncogenic activities. Ectopic expression of cyclin D1b promoted cell invasiveness and anchorage-independent growth of the cancer cells. On the other hand, cyclin D1-T286A enhanced anchorage-independent growth, but did not promote cell invasiveness. The amount of nuclear-localized cyclin D1b and cyclin D1-T286A was higher than that of nuclear-localized cyclin D1a. In addition, introduction of siRNA specific for cyclin D1b into cells of the T24 bladder cancer cell line, in which cyclin D1b transcript was expressed, significantly suppressed cell invasiveness. Immunoprecipitation analysis revealed that cyclin D1a and cyclin D1-T286A could bind to cyclin-dependent kinase 4 (CDK4) but cyclin D1b has lost its capacity to associate with CDK4. Unlike cyclin D1a and cyclin D1-T286A, expression of cyclin D1b did not enhance phosphorylation of Rb protein in SBT991 cells. These results indicate that cyclin D1b promotes cell invasiveness independent of binding to CDK4 to enhance Rb phosphorylation.

Cyclin D1 downregulation is important for permanent cell cycle exit and initiation of differentiation induced by anchorage-deprivation in human keratinocytes.

To understand the relationship between permanent cell cycle exit and differentiation the immortalized keratinocyte cell line, SIK and the squamous cell carcinoma, SCC9 were compared during differentiation induced by anchorage-deprivation. The SIK cells when placed in suspension culture promptly lost almost all ability to reinitiate growth by 2 days concomitantly expressing the differentiation specific proteins, transglutaminase (TGK) and involucrin. These cells rapidly underwent G1 cell cycle arrest with complete disappearance of phosphorylated RB. In contrast SCC9 cells neither showed TGK expression nor increase in involucrin. They decreased their colony-forming ability much more slowly, which coordinated well with a gradual decrease in phosphorylated RB, demonstrating the significant resistance to loss of colony-forming ability and cell cycle exit. In accordance, cyclin D1, a positive regulator of cyclin-dependent kinase (CDK) 4/6 which phosphorylates RB decreased drastically in anchorage deprived SIK but not in SCC9 cells. Endogenous cyclin D1 knockdown in SCC9 cells by siRNA enhanced loss of the colony-forming ability during anchorage-deprivation. Conversely enforced expression of cyclin D1 in SIK cells and in another immortalized keratinocyte cell line, HaCaT, partly prevented loss of their colony-forming abilities. Cyclin D1 overexpression antagonized Keratin 10 expression in suspended HaCaT cells. The result demonstrates the importance of cyclin D1 down regulation for proper initiation of keratinocyte differentiation.

An acidic environment changes cyclin D1 localization and alters colony forming ability in gliomas.

The human glioma cell lines, U87 and T98G, were evaluated for their ability to survive and form colonies in an acidic environment of pH(ext) 6.0. In contrast to U87, which showed an 80-90% survival rate, only 40% of T98G cells survived 6 days at pH(ext) 6.0 and lost their colony forming ability when returned to a normocidic environment. Although both U87 and T98G cells maintain an intracellular pH (pH(i)) of 7.0 at pH(ext) 6.0 and arrest mostly in G1 phase of the cell cycle, only T98G demonstrated a major loss of cyclin D1 that was prevented by the proteasome inhibitor MG132. Colony forming ability was restored by stably transfecting T98G cells with a cyclin D1-expressing plasmid. Both U87 and T98G cells demonstrated increased cytoplasmic localization of cyclin D1 during exposure at pH(ext) 6.0. Upon prolonged (24 h) incubation at pH(ext) 6.0, nuclear cyclin D1 was nearly absent in T98G in contrast to U87 cells. Thus, an acidic environment triggers cytoplasmic localization and proteasomal degradation of cyclin D1.

Elimination of radiation-induced gamma-H2AX foci in mammalian nucleus can occur by histone exchange.

Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX (gamma-H2AX) and formation of large nuclear gamma-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of gamma-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced gamma-H2AX foci. Maximal number of gamma-H2AX foci is observed one hour after irradiation, when approximately 20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of gamma-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of gamma-H2AX. This indicates that elimination of gamma-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange.

Glycogen synthesis correlates with androgen-dependent growth arrest in prostate cancer.

BACKGROUND: Androgen withdrawal in normal prostate or androgen-dependent prostate cancer is associated with the downregulation of several glycolytic enzymes and with reduced glucose uptake. Although glycogen metabolism is known to regulate the intracellular glucose level its involvement in androgen response has not been studied. METHODS: We investigated the effects of androgen on glycogen phosphorylase (GP), glycogen synthase (GS) and on glycogen accumulation in the androgen-receptor (AR) reconstituted PC3 cell line containing either an empty vector (PC3-AR-V) or vector with HPV-E7 (PC3-AR-E7) and the LNCaP cell line. RESULTS: Androgen addition in PC3 cells expressing the AR mimics androgen ablation in androgen-dependent prostate cells. Incubation of PC3-AR-V or PC3-AR-E7 cells with the androgen R1881 induced G1 cell cycle arrest within 24 hours and resulted in a gradual cell number reduction over 5 days thereafter, which was accompanied by a 2 to 5 fold increase in glycogen content. 24 hours after androgen-treatment the level of Glucose-6-P (G-6-P) had increased threefold and after 48 hours the GS and GP activities increased twofold. Under this condition inhibition of glycogenolysis with the selective GP inhibitor CP-91149 enhanced the increase in glycogen content and further reduced the cell number. The androgen-dependent LNCaP cells that endogenously express AR responded to androgen withdrawal with growth arrest and increased glycogen content. CP-91149 further increased glycogen content and caused a reduction of cell number. CONCLUSION: Increased glycogenesis is part of the androgen receptor-mediated cellular response and blockage of glycogenolysis by the GP inhibitor CP-91149 further increased glycogenesis. The combined use of a GP inhibitor with hormone therapy may increase the efficacy of hormone treatment by decreasing the survival of prostate cancer cells and thereby reducing the chance of cancer recurrence.

Inhibition of glycogen phosphorylase (GP) by CP-91,149 induces growth inhibition correlating with brain GP expression.

The role of glycogenolysis in normal and cancer cells was investigated by inhibiting glycogen phosphorylase (GP) with the synthetic inhibitor CP-91,149. A549 non-small cell lung carcinoma (NSCLC) cells express solely the brain isozyme of GP, which was inhibited by CP-91,149 with an IC(50) of 0.5 microM. When treated with CP-91,149, A549 cells accumulated glycogen with associated growth retardation. Treated normal skin fibroblasts also accumulated glycogen with G1-cell cycle arrest that was associated with inhibition of cyclin E-CDK2 activity. Overall, cells expressing high levels of brain GP were growth inhibited by CP-91,149 correlating with glycogen accumulation whereas cells expressing low levels of brain GP were not affected by the drug. Analyses of 59 tumor cell lines represented in the NCI drug screen identified that every cell line expressed brain GP but the profile was dominated by a few highly GP expressing cell lines with lower than mean GP-a enzymatic activities. The correlation program, COMPARE, identified that the brain GP protein measured in the NCI cell lines corresponded with brain GP mRNA expression, ADP-ribosyltransferase 3, and colony stimulating factor 2 receptor alpha in the 10,000 gene microarray database with similar correlation coefficients. These results suggest that brain GP is present in proliferating cells and that high protein levels correspond with the ability of CP-91,149 to inhibit cell growth.

Cell shape change precedes staurosporine-induced stabilization and accumulation of p27kip1.

The requirement of an intact cytoskeleton organization for G1/S cell cycle progression has been demonstrated in cultured cells. In the non-small-cell lung carcinoma cell line A549, the kinase inhibitor staurosporine induced G1 cell cycle arrest with an accumulation of the cyclin-dependent kinase inhibitor p27kip1. Staurosporine induced also a drastic change in cell shape that was accompanied by changes in the actin cytoskeleton. The cytoskeleton disruption agents, cytochalasin D (cyto D) and 2,3-butanedione 2-monoxime (BDM), also induced G1 cell cycle arrest in A549 cells but without an accumulation of p27kip1. A comparison of the cell shape changes caused by these agents revealed that a conversion from an epithelial polygonal shape to an elongated fibroblast-like shape was specific for staurosporine. The shape change induced by staurosporine preceded the accumulation of p27kip1 by about 4 h. The accumulation of p27kip1 was not due to enhanced transcription but to stabilization of the protein resulting from the inhibition of proteolytic degradation. Staurosporine, however, did not inhibit directly the proteasome that was involved in the cell-cycle-dependent p27kip1 degradation. The results indicate that the cell shape change caused by staurosporine correlates with the accumulation of p27kip1 and that staurosporine interferes with the p27kip1-specific proteolysis activity.