Frequency and resistance of CD95 (Fas/Apo‐1) gene‐transfected tumor cells to CD95‐mediated apoptosis by the elimination and methylation of integrated DNA

Abstract

It is important for more effective gene therapies to clarify the mechanisms by which cDNA integrated into cells can maintain or lose its function in vivo. We evaluated genetic and epigenetic events leading to alternation of the introduced CD95 (Fas/Apo-1) gene as a model of gene therapy. Solid tumors formed by CD95 cDNA-transfected hepatoma cells (F6b) were almost completely cured by a single treatment of anti-CD95 monoclonal antibody (mAb) but recurred in gld/gld lpr/lpr mice after initial complete response. Recurred tumors were resistant to repeated mAb treatment. The ratio of resistant cells in tumors was estimated as 4.2 cells per 10 6 cells. The CD95-resistant tumor contained CD95-vanished and CD95-decreased cells. CD95-vanished cells were due to the deletion of CD95cDNA. However, CD95-decreased cells retained CD95cDNA, which was highly methylated when determined with methylation-dependent enzymes and a demethylation reagent, indicating that DNA methylation was responsible for the reduced CD95 expression and resistance to mAb. CD95-decreased cells reduced the CD95 expression further but did not delete cDNA after a second in vivo treatment with anti-CD95 mAb, suggesting that the elimination of cDNA is not induced after its methylation and that cells containing methylated genes became more resistant by further methylation. Thus, the elimination and methylation of integrated cDNA appear to occur through different mechanisms. Our study of resistant tumor cells, which arose by both mutational and epigenetic modifications of the introduced CD95 plasmid, provides important and fundamental information about the fate of introduced cDNA, augmenting the efficiency of gene therapy. ' 2006 Wiley-Liss, Inc.Key words: CD95/Fas/Apo-1; apoptosis; epigenetics; mutation frequency; cDNA methylation; cDNA deletion CD95/Fas/Apo-1 is a type I membrane protein that plays a crucial role in the regulation of cellular homeostasis. [1][2][3][4] In the immune system, CD95 and the CD95 ligand (CD95L) are involved in the induction of apoptosis by T-cell-mediated cytotoxicity 5,6 and the development of T cells, 7 thus, CD95 is a good target for tumor cell gene therapy. 8,9 However, a study of CD95-mediated tumor therapy in the syngeneic system has been hampered by lethal liver damage induced by antibodies to CD95 in wild type mice. 10 Most mice died within 6 hr after an intraperitoneal (i.p.) injection of 100 lg anti-CD95 monoclonal antibody (mAb). Thus, whether murine CD95-mediated apoptosis is indeed involved in the death pathway of murine tumor cells remains to be addressed. On the other hand, mutant mice, lpr and gld, carry a loss-of-function in CD95 and CD95L, respectively. 11 We have overcome this problem by using double-mutant mice which carry both gld and lpr mutations in a homogenous state and therefore express neither functional CD95L nor CD95. 12 These mice are completely resistant to the administration of anti-CD95 mAb. Moreover, they have the advantage of avoiding the possible influences of endogenous CD95L on CD95 1 tumor c...

Enhancement of the Proapoptotic Properties of Newcastle Disease Virus Promotes Tumor Remission in Syngeneic Murine Cancer Models

“…Melanoma and colon carcinoma are some of the human cancers with the worst prognosis due to their metastatic capacity and resistance to chemotherapeutic drugs in which defects on the Fas/FasL system are known to be responsible for the tumor progression (35, 48, 49). The list of malignant tumors in which Fas/FasL deficits have been implicated also includes pancreatic, thyroid and lung carcinomas, breast and ovarian cancer, and blood cancers, among others (35, 50).…”

Identification of a methylation hotspot in the death receptor Fas/CD95 in bladder cancer

“…This pathway facilitates the elimination of unwanted cells and therefore provides an important means of tumor immune surveillance. Loss of Fas protein has been implicated in tumorigenesis (3)(4)(5); reduced Fas expression has been associated with higher stage and grade bladder tumors (6,7) and introduction of Fas cDNA has been considered as a therapeutic option (8).…”