OBJECTIVETo investigate the effects of immunologic effector cells to enhance apoptosis induced by adriamycin (ADR) in multi-drug resistant human breast cancer cell line MCF7/ADR.

METHODSThe immunologic effector cells were induced and expanded by IFN-gamma, McAb CD3, IL-1 and IL-2. The expression of P-glycoprotein (P-gp) and its relation to apoptosis in target cells were detected by TUNEL technique and immunohistochemical staining. Flow cytometry (FCM) was carried out to determine the expression level of human breast cancer related P185 antigen and the positive rate of Annexin V-FITC/PI expression. The subcellular distribution of ADR and Annexin V expression in the target cells were detected by fluorescence microscopy.

RESULTSThe immunologic effector cells down-regulated the expression of P185 and P-gp in MCF7/ADR cells. The accumulation and subcellular distribution of ADR in MCF7/ADR cells were increased after co-culture with the immunologic effector cells. After treatment with the immunologic effector cells in combination with ADR, apoptosis rate of the target cells was 10 times higher than that induced by ADR alone, and 13 times higher than that induced by the immunologic effector cells alone.

CONCLUSIONImmunologic effector cells can simultaneously down-regulate the expression of P185 and P-gp in MCF7/ADR cell line, and increase the apoptosis rate of MCF7/ADR cells in combination with ADR.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; immunology ; metabolism ; pathology ; Cell Line, Tumor ; Down-Regulation ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Female ; Humans ; Killer Cells, Lymphokine-Activated ; immunology ; Receptor, ErbB-2 ; metabolism

This study was aimed to investigate the proliferation activities and phenotype changes of DC, CIK and DC-CIK, and their cytotoxicity against hepatocarcinoma cells in co-culture of DC with CIK. Peripheral blood mononuclear cells (PBMNC) were isolated from healthy adult donors. After incubation of PBMNC for 2 hours, DCs were induced from adherent cells by some cytokines and CIKs were generated from non-adherent cells. Mature DCs were harvested after incubation for 9 days, and then were co-cultured with CIK at ratio of 1:5 for 3 days. The cytotoxicity activity against SMMC-7721 hepatocellular carcinoma cell line was detected by MTT assay. The results showed that CIK cells were able to lyse SMMC-7721 hepatocellular carcinoma cells at low ratios of effector to target. This effect was significantly enhanced by co-culture with DCs. It is concluded that CIK cells have high lytic activity against 7721 hepatocellular carcinoma cell line, which can be enhanced by co-culture with DC. DC-CIK cells are highly effective immune cells.
Carcinoma, Hepatocellular ; immunology ; pathology ; Cells, Cultured ; Coculture Techniques ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; Humans ; Killer Cells, Lymphokine-Activated ; cytology ; immunology ; Liver Neoplasms ; immunology ; pathology

Cell Line ; Cytotoxicity, Immunologic ; Hepatitis B ; immunology ; Histocompatibility Antigens Class I ; blood ; Histocompatibility Antigens Class II ; blood ; Humans ; Killer Cells, Lymphokine-Activated ; immunology ; Lymphocytes ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

The purpose was to investigate the effect of phytohemagglutinin (PHA) on proliferation and cytotoxicity of cytokine-induced killer (CIK). Peripheral blood mononuclear cells (PBMNCs) from healthy donors were divided into two groups. Cells were resuspended and maintained in complete medium containing of 10% autologous plasma. CIK cells were cultured by traditional method in group one. The other group cells were added PHA to stimulate PBMNCs for 24 hours, then cultured like incubating CIK cells. Their cytotoxicity to different target cells was evaluated by (51)Cr release assay. The results showed that the proliferation multiples of CIK and PHA-CIK cells were both high, however, the latter was much higher than CIK with significance (P < 0.05). Cells in each group cells showed high cytotoxicity. At the same high effector/target ratio PHA-CIK cells cytotoxicity was stronger than CIK cells when targets were K562 cells or acute leukemia cells (P < 0.05). In conclusion, PHA-CIK cells exhibit stronger proliferation and cytotoxicity than CIK cells, and the result provides an experimental basis for biotherapy.
Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; drug effects ; Humans ; K562 Cells ; Killer Cells, Lymphokine-Activated ; cytology ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Phytohemagglutinins ; pharmacology

Total RNA was extracted from human LAK cell, and a cDNA encoding mature peptide HMG-17 and its alpha helix domain was amplified by RT-PCR. The recombinant prokaryotic expression vector pGEX-1lambdaT-HMG-17 and pGEX-1lambdaT HMG-17alpha helix was constructed. Using affinity chromatography, thrombin cleaving and AU-PAGE elution, we obtained the purified HMG-17. Analyses of MIC, MEC and MBC indicated that HMG-17 and HMG-17alpha had strong antibacterial activity. MIC of the alpha-helic domain was almost the same as that of HMG17, suggesting that the alpha-helic structure would be essential for the antibacterial activity of HMG-17.
Anti-Bacterial Agents ; biosynthesis ; pharmacology ; Escherichia coli ; genetics ; metabolism ; HMGN2 Protein ; biosynthesis ; genetics ; pharmacology ; Humans ; Killer Cells, Lymphokine-Activated ; chemistry ; Peptides ; genetics ; pharmacology ; Prokaryotic Cells ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology

OBJECTIVESTo evaluate the effects of K562-dendritic cell (DC) and Raji-DC fusion vaccines on the cytotoxicity of cord blood (CB) derived cytokine-induced killer/natural killer (CIK/NK) cells.

METHODSDC and CIK/NK cells were derived from CB mononuclear cells. CB-DC were fused with inactivated K562 or Raji cells by PEG to form K562 or Raji-DC fusion vaccine. The CIK/NK cells stimulated by different co-culture antigens were three groups: K562-DC or Raji-DC fusion vaccine group, inactivated K562 or Raji plus DC group, and CB-DC alone group. The cytotoxicity of CIK/NK cells stimulated by different co-culture antigens was measured by MTT test.

RESULTSAll the antigens used for stimulation could enhance the cytotoxicity of CB-CIK/NK cells, with no specificity difference. At 20:1 effector-target ratio, the cytolytic activities of K562-DC and Raji-DC fusion vaccine groups against Raji cells were (75.44 +/- 4.19)% and (81.33 +/- 4.18)% respectively (P < 0.05); and that of inactivated K562 + DC and Raji + DC group against Raji cells were (73.12 +/- 4.22)% and (80.49 +/- 4.27)%, respectively (P < 0.01). There was no significant difference in the cytotoxicity to K562 cells between the two fusion vaccine groups (P > 0.05). The cytotoxicity of CB-CIK/NK cells immunized by Raji cells was higher than that by K562 cells. In CIK/NK cells co-stimulated by the same tumor antigen, there was no significant difference in the cytotoxicity between DC fusion vaccine group and inactivated cells plus DC group to different tumor cells.

CONCLUSIONSThe cytotoxicity of CB-CIK/NK cells to tumor cells was not specific. There was no significant difference in the cytotoxic activity of CB-CIK/NK cells between the DC fusion vaccine group and inactivated cells plus DC group.

Cancer Vaccines ; immunology ; pharmacology ; Cells, Cultured ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Fetal Blood ; cytology ; Humans ; K562 Cells ; Killer Cells, Lymphokine-Activated ; immunology ; Killer Cells, Natural ; immunology

OBJECTIVETo investigate whether dendritic cells pulsed with whole tumor lysates (WTL) could in vitro elicit antitumor T cell responses in patients with non-small-cell lung cancer (NSCLC).

METHODSMonocyte-derived immature DCs (imDCs) generated in the presence of human recombinant granulocyte-macrophage colony stimulating factor and interleukin-4 from peripheral blood mononuclear cell of NSCLC patients, and then were induced to mature by pulsing autologous WTL (DCs/WTL) or by the addition of TNF-alpha(TNF/DCs). FACS and MLR assay were used to monitor their phenotypic changes and capacity to stimulate allogeneic and autologous T cell proliferation. DCs/WTL activated with TNF-alpha (* DCs/WTL) were cocultured in vitro with autologous T cells for eliciting antitumor CTLs. T cell mediated antitumor responses were measured by IFN-gamma enzyme-linked immunospot (ELISPOT) assay for WTL-specific IFN-gamma releasing T cells and by lactate dehydrogenase release (LDH) assay for lysis of autologous tumor cells, respectively.

RESULTSWhen monocytes-derived imDCs from the patients with NSCLC (n = 10) were pulsed with autologous WTL for a day at 30 microg total protein of WTL per 10(6) DCs/ml, this led to up-regulation of CD1a, CD83 and CD86 as well as HLA-DR, and also led to marked stimulation of allogeneic T cell proliferating activity, which was comparable to that of TNF/DCs. However, their capacity of stimulating autologous T cell proliferation in vitro was significantly more potent than those of TNF/DCs (P < 0.05). The numbers of WTL-specific IFN-gamma releasing T cells in 1/3 cultures after one week exposure to * DCs/WTL was increased significantly compared with those pulsing with TNF/DCs plus IL-2 or IL-2 alone (P = 0.05). T cells derived by priming of non-adherent PBMCs with * DCs/WTL after 14 days in vitro stimulation were significantly more responsive to autologous tumor cells compared with LAK (n = 3, P < 0.05), but its cytotoxicity against K562 cells was also comparable to LAK cells.

CONCLUSIONMonocyte-derived DCs from NSCLC patients could serve as functional APC. The * DCs/WTL may effectively elicit T cell-mediated antitumor response in vitro and enhance NK killing activity.

Antigens, CD1 ; metabolism ; Carcinoma, Non-Small-Cell Lung ; immunology ; Cell Culture Techniques ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; HLA-DR Antigens ; metabolism ; Humans ; Interferon-gamma ; secretion ; K562 Cells ; Killer Cells, Lymphokine-Activated ; immunology ; Leukocytes, Mononuclear ; immunology ; pathology ; Lung Neoplasms ; immunology ; Lymphocyte Culture Test, Mixed ; T-Lymphocytes, Cytotoxic ; immunology ; pathology ; Tumor Necrosis Factor-alpha ; pharmacology

To investigate the specific antileukemia effect of dendritic cells (DC) pulsed with chronic myelogenous leukemic lysate antigen (CLA), dendritic cells from patients with chronic myelogenous leukemia (CML) were pulsed by CLA, and then cocultured with cytokine-induced killer (CIK) cells from CML patients (CIK + CLA-DC group). The cytotoxic activity in vitro was measured by using a lactate dehydrogenase release assay, and compared with CIK + DC, CIK and CIK + CLA groups. The results showed that under an effector-target ratio of 25:1, the cytotoxic activity of CIK + CLA-DC, CIK + DC, CIK and CIK + CLA groups against autologous CML cells was (68.8 +/- 14.2)%, (52.5 +/- 9.4)%, (20.6 +/- 7.5)% and (24.2 +/- 8.7)%, respectively. CIK + CLA-DC group displayed a strongest cytotoxic activity. When K562 and Raji cells acted as target cells and CIK as effectors, the cytotoxic activity against autologous CML cells in CIK + CLA-DC group (68.8 +/- 14.2)% was much higher than that against K562 cells (14.6 +/- 6.2)% and Raji cells (12.7 +/- 10.2)%, respectively. In conclusion, coculture of CIK cells with DC led to a significant increase in cytotoxic activity. The cytotoxicity could be further increased by DC pulse with CML cell lysate antigen, and cytotoxicity mediated by CML lysate antigen possess stronger specificity.
Antigens, Neoplasm ; immunology ; CD3 Complex ; analysis ; CD4 Antigens ; analysis ; CD56 Antigen ; analysis ; CD8 Antigens ; analysis ; Cell Division ; immunology ; Coculture Techniques ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; Humans ; Immunophenotyping ; K562 Cells ; Killer Cells, Lymphokine-Activated ; cytology ; immunology ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; pathology ; Tumor Cells, Cultured

In order to investigate whether cytokine-induced killer (CIK) cells can induce apoptosis of bcr-abl(+) K562 cells, apoptosis of K562 cells and CEM cells induced by CIK cells, etoposide or camptothecin was detected with flow cytometry DNA assay. RT-PCR showed that K562 cells expressed the bcr-abl fusion gene, K 562 cells, K562 cells/etoposide or K562 cells/camptothecin groups showed no sub-G(1) peak. K562 cells/CIK cells group showed sub-G(1) peak (38.1%). CEM cells showed no sub-G(1) peak. CEM cells/camptothecin or CEM cells/etoposide groups showed sub-G(1) peak (23.5% or 32.3% respectively). CEM cells/CIK cells group showed sub-G(1) peak (45.4%). Etoposide or camptothecin did not induce apoptosis of K562 cells. CIK cells induce apoptosis of K562 cells. Bcr-abl fusion gene prevented apoptosis induced by etoposide or camptothecin, but did not prevent apoptosis induced by CIK cells. This property may support the observed adoptive immunologic effect of allogeneic bone marrow transplantation and donor lymphocyte transfusions of CML case relapsing after allogeneic bone marrow transplantation.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; immunology ; Camptothecin ; pharmacology ; Coculture Techniques ; Cytotoxicity, Immunologic ; Etoposide ; pharmacology ; Flow Cytometry ; Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; K562 Cells ; drug effects ; immunology ; metabolism ; Killer Cells, Lymphokine-Activated ; cytology ; immunology

OBJECTIVETo study the immunoprotective effect of IL-2 and B7-1 gene co-transfected liver cancer vaccine on hepatocarcinogenesis in mice.

METHODSThe murine liver cancer cell strain Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenovirus vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with the vaccines and challenged with the parental Hepal-6 cells afterwards. The immunoprotection was investigated and the reactive T cell line was assayed.

RESULTSThe effect of the Hep6-IL2/B7 vaccine on the onset of tumor formation was the strongest. The media survival time of the mice was the longest (68 days, P<0.05) and the implanted tumor was the smallest (P<0.05). The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the co-transfected gene group with the mean survival time being 59 and 54 days, respectively. The mean survival time of wild or BGFP gene modified vaccine immunized group was 51 and 48 days, respectively. The control group all died within 38 days and the implanted tumor was the largest (P<0.05). The cellular immunofunction test and cytotoxicity study showed that the mice immunized with the Hep6-IL2/B7 vaccine gained significantly increased NK, LAK and CTL activity (29.0% +/- 2.5%, 65.0% +/- 2.9%, 83.1% +/- 1.5% respectively, compared with other groups P<0.05).

CONCLUSIONSThe IL-2 and B7-1 gene co-transfected liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the vaccines may become a novel potential therapy for recurrence and metastases of HCC.

Adenoviridae ; genetics ; Animals ; B7-1 Antigen ; genetics ; immunology ; Cancer Vaccines ; administration & dosage ; genetics ; immunology ; Cell Division ; immunology ; Female ; Genetic Vectors ; administration & dosage ; genetics ; immunology ; Humans ; Interleukin-2 ; genetics ; immunology ; Killer Cells, Lymphokine-Activated ; immunology ; Killer Cells, Natural ; immunology ; Liver Neoplasms, Experimental ; immunology ; pathology ; prevention & control ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Survival Analysis ; T-Lymphocytes, Cytotoxic ; immunology ; Time Factors ; Transfection ; Tumor Cells, Cultured