Isolation and properties of gene-modified mouse bcr-abl-transformed cells expressing various immunostimulatory factors

Abstract

B210 cells are murine (BalB/c) cells transformed by bcr-abl fusion gene. after intravenous administration they are capable of inducing leukaemia-like disease in syngeneic mice. From these cells a thymidine-kinase less subline was derived. It was significantly less pathogenic than the parental cells. However, a highly pathogenic clone denoted B210cTK -/cl-2 was isolated from its population. as determined by Western blotting, these cells produced more p210 bcr-abl protein than the parental B210 cells. To successfully transfect these cells a modified electroporation method was introduced. Bicistronic plasmids carrying gene for herpes simplex thymidine kinase (HsV TK) and the gene for either granulocyte-monocyte colony stimulation factor (Gm-CsF), interleukin-2 (Il-2) or interleukin 12 (Il-12) were used for the transfection experiments. Gradually, cell lines producing these cytokines were isolated in media supplemented with hypoxantin, aminopterin and thymidine (HaT). all of them were highly sensitive to ganciclovir in vitro confirming that the cells produced HsV TK. The genetic modification of B210cTK-/cl-2 was associated neither with the alteration of p210 bcr-abl production nor with any changes in expression of mHC class I molecules. From populations of each of the three lines several cell clones were isolated and tested for the production of the respective cytokines. The original uncloned population and several clones differing in the cytokine production were administered intravenously into mice. all animals survived without symptoms of the disease suggesting that the gene-modification was associated with the loss of pathogenicity.

Unexpected properties of endostatin-producing mouse BCR-ABL-transformed cells

“…Mouse (Balb/c) B210 cells transformed by the BCR-ABL fusion gene, were the same as in previous experiments (12)(13)(14). These cells had been derived from the Ba/F3 cells of an early B lineage by a retroviral vector carrying the human BCR-ABL fusion gene (15).…”

Properties of bcr-abl-transformed mouse 12B1 cells secreting interleukin-2 and granulocyte-macrophage colony-stimulating factor: I. Derivation, genetic stability, oncogenicity and immunogenicity

“…The 12B1 TK-less cells grew well at a concentration of 100 µg BrdU/ml. The derivation and properties of IL-2-and GM-CSF-secreting B210 cells, designated B210/2/IL-2/cl-21 (producing 30 ng of the cytokine per 10 6 cells/24 h) and B210/2/GM-CSF/cl-1 (producing 40 ng of the cytokine per 10 6 cells/24 h) have been described elsewhere (26). These cells were derived from clone no.…”

“…Plasmids pTR-UF2, pTR-GM-CSF-IRES-TK and pTR-IL-2-IRES-TK were used in non-linearized form. Electroporation was performed using the Gene Pulser Electroporation system (Bio-Rad, Hercules, CA) as described previously (26). Electroporation was carried out at room temperature exposing the cells to 280 V, 1050 µF.…”

“…Cell lysates were prepared and Western blotting with anti-c-ABL monoclonal antibody (Ab-3, Oncogene Research Products, Boston, MA) was performed as described previously (26). Then the membrane was washed 2 times in PBS with 0.1% Tween and reprobed with anti-β-tubulin monoclonal antibody (Sigma-Aldrich, Steinheim, Germany).…”

“…The key element of these studies was the development of a variety of vaccines aimed at inducing protection against challenge with these cells (24,25) and at testing their immunotherapeutic potential (13). In the previous paper of ours (26), we showed that bcr-abl-transformed mouse (Balb/c) B210 cells, which had been gene-modified by transfection of mouse genes for IL-2, IL-12 or GM-CSF immunostimulatory factors, had lost their capability of inducing leukaemia in syngeneic animals. It was of interest to determine how a similar genetic modification would influence the pathogenicity and immunogenicity of another mouse (Balb/c) bcr-abl-transformed cell line, namely 12B1 cells, which are more oncogenic than the B210 cells after intravenous administration and, in addition, form solid lymphoma-like tumours after subcutaneous (s.c.) administration.…”