Soluble CD40 ligand (sCD40L) provides a new delivery system for targeted treatment: sCD40L-caspase 3 chimeric protein for treating B-cell malignancies.
BACKGROUND
A wide range of hematologic malignancies arises from numerous cell types. In an attempt to offer a new target for treating B-cell malignancies, in this study, the authors tested the possibility of using the CD40/CD40L system as a common targeting system for the various malignancies in this group.


METHODS
Two chimeric proteins, soluble CD40 ligand (sCD40L)-caspase 3 (sCD40L-l-Caspase3) and sCD40L-pseudomonas exotoxin 38 (PE38) (sCD40L-l-PE38), were constructed, expressed, and partially purified. The ability of the chimeric proteins to kill tumor cells that expressed CD40 was tested by using proliferation assays. In addition, the induction of apoptosis in treated cells was followed by measuring expression levels of apoptotic proteins using real-time polymerases chain reaction analysis, caspase 3 enzymatic activity, and tracking changes in the cell cycle with fluorescence-activated cell-sorting analysis.


RESULTS
The chimeric proteins exhibited concentration-dependent and time-dependent killing ability. The new chimeric proteins had no effect in several carcinoma cell lines that did not express the CD40 receptor. Treating tumor cells with sCD40L-based chimeric proteins led to internalization of the fusion proteins into the cell cytoplasm of B cells. Shortly after treatment, a sharp rise in B-cell chronic lymphocytic leukemia/lymphoma 2 (Bcl2) expression levels occurred. Approximately 36 hours after the initiation of treatment, Bcl2 levels dropped, whereas Bcl2-associated X protein (Bax) expression levels rose, pushing the cells toward apoptosis. Concomitantly, caspase 3 RNA levels rose.


CONCLUSIONS
sCD40L-based chimeric proteins were able to bind and internalize into B cells that expressed the CD40 receptor and specifically and efficiently induced apoptotic death. Moreover, the current results validated for the first time the ability of sCD40L to serve as a direct delivery system for targeted molecules. sCD40L-based chimeric cytotoxic proteins offer a new weapon in the everlasting war against cancer.
Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Rotem
R
Kedar
Ofra
O
Sabag
Michal
M
Licthenstein
Haya
H
Lorberboum-Galski
D017209:Apoptosis; D001402:B-Lymphocytes / Q000473:pathology*; D015153:Blotting, Western; D019013:CD40 Antigens / Q000235:genetics / Q000378:metabolism; D023201:CD40 Ligand / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D005098:Exotoxins / Q000378:metabolism; D006801:Humans; D015448:Leukemia, B-Cell / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D016393:Lymphoma, B-Cell / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
C494826:BAX protein, human; D019013:CD40 Antigens; D005098:Exotoxins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051028:bcl-2-Associated X Protein; D023201:CD40 Ligand; D053148:Caspase 3