Modiano Test Responses Tim Pearce 1) The overall purpose of figure 4 is to display the effects of the transgenes MALT1 and API2-MALT1 on FAS induced apoptosis in BJAB cells. The authors use 3 cell lines to obtain the data for the figure. One cell line is mock infected and is designated as wild type (WT) while the other two are transfected to express either MALT1 or API2-MALT1. As a control (fig 4D) the authors present flow cytometry data analyzing the FAS receptor expression of each cell line. The results show all three cell lines have similar levels of FAS expression and therefore the differences between cell line responses to FAS ligands cannot be attributed to FAS receptor expression. Figure 4A shows cell viability of WT, MALT1, and API2-MALT1 cell types as a function of FAS ligand concentrations between 1-1000 ng/ml after 16 hours of culture. Both MALT1 and API2-MALT1 cell lines show apoptotic resistance while the WT line shows marked cell death. Addition of CD40, a stimulatory protein which activates a proliferative pathway, to the culture mostly rescues the WT cells from apoptosis but has no significant affect on either MALT1 or API2-MALT1 cell lines (fig 4B). Blocking CD40 with the addition of an antibody to the CD40 positive culture shifted the percent of apoptotic cells back to that of the untreated WT cell line (fig 4C). Figure 4 is meant to show the abilities of MALT1 and API2-MALT1 cell lines to resist apoptosis as compared to the wildtype cell line. Figure B,C are meant to highlight the mechanism by which the WT cell evades apoptosis (by CD40 pathway stimulation) and suggest that MALT1 and API2-MALT1 supplement this pathway. 2) The question I would ask is: Does either MALT1 or API2-MALT1 overexpression overcome a downregulation of BCL10 or CARMA1 mediated immune response? The introduction of the paper suggests that it is probable that MALT1 deregulation and API2-MALT1 fusion maintain and/or enhance cytoprotection after initial CD40 signaling. The authors present a model where MALT1 or API2-MALT1 overexpression results in elevated constitutive and CD40 stimulated IKK activity favoring survival and proliferation but never explicitly prove the necessity of CD40 signaling. It may be possible that either MALT1 or API2-MALT1 alone, or in concert with low levels of MALT1-BCL10-CARMA1 regulated signaling, is sufficient to provide the cell with anti-apoptotic signals. To test this hypothesis it will be necessary to obtain knockdown and knockout cell lines for BCL10 and CARMA1. Ideally, the expression of the proteins will be under the control of an inducing agent which can be supplied to the culture medium allowing the cell to be turned-on or turned-off/down. As in figure 4, an initial experiment should be done to determine the level of FAS receptor expression while the cells are knocked out/down as well as when they are untreated. A second control experiment should be done to determine the amount of MALT1, API2-MALT1, BCL10, and CARMA1 protein expression per time when the various genes are upregulated or downregulated. These control experiments should be done to ensure the general expression levels are not significantly different and thus affecting the cellular response. To test whether constitutively high levels of either MALT1 or API2-MALT1 can induce the IKK anti-apoptotic pathway in the absence of BCL10 or CARMA1, six sets of experiments should be run. Experiment one should use high expression of MALT1 (MALT1 +) with BCL10 absent (BCL10 -) and CARMA1 at a normal level (CARMA1). Experiment 2: MALT1 +, CARMA1 -, BCL Experiment 3: API2-MALT1 +, BCL10 -, CARMA1 Experiment 4: API2-MALT1 +, CARMA1 -, BCL10 Experiment 5: MALT1 + , BCL10 -, CARMA1 - Experiemnt 6: API2-MALT1 +, BCL10 -, CARMA1 ? Measurements of cell viability should be done after 16 hours of FAS ligand culture as in fig 4. I expect that MALT1 overexpression in the absence of the entire MALT1-CARMA1-BCL10 complex will not be sufficient to stimulate the IKK complex pathway. I think it is probable that overexpression of MALT1 as seen in the original experiment acted to stimulate the CD40 pathway enough to overcome lack of CD40 specific signaling but that MALT1 expression itself is not enough to signal IKK complex activation and downstream signaling. On the other hand, I expect that API2-MALT1 overexpression will be sufficient to stimulate this pathway. I believe this to be the case because of the lack of location for this fusion gene product to interact within the currently known pathway. I foresee two possible problems with the current experiment. One, the MALT1-CARMA1-BCL10 complex could prove to be critical in other downstream signaling and the removal of this complex could be lethal to the cell. A second problem is the amount of time it will take to obtain the knockout/knockdown cell lines. With only 5 weeks to go, I hope the process is quick or the PI has these available.
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