Weak by the machines: muscle motor protein dysfunction - a side effect of intensive care unit treatment.

Intensive care interventions involve periods of mechanical ventilation, sedation and complete mechanical silencing of patients. Critical illness myopathy (CIM) is an ICU-acquired myopathy that is associated with limb muscle weakness, muscle atrophy, electrical silencing of muscle and motor proteinopathy. The hallmark of CIM is a preferential muscle myosin loss due to increased catabolic and reduced anabolic activity. The ubiquitin proteasome pathway plays an important role, apart from recently identified novel mechanisms affecting non-lysosomal protein degradation or autophagy. CIM is not reproduced by pure disuse atrophy, denervation atrophy, steroid-induced atrophy or septic myopathy, although combinations of high-dose steroids and denervation can mimic CIM. New animal models of critical illness and ICU treatment (i.e. mechanical ventilation and complete immobilization) provide novel insights regarding the time course of protein synthesis and degradation alterations, and the role of protective chaperone activities in the process of myosin loss. Altered mechano-signalling seems involved in triggering a major part of myosin loss in experimental CIM models, and passive loading of muscle potently ameliorates the CIM phenotype. We provide a systematic overview of similarities and distinct differences in the signalling pathways involved in triggering muscle atrophy in CIM and isolated trigger factors. As preferential myosin loss is mostly determined from biochemistry analyses providing no spatial resolution of myosin loss processes within myofibres, we also provide first results monitoring myosin signal intensities during experimental ICU intervention using multi-photon Second Harmonic Generation microscopy. Our results confirm that myosin loss is an evenly distributed process within myofibres rather than being confined to hot spots.

Differential impact of Cetuximab, Pertuzumab and Trastuzumab on BT474 and SK-BR-3 breast cancer cell proliferation.

OBJECTIVES: The potential of epidermal growth factor receptor (EGFR)- and Her2-targeted antibodies Cetuximab, Pertuzumab and Trastuzumab, used in combination to inhibit cell proliferation of breast cancer cells in vitro, has not been extensively investigated. It is anticipated that there would be differences between specific erbB receptor co-expression profiles that would affect tumour cell growth. MATERIALS AND METHODS: We have examined the effects of Cetuximab, Pertuzumab and Trastuzumab, applied separately or in combination, on cell proliferation of BT474 and SK-BR-3 breast cancer cell lines. Cell cycle progression of BT474 and SK-BR-3 cells was statically and dynamically assessed using flow cytometry. In order to discover a potential influence of differential EGFR co-expression on sensitivity to antibody treatment, EGFR was down-regulated by siRNA in SK-BR-3. An annexinV/propidium iodide assay was used to identify potential induction of apoptosis. RESULTS: Treatment with Pertuzumab and Trastuzumab, both targeted to Her2, resulted in a reduced fraction of proliferating cells, prolongation of G(1) phase and a great increase in quiescent BT474 cells. Cetuximab had no additional contribution to the effect of either Pertuzumab or Trastuzumab when administered simultaneously. Treatment with the antibodies did not induce an appreciable amount of apoptosis in either BT474 or SK-BR-3 cells. In contrast to SK-BR-3, the BT474 cell line appears to be more sensitive to antibody treatment due to low EGFR content besides Her2 overexpression. CONCLUSION: The extent of decelerated or blocked cell proliferation after antibody treatment that is targeted to EGFR and to Her2 depends both on EGFR and Her2 co-expression and on antibody combination used in the treatment setting. Cetuximab did not enhance any inhibitory effect of Trastuzumab or Pertuzumab, most probably due to the dominant overexpression of Her2. Cell susceptibility to Trastuzumab/Pertuzumab, both targeted to Her2, was defined by the ratio of EGFR/Her2 co-expression.

Exposure to continuous bromodeoxyuridine (BrdU) differentially affects cell cycle progression of human breast and bladder cancer cell lines.

Incorporation of bromodeoxyuridine (BrdU) during DNA replication is frequently used for cell cycle analysis. The flow cytometric BrdU/Hoechst quenching technique is conducive to high-resolution assessment of cell cycle kinetics, but requires continuous BrdU treatment, which may have cytostatic or cytotoxic effects. Here, we have examined the impact of BrdU on the proliferation of BT474 and SK-BR-3 breast cancer cell lines and compared the observed effects with cell proliferation of RT4 and J82 bladder carcinoma cells, previously described to be sensitive and insensitive to BrdU, respectively. Both uni- and bi-parametric DNA measurements were performed to identify BrdU-induced alterations in the S-phase fraction and in cell cycle progression. An annexinV/propidium iodide (PI) assay was used to identify potential induction of apoptosis by BrdU. Proliferative activity in BT474, SK-BR-3, and RT4 cultures was reduced in different cell cycle phases due to continuous treatment with 60, 5.0, and 3.5 micro m BrdU. This effect, which was not found in J82 cultures, was dependent on exposure time (96 versus 48 h) and was also dose-dependent for RT4 and SK-BR-3. BrdU application does not induce apoptosis or necrosis as revealed with the annexin V/PI assay. We concluded that continuous BrdU treatment did not affect cell viability, but essentially alters cell cycle progression in three out of four cell lines tested. Cell-type specific validation of the feasibility of the powerful BrdU/Hoechst quenching technique is required and recommended.