Influence of Systemic Chemotherapy on Anti-P-glycoprotein Antibody-dependent Cell-mediated Cytotoxicity in Patients with Small Cell Lung Cancer

Anti-P-glycoprotein antibody (MRK-16)-dependent cell-mediatedcytotoxicity (ADCC by blood mononuclear cells (MNC was examinedin patients with small cell lung cancer (SCLC) before and aftersystemic chemotherapy. The effect of in vitro treatment of MNCwith interleukin (IL)-2 and macrophage-colony-stimulating factor(M-CSF) was also examined. The ADCC reaction was assessed bya 6 h ⁵¹ Cr-release assay using a multidrug-resistant (MDR)SCLC cell line (H69/VP cells). The MRK-16 monoclonal antibodywas able to augment spontaneous cytotoxicity by MNC, even inSCLC patients. Pretreatment of MNC with IL-2 significantly augmentedtheir ADCC ability in SCLC patients, while M-CSF had no effecton ADCC activity. After the first cycle of systemic chemotherapy,the ADCC activity tended to decline, but ADCC of MNC pretreatedwith IL-2 was not affected. The results suggest that anti-P-glycoproteinantibody, in combination with a cytokine such as IL-2, may betherapeutically useful against human SCLC resistant to chemotherapeuticdrugs.     

Monocyte chemoattractant protein-1 gene modification of multidrug-resistant human lung cancer enhances antimetastatic effect of therapy with anti-P-glycoprotein antibody in SCID mice

Distant metastases and multidrug resistance are critical problems in the therapy of human small cell lung cancer (SCLC). In this study, we investigated whether transduction of the monocyte chemoattractant protein-1 (MCP-1) gene into multidrug-resistant (MDR) human lung cancer cells affected the formation of metastases or their inhibition by the anti-P-glycoprotein (P-gp) monoclonal antibody (MAb) MRK16. MDR human SCLC (H69/VP) cells were transduced with the human MCP-1 gene inserted into the expression vector BCMGSNeo. MCP-1 gene transduction had no effect on drug sensitivity, the expression of surface antigens or the in vitro proliferation of H69/VP cells. Using the metastatic model of NK cell-depleted SCID mice, H69/VP cells transduced with the MCP-1 gene were inoculated intravenously (i.v.) and formed metastatic colonies in the liver, kidneys and lymph nodes, similar to those formed by parent or mock-transduced cells. However, systemic treatment of the mice with MRK16 reduced the metastases of H69/VP cells in the liver, kidneys and lymph nodes, and was significantly more effective in inhibiting the metastases of MCP-1 producing H69/VP than those of mock-transduced cells. MCP-1 gene transduction significantly prolonged the survival of tumor-bearing mice treated with MRK16. Our findings suggest that local production of MCP-1 in the tumor site increases the anti-P-gp antibody-dependent cell-mediated cytotoxicity, and the MCP-1 gene-induced modification of MDR human SCLC cells thereby enhances the antimetastatic effect of therapy with anti-P-gp antibody. Thus, the accumulation of effector cells in the tumor site is a very important factor in the therapy using the anti-P-gp antibody.     

A new quinoline derivative MS-209 reverses multidrug resistance and inhibits multiorgan metastases by P-glycoprotein-expressing human small cell lung cancer cells.

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3 / ADM and H69 / VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3 / ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3 / ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3 / ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3 / ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3 / ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3 / ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3 / ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3 / ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.     

Combined therapy of multidrug-resistant human lung cancer with anti-P-glycoprotein antibody and monocyte chemoattractant protein-1 gene transduction: The possibility of immunological overcoming of multidrug resistance

We determined whether transduction of the monocyte chemoattractant protein-I (MCP-I) gene into MDR human lung cancer cells affected their tumorigenicity and sensitivity to antibody-dependent cellular cytotoxicity (ADCC) reaction mediated by the anti-P-glycoprotein (P-gp) monoclonal antibody MRK 16. The human MCP-I gene inerted into an expression vector (BCMGSNeo) was transfected into MDR human small-cell lung cancer (H69/VP) cells. Monocyte chemotactic activity was found in culture supernatants collected from MCP-I-transfected H69/VP cells, but not in supernatants of parent and mock-transfected cells. In an in vitro experiment, recombinant MCP-I did not affected monocytemediated ADCC against H69/VP cells when added to the monocyte culture in either the activation or the effector phase at sufficient concentrations to attract and activate monocytes. Tumorigenicity and growth rates of MCP-I-producing H69/VP cells in nude mice were similar to those of parental cells and mock-transfected cells. However, systemic treatment with MRK16 was move effective in inhibiting the formation of tumors by MCP-I-gene-transfected cells than by mock-transfected cells. Systemic treatment with MRK16 also inhibited the growth of a mixture (1:1) of MCP-I-producing cells and mock-transfected cells. These results suggest that combination therapy with MRK16 and MCP-I gene transduction may be a useful immunological strategy to inhibit the growth of human MDR cancer cells expressng P-gp. Int. J. Cancer 71:170–177, 1997. © 1997 Wiley-Liss, Inc.     

A New Quinoline Derivative MS-209 Reverses Multidrug Resistance and Inhibits Multiorgan Metastases by P-glycoprotein-expressing Human Small Cell Lung Cancer Cells

Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3/ADM and H69/VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3/ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro , compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3/ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3/ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3/ ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3/ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3/ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3/ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3/ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases.     

Circumvention of multidrug resistance by a quinoline derivative, MS-209, in multidrug-resistant human small-cell lung cancer cells and its synergistic interaction with cyclosporin A or verapamil

Purpose and methods: To develop a clinically useful approach to circumvent P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MDR human small-cell lung cancer (SCLC), we examined the ability of a novel quinoline compound, MS-209, to reverse MDR by inhibition of P-gp function in combination with other MDR-reversing drugs using a cytotoxicity assay. Results: We established MDR human SCLC cells by culture in medium with gradually increasing concentrations of adriamycin (ADM). Compared with the parental human SCLC cells, SBC-3, the MDR variant SBC-3 cells obtained (SBC-3/ADM) were highly resistant to various chemotherapeutic agents due to P-gp expression. MS-209 reversed the resistance to ADM and vincristine (VCR) of SBC-3/ADM and H69/VP cells in a dose-dependent manner. Moreover, MS-209 in combination with cyclosporin A (CsA) or verapamil (VER) synergistically enhanced the antitumor effects of ADM and VCR on SBC-3/ADM cells. MS-209 restored ADM incorporation and this effect was enhanced by CsA and VER, suggesting that these synergistic effects were due to competitive inhibition of P-gp function. Conclusion: MS-209 in combination with CsA or VER might increase the efficacy of these chemotherapeutic agents against MDR human SCLC cells. Received: 10 December 1997 / Accepted: 16 April 1998     

Therapeutic efficacy of a new topoisomerase I and II inhibitor TAS-103, against both P-glycoprotein-expressing and -nonexpressing drug-resistant human small-cell lung cancer

We examined the effect of a novel topoisomerase I and II (topo I and II) inhibitor, TAS-103, on P-glycoprotein (P-gp)-expressing and -nonexpressing drug-resistant human small-cell lung cancer (SCLC) cells in vitro and in vivo. We observed that TAS-103 was effective in inhibiting in vitro proliferation of human SCLC (SBC-3 and H69) cells and their drug-resistant variants SBC-3/ADM or SBC-3/CDDP and H-69/VP, respectively. SBC-3/ADM and H-69/VP expressed high P-gp, whereas SBC-3/CDDP did not. TAS-103 also effectively reduced the tumor growth (more than 50% inhibition) of the parental as well as MDR SCLC cells grown SC in nude mice. Adriamycin (ADM) and cisplatin (CDDP), on the other hand, were effective only against the parental cells, while these drugs failed to inhibit the respective drug-resistant variants in vitro or in vivo. TAS-103 was observed to induce apoptosis dose dependently in the parental as well as drug-resistant SCLC cells as analyzed after 48 h of in vitro treatment, suggesting that the stabilization of cleavable topo I- or II-DNA complexes by topo I and II inhibitors like TAS-103 is followed by apoptosis of the cells. Overall, our study suggests that TAS-103 may have clinical application against drug-resistant human SCLC.     

Effector Cell Analysis of Human Multidrug-resistant Cell Killing by Mouse-Human Chimeric Antibody against P-Glycoprotein

A mouse-human chimeric monoclonal antibody (mAb), MH162, against P-glycoprotein was previously found to be more effective than an all-mouse mAb (MRK16) in lysis of multidrug-resistant (MDR) tumor cells by blood mononuclear cells. The present study was performed to identify the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against MDR cells. The ADCC reaction was assessed by a 6-h ⁵¹Cr release assay. Highly purified lymphocytes (>99%), monocytes (>99%) and neutrophils (>96%) were obtained from peripheral blood of the same healthy donors. A comparison of these three effector cell populations showed no difference between MH162 and its all-murine counterpart MRK16 in MDR cell lysis by monocytes or neutrophils. But MH162 was more effective than MRK16 in lymphocyte-mediated lysis of the MDR cells. The lymphocytes responsible for this ADCC had CD16+ Fc receptors. Pretreatment of monocytes with colony-stimulating factors (IL-3, GM-CSF and M-CSF) caused significant increase in their MH162-mediated lysis of MDR cells. Another anti-P-glycoprotein chimeric mAb (MH171) was also more effective than its murine counterpart MRK17 in lymphocyte-mediated lysis of MDR cells. These findings suggest that mouse-human chimeric mAbs may be useful therapeutically for in vivo destruction of MDR cancer cells by the ADCC reaction.     

Effector cell analysis of human multidrug-resistant cell killing by mouse-human chimeric antibody against P-glycoprotein.

A mouse-human chimeric monoclonal antibody (mAb), MH162, against P-glycoprotein was previously found to be more effective than an all-mouse mAb (MRK16) in lysis of multidrug-resistant (MDR) tumor cells by blood mononuclear cells. The present study was performed to identify the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against MDR cells. The ADCC reaction was assessed by a 6-h 51Cr release assay. Highly purified lymphocytes (greater than 99%), monocytes (greater than 99%) and neutrophils (greater than 96%) were obtained from peripheral blood of the same healthy donors. A comparison of these three effector cell populations showed no difference between MH162 and its all-murine counterpart MRK16 in MDR cell lysis by monocytes or neutrophils. But MH162 was more effective than MRK16 in lymphocyte-mediated lysis of the MDR cells. The lymphocytes responsible for this ADCC had CD16+ Fc receptors. Pretreatment of monocytes with colony-stimulating factors (IL-3, GM-CSF and M-CSF) caused significant increase in their MH162-mediated lysis of MDR cells. Another anti-P-glycoprotein chimeric mAb (MH171) was also more effective than its murine counterpart MRK17 in lymphocyte-mediated lysis of MDR cells. These findings suggest that mouse-human chimeric mAbs may be useful therapeutically for in vivo destruction of MDR cancer cells by the ADCC reaction.     

微型双功能抗体介导人T细胞杀伤耐药实体瘤细胞

目的 探讨抗P-糖蛋白（P-gP）／抗CD3微型双功能抗体介导人T细胞杀伤耐药实体瘤细胞的作用。方法 采用抗E-tag亲和层析柱分离纯化抗P-gp／抗CD3微型双功能抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定;采用^51Cr释放实验,测定抗P-gP／抗CD3微型双功能抗体介导的人T细胞体外靶向杀伤活性;采用多药耐药（MDR）细胞系KB／MDR、敏感KB细胞裸鼠移植瘤模型,测定该微型双功能抗体介导的体内靶向杀伤活性。结果 纯化的抗P-gp／抗CD3微型双功能抗体,在相对分子质量28000和26000处各有1条蛋白带。在抗P-gp／抗CD3微型双功能抗体存在的情况下,激活的T淋巴细胞能够裂解KB／MDR细胞,且随着效靶比的增高而增高。抗P-gp／抗CD3微型双功能抗体联合人T淋巴细胞能有效抑制KB／MDR细胞裸鼠移植瘤的生长,而对敏感KB细胞移植瘤的生长无抑制作用。结论 抗P-gP／抗CD3微型双功能抗体在体内外均能介导人T淋巴细胞杀伤表达P-gP抗原的KB／MDR细胞,是有望用于耐药实体瘤临床治疗的双特异性抗体。     

Novel metastasis model of human lung cancer in SCID mice depleted of NK cells

Metastasis is a critical problem in the treatment of human lung cancer. Thus, a suitable animal model of metastasis of human lung cancer is required for in vivo biological and preclinical studies. In this study, we tried to establish a suitable model for this, using SCID mice. Neither human SCLC H69/VP cells (5 × 10⁶) nor squamous-cell carcinoma RERF-LC-AI cells (1 × 10⁶), injected through a tail vein, formed metastases in untreated SCID mice. Pre-treatment of SCID mice with anti-asialo GM₁ serum resulted in only a few metastases of H69/VP cells, but pre-treatment with anti-mouse IL-2 receptor β chain Ab (TM-β1) resulted in numerous lymph-node metastases 56 days after tumor inoculation. H69/VP-M cells, an in vivo-selected variant line, formed significant numbers of lymph-node metastases even in SCID mice pre-treated with anti-asialo GM₁ serum. SCID mice depleted of NK cells by treatment with TM-β1 showed different patterns of metastasis when inoculated intravenously with the 2 different human lung cancer cell lines (H69/VP and RERF-LC-AI cells): H69/VP cells formed metastases mainly in systemic lymph nodes and the liver, whereas RERF-LC-AI cells formed metastases mainly in the liver and kidneys, with only a few in lymph nodes. A histopathological study showed that the metastatic colonies consisted of cancer cells. The numbers of metastatic colonies formed by the 2 cell lines increased with the number of cells inoculated. TM-β1 treatment of SCID mice efficiently removed NK cells from peripheral blood for at least 6 weeks, whereas, after treatment of the mice with anti-asialo GM₁ serum, NK cells were recovered within 9 days. These findings suggest that NK-cell-depleted SCID mice may be useful as a model in biological and pre-clinical studies on metastasis of human lung cancer. © 1996 Wiley-Liss, Inc.     

Enhanced tumor growth and metastatic spread of an MH134 variant lacking a part of the MM antigen: a possible role of antibody-dependent cellular cytotoxicity in control of tumor growth and metastases

The role of antibody-dependent cellular cytotoxicity (ADCC) in host defense against tumor growth and metastasis was investigated with MH134, an MM antigen-positive murine hepatoma, and MH134-M, a variant with enhanced tumorigenesis and metastasis, in syngeneic C3H/HeN mice. When inoculated subcutaneously into C3H/HeN mice, MH134-M tumors grew more rapidly than did MH134 tumors and consistently metastasized to the draining lymph nodes, whereas MH134 tumors did not. Also, MH134-M exhibited a significantly greater lung colonization potential than did MH134, when inoculated intravenously into C3H/HeN mice. In BALB/c nu/nu mice, however, solid MH134 tumors grew and metastasized to the same extent as MH134-M, indicating that there is no significant intrinsic difference between these two tumor lines in proliferative or metastatic capacity. Enzyme-linked immunosorbent assay (ELISA) and immunoblotting, performed after SDS-PAGE analysis of cellular extracts with a monoclonal antibody (MAb) that recognizes a part of the MM-antigen, revealed that the cells of MH134-M lack at least a part of the MM antigen. Sera of C3H/HeN mice bearing solid MH134 tumors were found to contain anti-MM-antigen antibodies, when tested by immunoblotting of SDS-PAGE-developed materials. Cytotoxicity testing in which thioglycollate-induced peritoneal macrophages were used as effector cells revealed that antibodies present in sera strongly induced ADCC to MH134 but not to MH134-M. On the other hand, sera of MH134-M tumor-bearing C3H/HeN mice neither contained anti-MM-antigen antibodies nor induced ADCC to MH134 or MH134-M tumor cells. Intravenous injection of carrageenan into C3H/HeN mice bearing solid MH134 tumors significantly enhanced tumor growth, whereas the growth of subcutaneously injected MH134-M tumors was not influenced by this treatment. These results suggest that the enhanced tumorigenesis and metastasis of the MH134-M line in C3H/HeN mice are based, at least in part, on significant loss of the MM antigen and the resultant inability to induce ADCC-triggering antibody production during tumor growth.     

M-CSF gene transduction in multidrug-resistant human cancer cells to enhance anti-P-glycoprotein antibody-dependent macrophage-mediated cytotoxicity

A human macrophage-colony-stimulating-factor(M-CSF) gene inserted into an expression vector (pRc/CMV-MCSF) was transfected into multidrug-resistant (MDR) human ovarian cancer cells (AD 10) to induce secretion of human M-CSF into the medium. The M-CSF level in the culture medium of the transfected cells reached 100 ng/ml after 7 days, and the ability of the cells to secrete M-CSF was stable for at least 3 months. Transfection of the M-CSF gene did not result in any change in expression of MDRI (P-glycoprotein), proliferation or chemosensitivity of the cells from those of the parent cells. There was also no difference between the transfected and the parent cells in susceptibility to NK cell- or interleukin-2-activated killer-cell-mediated cytotoxicity. Human blood monocytes that had been incubated for 4 days in medium with the culture supernatant of MH-AD 10 cells exhibited higher ADCC activity than untreated monocytes against MDRI-positive cancer cells. This effect of the supernatant of AD 10 cells was completely abolished by its treatment with a monoclonal anti-M-CSF antibody (MAb). When transfected human MDR cells were injected into nude mice, an inverse correlation was seen between the ability of the cells to produce M-CSF and their tumorigenicity. Thus, gene modification of MDR cancer cells seems hopeful as a therapeutic method for enhancing anti-MDRI -MAb-dependent macrophage-mediated cytotoxicity against human MDR cancer cells.     

Anti-CD19 antibodies inhibit the function of the P-gp pump in multidrug-resistant B lymphoma cells.

After chemotherapy, tumor cells with multidrug resistance (MDR) often emerge. MDR is attributable to the expression of membrane transport proteins that inhibit the cellular influx and increase the efflux of many chemotherapeutic drugs. One such protein is P-glycoprotein (P-gp), which functions as an ATP-dependent active transporter. Recently, an anti-P-gp monoclonal antibody (MAb) that inhibits P-gp has been described. Previous studies from our laboratory using the anti-CD19 B-cell lymphoma-reactive MAb, HD37, have suggested that HD37 may also influence MDR. To test this directly, we used Namalwa/MDR1 cells to study the effect of HD37 on the efflux of rhodamine 123 from these cells. We found that HD37 and three other anti-CD19 MAbs inhibited the efflux of rhodamine 123 from Namalwa/MDR1 cells with approximately 50% of the efficiency of the well-known chemosensitizer, verapamil. In contrast, MAbs against seven other molecules expressed on these cells were ineffective. The inhibitory activity of HD37 did not require an Fc portion; F(ab')2 fragments were effective, but Fab' fragments were not, suggesting that higher avidity binding and/or cross-linking of CD19 are necessary. We could find no evidence that HD37 recognizes a cross-reactive epitope on P-gp, modulates P-gp from the cell surface, or enhances the ATPase activity of membranes from treated cells.     

High cytotoxicity of alpha-tocopheryl hemisuccinate to cancer cells is due to failure of their antioxidative defense systems

Multiple drug resistance is a significant problem in small-cell lung cancer (SCLC). Artemisinin (ART) is a natural product used to treat drug-resistant malaria. The drug is effective because the Fe2+ present in infected erythrocytes acts non-enzymatically to convert ART to toxic products. We tested the effects of ART on drug-sensitive (H69) and multi-drug-resistant (H69VP) SCLC cells, pretreated with transferrin (TF) to increase the intracellular Fe2+ level. Antibody staining followed by flow cytometry analysis showed twice the level of TF receptors on the H69VP as compared to the H69 cells. Low doses of ART were cytotoxic to SCLC cells. The cytotoxicity of ART for H69VP cells (IC50=24 nM) was ten-fold lower than for H69 cells (IC50=2.3 nM), indicating that ART is part of the drug resistance phenotype. Pretreatment of H69 cells with 220-880 nM TF did not alter the IC50 for ART. However, in the ART-resistant H69VP cells, pretreatment with TF lowered the ART IC50 to near drug-sensitive levels (IC50=5.4 nM after 4 h pretreatment with 880 nM TF). Desferrioxamine (5 microM) inhibited the effect of TF on the IC50 for ART in drug-resistant cells but did not have an effect on ART cytotoxicity in drug-sensitive cells. DNA fragmentation as measured by ELISA occurred within ART-treated cells, with kinetics indicating apoptosis rather than necrosis. This was confirmed by TUNEL staining. These data indicate the potential use of ART and TF in drug-resistant SCLC.     

Amplification and expression of mdr1 gene in a multidrug resistant variant of small cell lung cancer cell line NCI-H69

Amplification and expression of the mdr1 gene encoding P-glycoprotein have been studied in H69/LX4 a multidrug resistant variant (MDR) of small cell lung cancer (SCLC) cell line NCI-H69. Recently a second independently derived MDR variant of this cell line designated H69/AR was found by others not to show amplification, rearrangement or over-expression of the mdr1 gene. The present study reports that in marked contrast to H69/AR, H69/LX4 shows amplification and expression of the P-glycoprotein gene and raises the possibility that P-glycoprotein hyperexpression may be a clinically relevant component of MDR in some SCLC tumours.     

Synergistic antitumor effects of BCG and monoclonal antibodies capable of inducing antibody-dependent cell-mediated cytotoxicity

Three monoclonal antibodies (MAbs) of different immunoglobulin subclasses against MH134 murine ascitic hepatoma cells, detecting the same antigenic determinant of tumor-associated antigen of the tumor cells, were tested for their ability to produce a synergistic antitumor effect with Mycobacterium bovis BCG in C3H/HeN mice. 12A2 (IgG2a) induced both antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the tumor cells. C3H/HeN mice were inoculated ip with MH134 tumor cells (day 0), and received an ip injection of BCG (day 1) and/or 12A2 (day 5). The combined therapy with BCG and 12A2 brought about a significant prolongation of the survival period, whereas either BCG or the MAb alone exhibited poor therapeutic effectiveness. 11G2 (IgG1), inducing ADCC but not CDC against MH134 tumor cells, was shown to exhibit antitumor effects as potent as those of 12A2, when used in combination with BCG. However, 7C2 (IgM), capable of inducing CDC but not ADCC to the tumor cells, produced no apparent synergistic effect with BCG. ADCC of BCG-induced peritoneal cells was mediated by the adherent cell population of the cells and abolished by the addition of carrageenan, suggesting that the effector cells of the cytotoxicity were macrophages. Moreover, carrageenan abolished the combined antitumor effect of BCG and these MAbs in the serological Winn assay. These results suggest that activated macrophages play a major role in the synergistic antitumor effect of BCG and MAbs capable of inducing ADCC against MH134 tumor.     

P-glycoprotein,expressed in multidrug resistant cells,is not responsible for alterations in membrane fluidity or membrane potential

Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to MDR in tumors. However, the physiological role of P-gp in normal tissues is not well understood. Previous studies on multidrug-resistant cells have suggested changes in membrane fluidity and membrane potential associated with P-gp expression, but interpretation of these studies is difficult, because most experimental cells have been selected for long periods in the presence of cytotoxic drugs and may have other host alterations. Therefore, we created two cell lines in which a transfected human MDR1 cDNA is repressed by tetracycline and induced in the absence of tetracycline. One cell line was derived from a mouse embryonic fibroblast cultured from a double (mdr1a/1b) knockout mouse, and the other was from a human HeLa cell line. Analysis of the kinetics of expression of P-gp showed that the mRNA had a half-life of approximately 4 h, and the protein had a half-life of approximately 16 h. P-gp cell surface expression (measured with monoclonal antibody MRK-16) and P-gp function (measured with a fluorescent substrate, rhodamine 123) was characterized by using fluorescence-activated cell sorting. No differences in membrane potential using the fluorescent probe oxonol or in membrane "fluidity" using fluorescent anisotropy probe or electron spin resonance probe were observed in the tet-repressible P-gp-expressing cells. In contrast, several drug-selected cells that express P-gp showed an increase in membrane fluidity and membrane potential. These results suggest that expression of P-gp per se has little effect on membrane fluidity or membrane potential, and it does not have H(+) pump activity. The changes in these parameters observed in drug-selected cells must reflect other host adaptations to drug selection.     

Differential cytotoxicity of 19 anticancer agents in wild type and etoposide resistant small cell lung cancer cell lines.

A panel of six ''wild type'' and three VP-16 resistant small cell lung cancer (SCLC) cell lines is used to evaluate to what extent in vitro sensitivity testing using a clonogenic assay can contribute to combine cytotoxic drugs to regimens with improved efficacy against SCLC. The resistant lines include (a) H69/DAU4, which is classical multidrug resistant (MDR) with a P-glycoprotein efflux pump (b) NYH/VM, which exhibits an altered topoisomerase II (topo II) activity and (c) H69/VP, which is cross-resistant to vincristine, exhibits a reduced drug accumulation as H69/DAU4 but is without P-glycoprotein. 19 anticancer agents were compared in the panel. The MDR lines demonstrated, as expected, cross-resistance to all topo II drugs, but also different patterns of collateral sensitivity to BCNU, cisplatin, ara-C, hydroxyurea, and to the topo I inhibitor camptothecin. The complete panel of nine cell lines clearly demonstrated diverse sensitivity patterns to drugs with different modes of action. Correlation analysis showed high correlation coefficients (CC) among drug analogues (e.g. VP-16/VM-26 0.99, vincristine/vindesine 0.89), and between drugs with similar mechanisms of action (e.g. BCNU/Cisplatin 0.89, VP-16/Doxorubicin 0.92), whereas different drug classes demonstrated low or even negative CC (e.g. BCNU/VP-16 -0.21). When the CC of the 19 drug patterns to VP-16 were plotted against the CC to BCNU, clustering was observed between drugs acting on microtubules, on topo II, alkylating agents, and antimetabolites. In this plot, camptothecin and ara-C patterns were promising by virtue of their lack of cross-resistance to alkylating agents and topo II drugs. Thus, the differential cytotoxicity patterns on this panel of cells can (1) give information about drug mechanism of action, (2) enable the selection and combination of non-cross-resistant drugs, and (3) show where new drugs ''fit in'' among established agents.