Expression of the Purinergic P2X7 Receptor in Murine MOPC315.BM Myeloma Cells

Round 1

Reviewer 1 Report

General Comments:

The authors of this study investigated the  P2X7 receptor function and expression  in murine MOPC315.BM myeloma cells,  a model for studying multiple myeloma and the associated bone disease. For comparison, they performed similar experiments in human myeloma cell line RPMI-8226, and primary osteoclasts derived from BALB/c mice. They found that ATP or BzATP application has no effect on calcium influx and YO-PRO-1 uptake in MOPC315.BM cells indicating that these cells lack functional. In agreement with this, they they found low P2rx7 but high P2rx4 gene expression in MOPC315.BM cells  They concluded that MOPC315.BM cells can be used as model to study the role of P2X3 and P2X4 in multiple myeloma and to investigate possible impact of P2X7R knock in on this disease,

Major Comments:

1.      Example records form calcium measurements and dye uptake experiments have to be shown.

2.      It is not understandable why the authors used  specific P2X7 antagonists in experiments on murine MOPC315.BM myeloma cells that did not respond to ATP or BzATP, but  not in experiments with human cell line RPMI-8226 that responded to BzATP. It is important to show that BzATP-induced responses in RPMI-8226 cells are inhibited P2X7 antagonists and thus  mediated by P2X7.

3.      Statistics is missing for expression experiments  (Fig.4)

4.      The reason for difference in P2X7 function and expression between  murine MOPC315.BM myeloma and  human myeloma cell line RPMI-8226 is not sufficiently discussed. Is it possible that species differences in structure and agonist sensitivity between mouse and human P2X7 isoforms could  play a role?

Minor Comments:

Line 2: Instead of “P2RX7”, I recommend to use the term "P2X7 receptor" which is widely used in purinergic literature for protein

Line 77:  …murine MOPC315.BM myeloma cells….

Line 83: The source of human myeloma cell line RPMI-8226 (ATCC) should be mentioned here

Line 136: “ primary osteoclasts derived from BALB/c mice“  These cells are not mentioned in section 2.1. Isolation of osteoclasts has to be described in Material and Method section.

Lines 182-183: „ ..antagonists A-438079 (10 µM) or A-740003 (1 µM) did not alter the calcium influx response induced by either 2.5 mM ATP or 300 µM BzATP.“ But ATP or BzATP did not stimulate any calcium influx. Thus, the sentence should be rephrased, maybe like follows:  ".. antagonists A-438079 (10 µM) or A-740003 (1 µM) did not alter calcium signal in the presence of 2.5 mMATP...“

 Line 184: instead of „c“ should be „e“

Line 205: Why 500 BzATP was used in dye uptake experiments? It is extremely high concentration even for human P2X7. Does it mean that no uptake was observed with 100 BzATP?

 Line 217: in MOPC315.BM cells

 Figure 1: concentration of antagonists is given in panel "c" and thus should be also given in panel "d".

 Figures 1e :  Involvement of P2X7 in BzATP stimulated responses in RPMI-8226 has be confirmed using P2X7R  antagonists.  Provided that these cells express P2Y receptors, BzATP-stimulated calcium signal could be due to calcium mobilization from intracellular sources.

 Figure 4: Differences in colors before and after treatment with BzATP are not always visible and statistics is missing. Please, insert full names of candidate genes involved in purinergic signaling pathways into the text of figure.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

  The manuscript "Expression of the purinergic receptor, P2RX7 in murine MOPC315.BM myeloma cells" is a preliminary study designed to investigate the expression of P2X7 receptors in a validated cellular model for studying MM, and to test its functionality by measuring influx of calcium and pore formation.   Overall, the manuscript is well-reasoned and the experimental design is clear. However, if the expression of other P2X and P2Y receptors had not been investigated, the results would only be negative.   The authors could have further explored the functionality of the P2X4 and P2X3 receptors and the article would have been more interesting.   Taking into account the results obtained, the title of the article could be changed.   The discussion can be improved and I have two main issues that I would like to see clarified in the discussion and conclusions:   1- How could the expression of P2X3 receptors in the MOPC315.BM cell line represent a potential target for pain modulation in the MM?   2- Can you justify the sentence "Gene modification strategies such as to generate a P2RX7 knock in could be employed with MOPC315.BM cells to help study the potential of P2RX7 as a druggable target in MM".   I do not understand this proposal taking into account that the cell line used is a validated model of MM and expresses other receptors that may be more relevant targets, and druggable, than the P2X7 receptors which was found to be underexpressed.   Best regards.

Good

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The mansucript was revised as adviced, I have no further comments.