Comparative analysis of antibodies to xCT (Slc7a11): Forewarned is forearmed.

The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-)) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges.

The natural compound forskolin synergizes with dexamethasone to induce cell death in myeloma cells via BIM.

We have previously demonstrated that activation of the cyclic adenosine monophosphate (cAMP) pathway kills multiple myeloma (MM) cells both in vitro and in vivo. In the present study we have investigated the potential of enhancing the killing of MM cell lines and primary MM cells by combining the cAMP-elevating compound forskolin with the commonly used MM therapeutic drugs melphalan, cyclophosphamide, doxorubicin, bortezomib and dexamethasone. We observed that forskolin potentiated the killing induced by all the tested agents as compared to treatment with the single agents alone. In particular, forskolin had a synergistic effect on the dexamethasone-responsive cell lines H929 and OM-2. By knocking down the proapoptotic BCL-2 family member BIM, we proved this protein to be involved in the synergistic induction of apoptosis by dexamethasone and forskolin. The ability of forskolin to maintain the killing of MM cells even at lower concentrations of the conventional agents suggests that forskolin may be used to diminish treatment-associated side effects. Our findings support a potential role of forskolin in combination with current conventional agents in the treatment of MM.

Death of multiple myeloma cells induced by cAMP-signaling involves downregulation of Mcl-1 via the JAK/STAT pathway.

There is a continuous search for new therapeutic targets for treatment of multiple myeloma (MM). Here we investigated the mechanisms involved in cAMP-induced apoptosis of human MM cells. cAMP-increasing agents rapidly inhibited activation of JAK1 and its substrate STAT3. In line with STAT3 being a regulator of Mcl-1 transcription, the expression of this pro-survival factor was rapidly and selectively reduced. Notably, exogenous interleukin-6 neither prevented the inhibition of JAK1/STAT3 nor the death of MM cells induced by cAMP. Our results suggest that cAMP-mediated killing of MM cells involves inhibition of the JAK/STAT pathway, making the cAMP-pathway a promising target for treatment of MM.

The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS.

The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules µm⁻³), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-D-aspartate (4-methoxy-7-nitroindolinyl-D-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and D-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.

Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity.

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.

Cyclic AMP induces apoptosis in multiple myeloma cells and inhibits tumor development in a mouse myeloma model.

BACKGROUND: Multiple myeloma is an incurable disease requiring the development of effective therapies which can be used clinically. We have elucidated the potential for manipulating the cAMP signaling pathway as a target for inhibiting the growth of multiple myeloma cells. METHODS: As a model system, we primarily used the murine multiple myeloma cell line MOPC315 which can be grown both in vivo and in vitro. Human multiple myeloma cell lines U266, INA-6 and the B-cell precursor acute lymphoblastic leukemia cell line Reh were used only for in vitro studies. Cell death was assessed by flow cytometry and western blot analysis after treatment with cAMP elevating agents (forskolin, prostaglandin E2 and rolipram) and cAMP analogs. We followed tumor growth in vivo after forskolin treatment by imaging DsRed-labelled MOPC315 cells transplanted subcutaneously in BALB/c nude mice. RESULTS: In contrast to the effect on Reh cells, 50 µM forskolin more than tripled the death of MOPC315 cells after 24 h in vitro. Forskolin induced cell death to a similar extent in the human myeloma cell lines U266 and INA-6. cAMP-mediated cell death had all the typical hallmarks of apoptosis, including changes in the mitochondrial membrane potential and cleavage of caspase 3, caspase 9 and PARP. Forskolin also inhibited the growth of multiple myeloma cells in a mouse model in vivo. CONCLUSIONS: Elevation of intracellular levels of cAMP kills multiple myeloma cells in vitro and inhibits development of multiple myeloma in vivo. This strongly suggests that compounds activating the cAMP signaling pathway may be useful in the field of multiple myeloma.