Biomedical subjects

B Bonavida

Publications and source records attributed to B Bonavida.

At least 55 records · Page 3Linked to original sources

PubMed1997

Immunosensitization of prostate carcinoma cell lines for lymphocytes (CTL, TIL, LAK)-mediated apoptosis via the Fas-Fas-ligand pathway of cytotoxicity.

Several reports suggest that immunotherapy mediated by cytotoxic lymphocytes is beneficial in the destruction of drug-resistant tumor cells. Cytotoxic T lymphocytes kill target cells by two main mechanisms, namely by the perforin pathway and by the Fas-ligand (Fas-L) pathway. The role of the Fas-L pathway in tumor cell killing is not clear because many Fas(+)-expressing tumor cells are resistant to the Fas-L agonist cytotoxic anti-Fas antibody. The human prostate tumor cell lines (PC-3, DU145, and LnCAP) express Fas on the cell surface but are resistant to killing by anti-Fas antibody. This study examined the sensitivity of prostate tumor cells to Fas-L-mediated cytotoxicity and sensitization of the tumor cells by drugs to Fas-L-mediated killing. All three prostate tumor cell lines are resistant to Fas-L killing as determined by the use of the murine CTL hybridoma PMMI that kills only through the Fas-L pathway. However, the addition of subtoxic concentrations of CDDP or VP-16 significantly sensitized the PC-3 and DU145, but not LnCAP, tumor cells to Fas-L killing and apoptosis by PMMI. The sensitization of tumor cells by drugs was inhibited by neutralizing anti-Fas antibody. These findings demonstrate that immunoresistant Fas(+)-expressing DU145 and PC-3 prostate tumor cells can be sensitized by drugs to Fas-L killing. We then examined the role of Fas-L killing by TIL and LAK cells. All three prostate tumor cell lines were sensitive to killing by TIL and LAK and cell killing was primarily mediated through the Ca(2+)-dependent perforin pathway because it was blocked by the addition of EGTA/MgCl2. Sensitization by CDDP or VP-16 did not significantly augment killing of untreated tumor cells by TIL or LAK cells. However, in the presence of EGTA/MgCl2, the addition of CDDP or VP-16 significantly augmented killing of PC-3 and DU145, but not LnCAP, by TIL and LAK, and killing was blocked by neutralizing anti-Fas antibody. These findings demonstrate that both TIL and LAK exhibit a Fas-L-mediated killing pathway that is revealed once the perforin pathway is blocked by the Ca2+ chelator EGTA/MgCl2. Altogether, these findings show that drug-resistant, Fas(+)-expressing PC-3 and DU145 prostate tumor cells can be sensitized by CDDP and VP-16 to killing by Fas-L-bearing CTL, TIL, and LAK cells. Sensitization of tumor cells by drugs may augment the efficacy of immunotherapy in the eradication of tumor cells that are resistant to Fas-L-mediated killing.

Animals↗

PubMed1997

Predictors of natural killer cell-mediated cytotoxicity deficiency in advanced heart failure secondary to either ischemic or idiopathic dilated cardiomyopathy.

The effect of psychologic variables (situational emotional state and psychiatric diagnosis) and physiologic variables (plasma norepinephrine, decreased cardiac exercise capacity, and elevated pulmonary capillary wedge pressure) on natural killer cell activity was evaluated in 19 patients with advanced heart failure of ischemic or idiopathic origin. Only peak exercise capacity was independently predictive of natural killer cell deficiency.

Cardiomyopathy, Dilated↗

PubMed1997

Concomitant killing in vitro of both gp120-coated CD4+ peripheral T lymphocytes and natural killer cells in the antibody-dependent cellular cytotoxicity (ADCC) system.

NK cells play an important immunoregulatory role in first line defense mechanisms against infection. As disease progresses, HIV-1 infected patients show loss of NK cytotoxic function, down-modulation and/or loss of expression of both CD16 and CD56 surface Ags on NK cells and a gradual loss of both CD4+ T cells and NK cell numbers. A potential mechanism by which these manifestations may occur in vivo was investigated. We hypothesized that NK-mediated Ab-dependent cellular cytotoxicity (ADCC), using gp120-coated CD4+ peripheral T lymphocytes as targets and anti-HIV serum, will result in the concomitant killing of the CD4+ T lymphocyte targets and the NK lymphocytes. This hypothesis was examined in an in vitro model system. The findings demonstrate that gp120-coated peripheral T lymphocytes can serve as targets and are killed in ADCC. Further, the NK cells that recover from the ADCC reaction show a loss of cytotoxic function, acquire the CD16(dim/-) CD56(dim/-) phenotype and a significant fraction is killed by activation-induced cell death or apoptosis. These findings are reminiscent of the properties of circulating NK cells in HIV-infected patients. The implication of these findings in the pathogenesis of AIDS is discussed.

Antibodies, Viral↗

PubMed1997

Doxorubicin sensitizes human bladder carcinoma cells to Fas-mediated cytotoxicity.

BACKGROUND: The resistance of bladder carcinoma to anticancer chemotherapeutic agents remains a major problem. Hence, several immunotherapeutic approaches have been developed to treat the drug-resistant cancer cells. Fas antigen (Fas) and Fas ligand participate in cytotoxicity mediated by T lymphocytes and natural killer cells. Like Fas ligand, anti-Fas monoclonal antibody (MoAb) induces apoptosis of the cells expressing Fas. This study examined whether bladder carcinoma cells are sensitive to cytotoxicity mediated by anti-Fas MoAb and whether anticancer agents synergize with anti-Fas MoAb in cytotoxicity. METHODS: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. RESULTS: The T24 human bladder carcinoma cell line constitutively expressed the Fas on the cell surface; however, T24 line was resistant to anti-Fas MoAb. Treatment of T24 cells with anti-Fas MoAb in combination with mitomycin C, methotrexate, or 5-fluorouracil did not overcome their resistance to these agents. However, treatment of T24 cells with a combination of anti-Fas MoAb and doxorubicin resulted in a synergistic cytotoxic effect. In addition, the doxorubicin-resistant T24 cells were sensitive to treatment with a combination of anti-Fas MoAb and doxorubicin. Synergy was also achieved in three other bladder carcinoma cell lines and four freshly derived human bladder carcinoma cells. Treatment with anti-Fas MoAb in combination with epirubicin or pirarubicin also resulted in a synergistic cytotoxic effect on T24 cells. The mechanisms of synergy were examined. Anti-Fas MoAb did not affect the intracellular accumulation of doxorubicin, the expression of P-glycoprotein, or the expression of the antioxidant glutathione S-transferase-pi mRNA. However, treatment with doxorubicin enhanced the expression of Fas on T24 cells. CONCLUSIONS: This study demonstrated that treatment of bladder carcinoma cells with doxorubicin sensitized the cells to lysis by anti-Fas MoAb. The synergistic effect obtained with established doxorubicin-resistant bladder carcinoma cells and freshly isolated bladder carcinoma cells suggests that drug-resistant bladder carcinoma cells can be sensitized by doxorubicin to Fas- and Fas ligant-mediated cytotoxicity by lymphocytes. Furthermore, the sensitization required low concentrations of doxorubicin, thus supporting the in vivo application of a combination of chemotherapy and immunotherapy in the treatment of drug-resistant and/or immunotherapy-resistant bladder carcinoma.

Antibiotics, Antineoplastic↗

PubMed1997

The participation of the Fas-mediated cytotoxic pathway by natural killer cells is tumor-cell-dependent.

Cytotoxic T lymphocytes and natural killer (NK) cells kill target cells by two main mechanisms, namely, the perforin/granzymes and the Fas ligand (Fas-L) pathways. The preferential activation of either of these two mechanisms by target cells is not known. This study examined whether various NK stimuli regulate preferentially the perforin/granzyme or the Fas pathways during the NK-cell-mediated cytotoxic reaction (NK-CMC). Purified peripheral-blood-derived NK cells were stimulated with interleukin-2 (IL-2), IL-12, or interferon alpha (IFN alpha) and their response was analyzed by the reverse transcriptase/polymerase chain reaction (RT-PCR) for NK-associated gene expression and by the 51Cr-release assay for cytotoxic function. RT-PCR data revealed that the perforin, granzyme A and granzyme B mRNAs were constitutively expressed in unstimulated NK cells and the level of perforin mRNA was augmented following activation. IL-2 enhanced the level of Fas-L mRNA in NK cells; however, the Fas-L level was much lower than that obtained in activated T cells. NK-CMC against Fas-sensitive cells was examined in the presence of neutralizing anti-(Fas antigen receptor) (Fas-R) antibody (ZB-4) or EGTA/Mg2+, which inhibits the perforin/granzyme pathway but not the Fas Fas-L interaction. The human colon adenocarcinoma HT-29 cells were sensitized to anti-Fas-R antibody (CH-11) cytotoxicity following treatment with IFN gamma. NK-CMC against untreated HT-29 cells was completely inhibited by EGTA/Mg2+ and was unaffected by ZB-4, while both EGTA/Mg2+ and ZB-4 partially inhibited NK-CMC against IFN gamma-treated HT-29 cells. Similar findings to those obtained with untreated NK cells were observed with NK cells stimulated with IL-2, IL-2 plus IL-12 or IFN alpha. In contrast to IFN gamma-treated HT-29 cells, the neutralizing anti-Fas antibody ZB-4 did not inhibit NK-CMC against Fas-sensitive U937, CEM or Jurkat tumor cells. These findings demonstrate that the Fas pathway is involved in NK-CMC against certain target cells but not all. Further, the data demonstrate that activation of NK cells by IL-2, IL-2 plus IL-12 or IFN alpha does not preferentially modulate the Fas-L-mediated killing by NK cells.

Cytotoxicity, Immunologic↗

PubMed1997

Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs.

More than 50% of patients with aggressive B lymphomas and the majority of patients with low grade lymphomas are not cured by current therapeutic strategies. The lymphomas express the B cell antigen CD20 on the cell surface and this antigen serves as target for antibody-directed therapies. Clinical studies with encouraging results have been underway with the use of a chimeric anti-CD20 antibody (IDEC-C2B8), consisting of human IgG1-6 constant regions and variable regions from the murine monoclonal anti-CD20 antibody IDEC-2B8. This study investigated the potential anti-tumor therapeutic value of combination treatment with anti-C2B8 and cytotoxic drugs. The in vitro study examined the sensitizing effect of C2B8 antibody on the DHL-4 B lymphoma line to various cytotoxic agents. Cytotoxicity was determined by the MTT assay. Surface and cytoplasmic proteins were determined by flow cytometry. Pretreatment of DHL-4 with C2B8 resulted in inhibition of cell proliferation and cell death and a fraction of the cells underwent apoptosis. While the DHL-4 tumor cells were relatively resistant to several cytotoxic drugs, pretreatment with C2B8 rendered the cells sensitive to TNF-alpha, ricin, diphtheria toxin (DTX), adriamycin and cisplatin but not to VP-16. Chemosensitization of DHL-4 tumor cells was not due to downmodulation of either the MDR-1 or bcl-2 gene products. However, treatment of DHL-4 with C2B8 inhibited TNF-alpha secretion. These findings demonstrate that C2B8 antibody potentiates the sensitivity of DHL-4 tumor cells to several cytotoxic agents. Further, the findings suggest that combination treatments with C2B8 antibody and drugs may be of clinical benefit in the treatment of patients with resistant aggressive B lymphomas.

Animals↗

PubMed1997

Chemosensitization of human prostate carcinoma cell lines to anti-fas-mediated cytotoxicity and apoptosis.

Androgen ablation has been an effective treatment in patients with advanced prostate cancer. However, most treated patients develop hormonally resistant disease and do not respond to conventional chemotherapy. Immunotherapy against prostate cancer is an alternative approach in overcoming hormonal/drug-resistant prostate cancer. Cytotoxic immune lymphocytes kill target cells via the perforin/granzyme and the Fas-ligand (Fas-L) pathways. We hypothesize that tumor cells respond poorly to immunotherapy by developing resistance to killing by the Fas-L mechanism. This study investigated whether prostate tumor cells are sensitive to Fas-mediated killing. The human prostate carcinoma cell lines DU145, PC-3, and LnCAP were examined for their sensitivity to killing and apoptosis by the Fas-L agonist anti-Fas antibody and CTLs. All three lines moderately expressed the Fas antigen on the cell surface; however, all three lines were relatively resistant to cytotoxicity mediated by anti-Fas (CH-11) antibody. Pretreatment of DU145 and PC-3 with subtoxic concentrations of drugs followed by anti-Fas antibody resulted in synergistic cytotoxicity and apoptosis, whereas only an additive effect was obtained with LnCAP. Chemosensitization with drugs and anti-Fas was completely blocked by the addition of neutralizing anti-Fas antibody. The murine CTL hybridoma, PMMI, which kills only via the Fas-L pathway, was able to kill chemosensitized PC-3 and DU145 but not LnCAP cells. Furthermore, this cytotoxicity was blocked by anti-Fas neutralizing antibody. Chemosensitization of PC-3 and DU145 prostate tumor cells was not due to up-regulation of Fas-receptor antigen expression. Treatment of tumor cells with cisplatin did not down-regulate the antiapoptotic genes bcl-2, FAP-1, and c-myc. Further, there was no induction by cisplatin of Fas-L on the tumor cells, thus ruling out Fas/Fas-L-mediated autologous killing. These findings demonstrate that pretreatment of drug-resistant/CTL-resistant prostate DU145 and PC-3 tumor cells with subtoxic concentrations of certain chemotherapeutic drugs sensitizes the tumor cells to Fas-mediated cytotoxicity. These findings suggest that chemosensitization of tumor cells should optimize the response to immunotherapeutic interventions in the treatment of hormone-resistant/drug-resistant prostate cancer.

Animals↗

PubMed1996

HTLV-1 gene expression in adult T-cell leukemia cells elicits an NK cell response in vitro and correlates with cell rejection in SCID mice.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). We have previously shown that the ATL cell line, RV-ATL, formed tumors when inoculated into severe combined immunodeficient (SCID) mice. In contrast, the HTLV-1 in vitro-transformed cell line, SLB-1, was nontumorigenic in SCID mice. ATL cells contain HTLV-1 proviral DNA sequences but lack detectable viral gene expression, in contrast to HTLV-1 in vitro-transformed cells, which express all viral gene products. We investigated the role of HTLV-1 gene expression in tumorigenesis by superinfecting RV-ATL cells with HTLV-1. The resulting cell line, HT-1RV, expressed HTLV-1. Injection of HT-1RV cells into SCID mice resulted in a reduced tumorigenic phenotype compared to the parental RV-ATL cells. In vitro natural killer (NK) cell cytotoxicity assays revealed that cell lines expressing HTLV-1 gene products, SLB-1 and HT-1RV, were sensitive to NK cell cytolysis. In contrast, nonexpressing RV-ATL cells were resistant to NK cell cytolysis. These studies indicate that lack of viral gene expression allows HTLV-1-infected cells to elude detection by murine NK cells and increases tumorigenicity in SCID mice. Thus the loss of HTLV-1 gene expression in ATL cells may be an important mechanism by which leukemic cells escape immune surveillance in humans.

Animals↗

PubMed1996

Resistance of AIDS-associated Kaposi's sarcoma cells to Fas-mediated apoptosis.

Escape of tumor cells from apoptotic-mediated stimuli results in tumor cell survival and resistance to cytotoxic mechanisms. Kaposi's sarcoma (KS) is the most common malignancy associated with AIDS, although its pathogenesis is not known. It is clinically important to determine whether AIDS-KS cells are resistant to apoptosis via the Fas system. Three isolates of AIDS-KS cells were studied. Although all KS cells express Fas on the cell surface, these cells were resistant to cytotoxic anti-Fas antibody (IgM, CH-11). Treatment of AIDS-KS cells with actinomycin D sensitized the tumor cells to anti-Fas cytotoxicity and apoptosis. Apoptosis was assessed by morphological changes and DNA fragmentation analysis. Three possible mechanisms related to AIDS-KS cells, resistance to anti-Fas cytotoxicity were examined. First, synthesis and secretion of soluble Fas by the tumor cells can neutralize antibody-induced cytotoxicity. However, none of the three types of KS cells expressed soluble Fas mRNA as determined by reverse transcription (RT)-PCR. Second, the expression of the proto-oncogene bcl-2 can protect cells from apoptotic signals. Analysis of bcl-2 mRNA by RT-PCR revealed that all three AIDS-KS cells express very low levels of bcl-2 mRNA. Third, the Fas-associated phosphatase-1 (FAP-1) is an antiapoptotic molecule reported to interact with Fas and can block transduction of the apoptotic signal. RT-PCR analysis revealed that all three types of AIDS-KS cells express high levels of FAP-1 mRNA, and treatment of KS cells with actinomycin D reduced the levels of FAP-1 mRNA significantly. These findings demonstrate that AIDS-KS cells are resistant to Fas-mediated apoptosis and suggest that FAP-1 may be involved in the acquisition of resistance of AIDS-KS to anti-Fas antibody-mediated apoptosis.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Involvement of the mitochondrion respiratory chain in the synergy achieved by treatment of human ovarian carcinoma cell lines with both tumor necrosis factor-alpha and cis-diamminedichloroplatinum.

BACKGROUND: Previous studies have demonstrated that treatment of human tumor cell lines with a combination of cis-diamminedichloroplatinum (CDDP) and tumor necrosis factor-alpha (TNF-alpha) results in additive/synergistic cytotoxic effects and reverses tumor cell resistance to TNF drugs. Free radical intermediates are induced by both TNF-alpha and CDDP; however, the role of free radicals in synergy is not known. This study investigated the effect of two inhibitors on synergy, phenoxan (Phe) and butylated hydroxyanisole (BHA), which inhibit Complex I and Complex I and II of the mitochondrion respiratory chain, respectively. METHODS: Three human ovarian carcinoma cell lines of different sensitivity to TNF-alpha and/or CDDP were selected for the study and consisted of 222, a TNF/CDDP-sensitive line, 222TR (TNF-resistant), a TNF-resistant, CDDP-sensitive line, and AD10, a TNF-sensitive, CDDP-resistant line. Cytotoxicity was determined by the microculture tetrazolium dye assay. RESULTS: Synergy in cytotoxicity was achieved in all three lines treated with a combination of TNF-alpha and CDDP. Cytotoxicity by either TNF-alpha or CDDP or by both TNF-alpha and CDDP was inhibited in the presence of either Phe or BHA. Pretreatment of tumor cells with either Phe or BHA for up to 4 hours, washed and followed by the addition of the cytotoxic agents (alone or combined), resulted in no inhibitory effect. Pretreatment of the cells with the cytotoxic agent for up to 2 hours, washed and then followed by the addition of Phe, resulted in significant inhibition of cytotoxicity. In contrast to Phe, the addition of BHA as late as 12 hours post pretreatment of the cells with the cytotoxic agent(s) still inhibited cytotoxicity. These results demonstrated that free radicals are involved in cytotoxicity mediated by a single agent, and in synergy with both agents. Further, the results demonstrated that Phe acts at an early stage of the cytotoxic pathway and that BHA acts at both an early and a late stage of the cytotoxic pathway. CONCLUSIONS: These results demonstrated that both TNF-alpha and CDDP rapidly stimulate the induction of free radicals but a lag of several hours was necessary to initiate the irreversible program of cell death. Further, the studies demonstrated that synergy and reversal of drug resistance in ovarian tumor cells by TNF-alpha and CDDP, used in combination, share the same pathway of cytotoxicity as that mediated by TNF-alpha or CDDP used as a single agent.

Antineoplastic Agents↗

PubMed1996

Target-induced inactivation and cell death by apoptosis in a subset of human NK cells.

Interaction of sensitive target cells with NK cells results in both positive and negative signaling. Positive signaling results in the induction of NK cytotoxicity and sensitization for IL-2-mediated proliferation and secretion of cytokines. Negative signaling prevents the NK cells from recycling for cytotoxicity. Functional inactivation is restricted to the NK-target conjugate subset sparing the nonconjugating free NK subset. The mechanism of target-induced inactivation of NK cells was examined in cell-sorted and purified conjugates. The conjugates were subdivided into two fractions; in one fraction the NK cells were dissociated from the target (NKDC), and in the other fraction the conjugates were not disturbed (NKC). After coculture overnight with IL-2, the cytotoxic function of NKC was not augmented although a subpopulation proliferated and secreted TNF-alpha and IFN-gamma into the supernatant. In contrast, NKDC cytotoxic activity was enhanced by IL-2, but proliferated poorly and did not secrete TNF-alpha or IFN-gamma following IL-2 activation. The phenotype of the inactive NKC was found to be CD16dim/- CD692+ CD11b2+. Target-mediated inactivation correlated with target cell sensitivity to NK cytotoxicity. Furthermore, a significant fraction of NK cells in the NKC was programmed for cell death by apoptosis. Altogether, these results demonstrate that sensitive targets inactivate NK cells for cytotoxicity resulting in loss of NK cells. Furthermore, the results suggest that signaling for cytotoxic function by target cells is not linked to signaling for proliferation and secretion of cytokines by NK cells.

Antigens, Surface↗

PubMed1996

Sensitization of human ovarian tumor cells by subtoxic CDDP to anti-fas antibody-mediated cytotoxicity and apoptosis.

Treatment of drug-sensitive or resistant tumor cells with combination of anti-Fas antibody and drugs (e.g., CDDP) results in augmented cytotoxicity and synergy. This study examined a possible underlying mechanism of synergy achieved by anti-Fas and CDDP. Three human ovarian tumor cell lines were selected for the studies, namely, the CDDP-sensitive A2780 and CDDP-resistant variants AD10 and C30 tumor cells. All three lines express Fas but are resistant to cytotoxicity by anti-Fas antibody. Treatment of tumor cells with monoclonal mouse antihuman Fas (IgM) antibody and CDDP resulted in significant augmentation of cytotoxicity and synergy in all three lines. The magnitude of synergy was a function of the concentrations of both the anti-Fas antibody and the CDDP used. Pretreatment of tumor cells first with CDDP, but not with anti-Fas and then treatment with anti-Fas, resulted in synergy, suggesting that CDDP sensitizes the cells to anti-Fas-mediated cytotoxicity. This was corroborated by inhibiting synergy by the addition of neutralizing anti-Fas (IgG) antibody. Sensitization of tumor cells by CDDP resulted in upregulation of surface Fas expression which was dependent on de novo protein synthesis. Findings similar to those obtained in cytotoxicity were also obtained in apoptosis as determined by DNA hypoploidy and DNA fragmentation. The effect of CDDP-mediated sensitization to anti-Fas and cytotoxicity was compared to CDDP-mediated toxicity. The addition of the antioxidant butylated hydroxyanisole (BHA) inhibited CDDP-mediated cytotoxicity in both AD10 and C30 but not in A2780 ovarian tumor cells. However, BHA did not inhibit upregulation of Fas expression by CDDP in all three lines and further BHA did not inhibit the synergy achieved with combination of subtoxic concentrations of CDDP and anti-Fas (IgM) antibody. These findings revealed that CDDP exerts its cytotoxic effect and its sensitization to Fas cytotoxicity by different mechanisms. Since cytotoxic T lymphocytes (CTL) express Fas ligand and kill Fas+ target cells, a significant potentiation of tumor cell killing will be achieved following sensitization of tumor cells to Fas signaling by subtoxic concentrations of CDDP. These findings suggest a new approach for augmenting CTL-mediated immune interventions in the therapy of resistant ovarian tumor cells.

Antibodies↗

PubMed1996

Differential secretion of TNF-alpha and IFN-gamma by human peripheral blood-derived NK subsets and association with functional maturation.

Natural killer cells can be separated into three major subsets (free, binder, and killer) based on their ability to bind and kill sensitive target cells. The nonbinder, nonkiller free cells are the most immature and can be activated to become binders and killers. Natural killer (NK) cells synthesize and secrete several cytokines that are intimately involved in NK activation. This study investigated the secretion of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) by purified NK cells and NK subsets following activation by various stimuli. K562 target cells stimulated secretion of both TNF-alpha and IFN-gamma by both the binder and the killer subsets but not by the free subset. IFN-alpha activated the secretion of IFN-gamma only, whereas IL-2 activated the secretion of both TNF-alpha and IFN-gamma by the binder and killer subsets and secretion was augmented by the addition of K562 to the cultures. Phorbol myristate acetate (PMA) and ionophore stimulated TNF-alpha and IFN-gamma secretion in both the binder and the killer subsets, though IFN-gamma secretion was more pronounced in the binder subset. Activation of TNF-alpha and IFN-gamma secretion was dependent on de novo protein synthesis. Analysis at the single-cell level demonstrated that the binder subset had the highest frequency of cells secreting IFN-gamma. These results demonstrate that both the binder and the killer subsets can be activated to secrete TNF-alpha and IFN-gamma, whereas the free NK subset secretes little or no TNF-alpha and IFN-gamma following activation. These data suggest that the ability of NK cells to secrete TNF-alpha and IFN-gamma following activation correlates with the functional stage of maturation of NK cells.

Cell Differentiation↗

PubMed1996

Significance of cytotoxic activity of peripheral blood lymphocytes against autologous tumor cells in patients with bladder cancer.

Cell-mediated immunity is an important and central mechanism of host resistance to cancer. Most reported studies have used cultured tumor cell lines as targets to assess antitumor cell-mediated cytotoxicity. However, it is difficult to translate the data generated from the cytotoxic activity against cultured tumor cell lines to cytotoxicity against autologous tumors. In a recent study, we have reported on the prognostic significance of circulating cytotoxic lymphocytes against autologous tumor cells in patients with bladder cancer. In this study, we examined whether established bladder cancer cell line like T24 or NK-sensitive K562 target cells can be substituted for autologous bladder cancer cells. The cytotoxic activity of peripheral blood lymphocytes (PBL) against freshly isolated autologous tumor cells, the T24 human bladder cancer cell line and the NK-sensitive K562 human myelogenous leukemia cell line was studied in 63 patients with primary initial bladder cancer by a 12-h 51Cr release assay. The mean percent cytotoxic activity of PBL directed against autologous tumor cells, T24 cells and K562 cells were 11.3%, 18.2% and 29.4%, respectively, using an E:T of 40:1. The cytotoxic activity against T24 cells in patients with bladder cancer was higher than that in normal individuals. The anti-K562 and the anti-T24 cytotoxic activities in patients with low-stage or low-grade bladder cancer were relatively higher than those in patients with high-stage or high-grade cancer, but not statistically significant. There was no correlation between the anti-autologous tumor cytotoxic activity and either the histologic grade or stage in patients with bladder cancer. The extent of the anti-autologous tumor cytotoxic activity was not paralleled with that of either the anti-K562 or the anti-T24 cytotoxic activity. In contrast, the anti-K562 cytotoxic activity correlated positively with the anti-T24 cytotoxic activity. Separation of PBL revealed that the anti-K562 and the anti-T24 cytotoxic activities were mediated mainly by the NK cells, whereas the anti-autologous tumor cytotoxic activity was mediated by both the NK cells and the T lymphocytes. These findings demonstrate that cytotoxicity against T24 or K562 cells is not of prognostic value. The magnitude of the anti-autologous tumor cytotoxic activity of PBL derived from bladder cancer patients might represent an independent and important immunological parameter to monitor disease progression.

Adult↗

PubMed1995

Natural killer cell immunodeficiency in HIV disease is manifest by profoundly decreased numbers of CD16+CD56+ cells and expansion of a population of CD16dimCD56- cells with low lytic activity.

Natural killer (NK) cells were enumerated by three-color immunofluorescence in 255 uninfected and 399 human immunodeficiency virus-infected adults. Several dramatic alterations were observed. First, the median number and percentage of CD16+CD56+ NK cells, the subset that comprises > 90% of the NK cells in healthy adults, were severely decreased (median, 175/mm3 in uninfected controls; 63/mm3 in HIV-infected non-AIDS subjects). Even subjects with > 800 CD4+ cells/mm3 had decreased CD16+CD56+ NK cell levels (97/mm3). Second, the number of CD16+CD56- cells, an NK population that is rare in healthy adults, was elevated (median, 20/mm3 in uninfected controls; 64/mm3 in HIV-seropositive non-AIDS subjects). Third, the expression of CD16 on the NK cells was markedly reduced; some CD56+ cells and virtually all CD56- cells were CD16dim. Fourth, fluorescence-activated cell-sorting studies revealed little NK- or antibody-dependent cellular cytotoxic activity in the CD16dimCD56- cell population. These results indicate that the pathogenesis of HIV disease includes numerical alterations in subpopulations of NK cells. A better understanding of how HIV infection causes this aspect of pathogenesis is needed.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Endogenous interleukin 6 is a resistance factor for cis-diamminedichloroplatinum and etoposide-mediated cytotoxicity of human prostate carcinoma cell lines.

Hormonal treatment of advanced prostatic cancer patients generally results in an initially beneficial response, but the treated patients develop hormonally resistant disease in which no curative therapy is currently available. Recent studies have revealed that interleukin 6 (IL-6) is a growth factor for myeloma, renal cell carcinoma, and certain T-cell lymphomas. Further, IL-6 has been shown to block apoptosis induced by p53, transforming growth factor beta, and certain cancer chemotherapeutic compounds. The objective of the present study was to determine whether IL-6 is a growth factor for two human prostate cancer lines and whether it protects the tumor cells from drug-induced cell death. Two hormone-independent prostate cell lines were used in this study, namely PC-3 and DU145, and these have been shown to be relatively resistant to cis-diamminedichloroplatinum (CDDP), etoposide (VP-16), and adriamycin (ADR). Both cell lines express IL-6 mRNA and secrete IL-6 constitutively. The addition of anti-IL-6 antiserum to the cell lines resulted in a significant inhibition of cell growth up to day 2, and when additional antibody was added at day 2 the inhibition persisted for 4 days. The coaddition of anti-IL-6 antiserum and CDDP or VP-16 resulted in synergy in cytotoxicity in both cell lines, whereas the combination of antibody and ADR or suramin resulted only in additive effects. Sequential treatment revealed that anti-IL-6 antibody was required to achieve synergy, whereas either sequence of pretreatment resulted in synergy with anti-IL-6 and CDDP but not with VP-16. CDDP treatment of tumor cells down-regulated IL-6 mRNA expression and IL-6 secretion. The present findings demonstrate that IL-6 is an autocrine/paracrine growth factor for DU145 and PC-3 prostate lines. Additionally, the secretion of this cytokine protects the tumor cells against the cytotoxic effect of CDDP and VP-16 and its neutralization sensitizes the cells to cytotoxicity. Overall, the studies suggest that agents that can down-regulate or inhibit protective factors in tumors may overcome drug resistance.

Antineoplastic Agents↗

PubMed1995

Sensitization of human renal cell carcinoma cells to cis-diamminedichloroplatinum(II) by anti-interleukin 6 monoclonal antibody or anti-interleukin 6 receptor monoclonal antibody.

Cytotoxic chemotherapy has shown little antitumor activity against renal cell carcinoma (RCC). It has been demonstrated that RCC cells secrete interleukin 6 (IL-6) and express IL-6 receptors (IL-6Rs). IL-6 inhibits apoptosis and enhances manganese superoxide dismutase expression. Several anticancer chemotherapeutic agents exert their cytotoxic activity in part through the induction of apoptosis and the production of free radicals. Thus, the resistance of RCC cells to the anticancer agents might correlate with IL-6 expression. The present study tested this hypothesis by examining the effect of anti-IL-6 mAb and anti-IL-6R mAb on the sensitivity of human RCC cells to anticancer chemotherapeutic agents. Treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb in combination with cis-diamminedichloroplatinum(II) (CDDP) or mitomycin C overcame their resistance to CDDP or mitomycin C. However, treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb in combination with Adriamycin, vinblastine or 5-fluorouracil did not overcome their resistance to these anticancer agents. Treatment of CDDP-resistant Caki-1 cells (Caki-1/DDP), two other RCC cell lines (ACHN and A704), and three freshly derived RCC cells with CDDP in combination with anti-IL-6 mAb or anti-IL-6R mAb reversed the resistance to CDDP in all these tumors. We then studied the effectiveness of other platinum derivatives. Treatment of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb enhanced their sensitivity to carboplatin, but not to trans-diamminedichloroplatinum(II). Several experiments investigated the mechanism of the antibody-mediated sensitization of RCC cells to CDDP. Incubation of Caki-1 cells with anti-IL-6 mAb or anti-IL-6R mAb did not change the intracellular accumulation of CDDP. The expressions of the multidrug resistant phenotype (gp170) and c-myc oncogene were not affected by the antibody-mediated sensitization. Treatment of Caki-1 cells with the anti-IL-6 mAb or anti-IL-6R mAb down-regulated the expression of glutathione S-transferase pi mRNA. This study demonstrates that treatment of RCC cells with CDDP in combination with anti-IL-6 mAb or anti-IL-6R mAb can overcome their CDDP-resistance and that the down-regulation of glutathione S-transferase pi expression by anti-IL-6 mAb or anti-IL-6R mAb might play a role in the enhanced cytotoxicity obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1995

Interferon-alpha activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-alpha secretion by immature human natural killer cells.

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon-alpha (IFN-alpha) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN-alpha activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN-alpha uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN-alpha resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN-alpha resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-gamma. Unlike IL-2, IFN-alpha did not stimulate the Free cells to proliferate or secrete TNF-alpha and activation of cytotoxicity and modulation of surface antigens by IFN-alpha were independent of TNF-alpha. The failure of IFN-alpha to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN-alpha and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF-alpha and proliferation. These results demonstrate that IFN-alpha serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN-alpha proceeds by TNF-alpha- dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.

Antigens, CD↗