Increased sensitivity of myeloid leukemia cell lines: potential of lovastatin as bone-marrow-purging agent.

Lovastatin reduces the isoprenylation of p21ras via suppression of mevalonic acid generation. Lovastatin has been shown to reduce tumor cell proliferation in a dose-dependent manner. Here, the potential of lovastatin for purging leukemia cells from bone marrow was investigated using the myeloblastic cell lines K562 and KG-1 as a model system, derived from an erythroleukemia and an acute myelogenous leukemia, respectively. Optimal purging conditions were determined using an MTT proliferation and a leukemia colony assay. Elimination of leukemia cells was time- and dose-dependent. Depletion of K562 was 2.5 logs for 100 microM of lovastatin at 72 h of incubation. Compared to another purging agent, 100 microg/ml mafosfamide had an activity comparable to 100 mM lovastatin. Interestingly, KG-1 acute myelogenous leukemia cells were even more sensitive to lovastatin than K562 cells. In clonogenic assays, 100 microM of lovastatin resulted in a 3- to 4-log reduction of K562 colonies. Lovastatin had a progressive effect on normal hematopoietic progenitor cells. At a concentration of 100 microM of lovastatin, CFU-GM colonies were reduced by 1-2 logs. In conclusion, a differential effect on leukemia and normal progenitor cells could be detected in a clonogenic assay. These results suggest that lovastatin deserves further study as an agent for ex vivo marrow purging.

Increase of proliferation rate and enhancement of antitumor cytotoxicity of expanded human CD3+ CD56+ immunologic effector cells by receptor-mediated transfection with the interleukin-7 gene.

Cytokine-induced killer (CIK) cells have been shown to eradicate established tumors in a SCID mouse-human lymphoma model. CIK cells depend on exogenous addition of cytokines such as interleukin-2 (IL-2), interleukin-7 (IL-7) or interleukin-12 (IL-12) for proliferation. In this study, we used the adenovirus-enhanced CD3 receptor-mediated gene transfer for transfection with the IL-7 gene. An episomally replicating plasmid was used containing cDNA of the human IL-7 gene under the control of a CMV promoter for transfection of CIK cells. Biosynthesis of IL-7 was demonstrated by RT-PCR, an enzyme-linked immunosorbent assay (ELISA) and using a bioassay. Transfected cells produced IL-7 in the range between 200 and 1100 pg/10(6) cells in 24 h. IL-7 was shown to be biologically active, since transfected CIK cells showed an improved proliferation rate as compared with nontransfected cells. Expression of IL-7 altered the secretion of other cytokines by CIK cells, in particular the production of TNF alpha increased after transfection. In contrast, nontransfected CIK cells fed with IL-7 showed no increase in TNF alpha secretion. No significant differences were found in expression of surface antigens linked to the cytotoxic activity of CIK cells. Cytotoxic activity against various tumor cell lines (eg renal cell carcinoma, malignant melanoma and colon carcinoma) was tested. Transfected cells possessed a significantly higher cytotoxic activity as compared with nontransfected cells. Receptor-mediated gene transfer effectively delivers expression plasmids for therapeutic genes into CIK cells and CIK cells transfected with an IL-7 gene expression construct may be valuable for adoptive immunotherapy.

Generation of cytokine-induced killer cells using exogenous interleukin-2, -7 or -12.

Immunologic effector cells termed cytokine-induced killer (CIK) cells are generated in vitro from peripheral blood lymphocytes by addition of interferon-gamma, interleukin (IL)-2, IL-1 and an antibody against CD3. CIK cells have been shown to eradicate established tumors in a SCID mouse/human lymphoma model. CIK cells are dependent on exogenous cytokines such as IL-2, IL-7, or IL-12. We studied the effect of these cytokines in detail. Cellular proliferation was analyzed using an MTT proliferation assay, surface antigen expression via flow cytometry, cytotoxic activity using an LDH release assay, and apoptosis via flow cytometric analysis. IL-2, IL-7 and IL-12 led to significant growth of lymphocytes. Cells grown in IL-2 and IL-7 showed higher proliferation rates than cells grown in IL-12 according to the MTT assay. Concerning surface antigen expression, exogenous IL-7 led to a decrease in IL-7 receptor expression (4.8% from 60.4%) and exogenous IL-2 to a decrease in IL-2 receptor expression (61.2% from 73.2%). CD28 expression was higher in cells grown in IL-7 (77.3%) than in cells grown in IL-2 (62.5%). IL-12 led to a decrease in ICAM-1 adhesion molecule expression (57.7% from 76.7%) and an increase in CD56 expression compared with exogenous IL-7. IL-7 led to higher number of CD4-positive cells than IL-2 (53.0% vs 49.5%). No significant difference was found between IL-2, IL-7 and IL-12 in cytotoxic activity measured in an LDH release assay. Small amounts of apoptotic cells were found with all cytokines. However, the percentage of necrotic cells was higher with exogenous IL-12 than with IL-2 or IL-7. In summary, CIK cells can be generated using exogenous IL-2, IL-7 or IL-12. No difference in cytotoxic activity was found. However, significant differences were found in cell proliferation rates, antigen expression and percentage of necrotic cells.

Sensitivity of multidrug-resistant tumor cell lines to immunologic effector cells.

The ability of malignant cells to survive exposure to cytotoxic agents is a major obstacle to cure in patients with cancer. Multidrug resistance and the expression of P-glycoprotein are emerging as a cause of chemotherapy failure. Immunologic effector cells such as lymphokine-activated killer (LAK) cells or cytokine-induced killer (CIK) cells are capable of killing a broad range of tumor cell lines or freshly isolated tumor cells. As demonstrated here, LAK, and CIK cells possess a high level of cytotoxic activity against tumor cell lines both resistant and sensitive to chemotherapeutic agents such as doxorubicin or vinblastine. CIK cells possessed a higher level of cytotoxic activity than LAK cells as determined by 51Cr release and a tumor colony assay. Monoclonal antibodies against P-glycoprotein did not block the lysis of tumor cells resistant to chemotherapy by CIK cells. In contrast, antibodies to LFA-1 and ICAM-1 blocked CIK cell-mediated tumor cell lysis. These data demonstrate that immunological approaches to cancer therapy may be useful in overcoming disease caused by drug resistance.