Mechanism Of Tumor Cell Killing Research Articles

A Novel Sphingosine Kinase Inhibitor Induces Autophagy in Tumor Cells

The sphingolipids ceramide, sphingosine, and sphingosine 1-phosphate (S1P) regulate cell signaling, proliferation, apoptosis, and autophagy. Sphingosine kinase-1 and -2 (SK1 and SK2) phosphorylate sphingosine to form S1P, shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-231 breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine, an inhibitor of autophagy, switched the mechanism of toxicity to apoptosis, but decreased the potency of the SK2 inhibitor, indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu-Leu-al (MG-132) or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts, daily administration of ABC294640 delayed tumor growth and elevated autophagy markers, but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy, which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently, ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.

The Critical Requirement of the NKG2D Receptor On CD8+ T Cells in Killing Myeloma Cells.

Abstract 4733Autologous stem cell transplant for myeloma improves survival, but the immunologic mechanisms accounting for this improvement are unknown. Our data indicate that NKG2D+CD8+T cells may be one reason for this benefit. NKG2D, one of four NK cell activating receptors, has been identified on some CD8+T cells that mediate TCR-independent and non-MHC restricted tumor cell killing.We identified methods for ex vivo expansion of cyclophosphamide – mobilized blood progenitor cells (BPCs) from myeloma patients, as part of a clinical trial (Cytotherapy 2008). Mobilized BPC are cultured in serum-free media with IL-2 (50 IU/ml) and OKT3 (50ng/ml). After 7 days, the CD8+ T cells are isolated, evaluated and tested. Myeloma cells from patients' marrow aspirates are used as targets in cytotoxicity assays. Phenotypic analysis of the expanded cells and the patients' primary myeloma cells was performed using flow cytometry. Ex vivo expansion of cyclophosphamide-mobilized BPCs induces CD8+ T cells that acquire the NKG2D receptor during ex vivo expansion (P <0.03). Three of the 6 known NKG2D ligands are strongly expressed on patients' myeloma cells, including MICA, ULBP1 and ULBP3. Using cytotoxicity assays, autologous effector cells recognize and kill the patient's autologous tumor cells (E:T 50:1, P<0.001). When using K562 leukemia cells as targets (lack MHC class I), the NKG2D+CD8+ T cells kill K562, supporting an MHC class I-independent mechanism of tumor cell killing. Blocking the NKG2D receptor on CD8+ T cells prevents killing of autologous myeloma cells (P <0.009). NKG2D-mediated cytotoxicity correlates with the amount of ligand expression on the target.The NKG2D receptor on CD8+T cells recognizes ligands expressed on autologous myeloma cells. The NKG2D+CD8+T cells aggressively kill myeloma cells in a non–MHC restricted and TCR-independent manner. Blocking NKG2D on CD8+ T cells prevents killing of autologous myeloma cells. Since tumor cells often down regulate MHC Class I expression, the ability of NKG2D+CD8+ T cells to recognize and kill autologous myeloma cells in a non-MHC restricted manner may contribute to the beneficial effects in the treatment of myeloma. Ongoing experiments are testing these methods in mouse models and defining the molecular mechanisms involved. Disclosures:Szczepiorkowski:Cersus Corporation: Research Funding; CaridianBCT: Research Funding; BASF: Research Funding; Terumo Corporation: Research Funding; Fenwel, Inc - Scientific Advisory Board: Research Funding. Meehan:Berlex Pharmaceutical: Research Funding.

Tumor Cell Killing Mechanisms of Epidermal Growth Factor Receptor (EGFR) Antibodies Are Not Affected by Lung Cancer-Associated EGFR Kinase Mutations

The epidermal growth factor receptor (EGFR) serves as a molecular target for novel cancer therapeutics such as tyrosine kinase inhibitors (TKI) and EGFR Abs. Recently, specific mutations in the EGFR kinase domain of lung cancers were identified, which altered the signaling capacity of the receptor and which correlated with clinical response or resistance to TKI therapy. In the present study, we investigated the impact of such EGFR mutations on antitumor cell activity of EGFR Abs. Thus, an EGFR-responsive cell line model was established, in which cells with tumor-derived EGFR mutations (L858R, G719S, delE746-A750) were significantly more sensitive to TKI than wild-type EGFR-expressing cells. A clinically relevant secondary mutation (T790M) abolished TKI sensitivity. Significantly, antitumor effects of EGFR Abs, including signaling and growth inhibition and Ab-dependent cellular cytotoxicity, were not affected by any of these mutations. Somatic tumor-associated EGFR kinase mutations, which modulate growth inhibition by TKI, therefore do not impact the activity of therapeutic Abs in vitro.

Blockade of CD200 in the Presence or Absence of Antibody Effector Function: Implications for Anti-CD200 Therapy

CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.

Gefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires BIM and Can Be Enhanced by BH3 Mimetics

BackgroundThe epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.Methods and FindingsWe performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.ConclusionsInhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.

G-Quadruplexes Induce Apoptosis in Tumor Cells

Several G-rich oligodeoxynucleotides (ODNs), which are capable of forming G-quadruplexes, have been shown to exhibit antiproliferative activity against tumor cell lines and antitumor activity in nude mice carrying prostate and breast tumor xenografts. However, the molecular basis for their antitumor activity remains unclear. In the current study, we showed that a variety of telomeric G-tail oligodeoxynucleotides (TG-ODNs) exhibited antiproliferative activity against many tumor cells in culture. Systematic mutational analysis of the TG-ODNs suggests that the antiproliferative activity depends on the G-quadruplex conformation of these TG-ODNs. TG-ODNs were also shown to induce poly(ADP-ribose) polymerase-1 cleavage, phosphatidylserine flipping, and caspase activation, indicative of induction of apoptosis. TG-ODN-induced apoptosis was largely ataxia telangiectasia mutated (ATM) dependent. Furthermore, TG-ODN-induced apoptosis was inhibited by the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125. Indeed, TG-ODNs were shown to activate the JNK pathway in an ATM-dependent manner as evidenced by elevated phosphorylation of JNK and c-Jun. Interestingly, a number of G-quadruplex ODNs (GQ-ODN) derived from nontelomeric sequences also induced ATM/JNK-dependent apoptosis, suggesting a possible common mechanism of tumor cell killing by GQ-ODNs.

Involvement of Sphingolipids in Apoptin-Induced Cell Killing

The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin–ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 μM) attenuated (30%; P<0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P<0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.

Cancer immunotherapy targeting Sp17: When should the laboratory findings be translated to the clinics?

Despite advances in chemotherapeutic agents, the prognosis for some cancers remains extremely poor, suggesting the need for other treatment modalities. Immunotherapy appears an ideal approach because the mechanisms of tumor cell killing induced by tumor vaccines are different from those from chemotherapy. Various investigations are ongoing to identify suitable targets for this purpose. Sperm protein 17 (Sp17) was originally identified by our group as a novel cancer-testis antigen in various malignancies, including multiple myeloma. Sp17 is a highly immunogenic protein and the observation that more than 90% of vasectomized males develop immunity against Sp17 suggests the opportunity and safety of Sp17 for tumor vaccines. Recent works by other workers suggest a low level of expression of Sp17 in some normal tissues, and investigators have questioned whether Sp17 is in fact a suitable target for immunotherapy. In this paper, we review the general principles of immunotherapy and provide evidence supporting the highly immunogenic nature of Sp17. We also address the discrepancies between the objectives of oncologists involved in treating cancer patients and their familiarity with acceptable levels of toxicity of any effective therapy and those of pure laboratory-based investigators. Finally, we present some early clinical data supporting the rationale for further investigations of Sp17 for tumor vaccines.

The Critical Role of the NKG2D Receptor in CD8+CTL and CD8+CD56+ NKT Cell Cytotoxicity.

We previously demonstrated the ability to grow and ex vivo expand mobilized peripheral blood mononuclear cells (PBMNC) from myeloma patients into aggressive cytotoxic effector cells (Blood 102; 422b, 2003). These experiments were designed to test the function and mechanism of tumor cell killing of these cells. Peripheral blood stem cells (PBSC) were collected from myeloma pts after mobilizing with cyclophosphamide and rhG-CSF and cultured in Aim-V serum-free medium at 37 and 5% CO2. After 2 hrs, the non-adherent cells were removed and placed in culture with Aim-V, IL-2 (50 IU/ml) and OKT-3 (50 ng/ml) for 7 days. Cytotoxicity of the expanded cells was tested on Day 0 and Day 7 using a chromium release assay. To identify the cytotoxic potential of cell subsets, cell populations were depleted using the Auto MACS Magnetic Cell Sorter (Miltenyi Biotec Auburn, CA) and cytotoxicity assays were repeated. Since the CD8+ cell(s) were the most cytotoxic, the CD8+ cells were isolated and their mechanisms of tumor cell killing were evaluated by testing killing through MHC Class I, T cell receptor or the NKG2D receptor. The ex vivo expanded population was extremely cytotoxic and killed RPMI 8226 myeloma cells at 60% lysis (+/− 1.6%) (E:T of 100:1) when compared to 3.9 % on day 0 (+/− 0.8%). CD8+ or CD8+CD56+ cell subsets contributed to > 83.3 % (+/− 1.5%) of the killing. Blocking the TCR pathway (Redirected Cytotoxicity Assay) had no effect on killing and blocking the MHC Class I molecules decreased cytotoxicity by only 6%. When the NKG2D receptor was blocked, cytotoxicity by the CD8+ cells decreased by 48% (+/− 2%), demonstrating the critical role of the NKG2D in these CD8+ cell populations. The expanded cytotoxic effector cells aggressively lyse myeloma tumor cells in a MHC and non-MHC restricted fashion. These ex vivo expanded CD8+ cells likely acquire the NKG2D receptor and kill tumor cells in a non-MHC restricted manner. Since MHC expression is often low or absent on myeloma cells and the NKG2D ligands are fairly specific to tumor cells, the infusion of these ex vivo expanded cells following transplant may improve clinical outcomes.

Lipoptosis: tumor-specific cell death by antibody-induced intracellular lipid accumulation.

A balanced lipid metabolism is crucial for all cells. Disturbance of this homeostasis by nonphysiological intracellular accumulation of fatty acids can result in apoptosis. This was proven in animal studies and was correlated to some human diseases, like lipotoxic cardiomyopathy. Some metabolic mechanisms of lipo-apoptosis were described, and some causes were discussed, but reagents, which directly induce lipo-apoptosis, have thus far not been identified. The human monoclonal IgM antibody SAM-6 was isolated from a stomach cancer patient by using the conventional human hybridoma technology (trioma technique). The addition of SAM-6 to tumor cells leads to an increase in the intracellular accumulation of neutral lipids, followed by tumor cell apoptosis. The antibody SAM-6 does not react with noncancerous human epithelial and fibroblastic cells, because the M(r) 140000 membrane molecule, recognized by the antibody, is specifically expressed on human malignant cells. The antibody is coded by the germ-line genes IgHV3-30.3*01 and IgLV3-1*01 and is a component of the innate immunity to cancer. In this article, we describe an antibody-induced tumor-specific cell death, named lipoptosis. This is, to our knowledge, the first description of this specific form of lipo-apoptosis as an antibody-mediated mechanism of tumor cell killing.

Combretastatin family member OXI4503 induces tumor vascular collapse through the induction of endothelial apoptosis.

The mechanism of tumor cell killing by OXI4503 was investigated by studying vascular functional and morphological changes post drug administration. SCID mice bearing MHEC5-T hemangioendothelioma were given a single dose of OXI4503 at 100 mg/kg. Tumor blood flow, measured by microsphere fluorescence, was reduced by 50% at 1 hr, and reached a maximum level 6-24 hr post drug treatment. Tumor vascular permeability, measured by Evan's blue and hemoglobin, increased significantly from 3 hr and peaked at 18 hr. The elevated tumor vessel permeability was accompanied by an increase in vascular endothelial growth factor (VEGF) from 1 hr post drug treatment. Immunohistochemical staining for CD31 and laminin showed that tumor blood vessels were affected as early as 3 hr but more prominent from 6 hr. From 12 hr, the vessel structure was completely destroyed. Histopathological and double immunohistochemical staining showed morphological change and induction of apoptosis in endothelial cells at 1-3 hr, followed by tumor cell necrosis from 6-72 hr. There were no statistically significant changes of Evan's blue and hemoglobin contents in liver tissue over the time course. These results suggest that OXI4503 selectively targets tumor blood vessels, and induces blood flow shutdown while it enhances tumor blood vessel permeability. The early induction of endothelial cell apoptosis leads to functional changes of tumor blood vessels and finally to the collapse of tumor vasculature, resulting in massive tumor cell necrosis. The time course of the tumor vascular response observed with OXI4503 treatment supports this drug for development as a stand alone therapy, and also lends support for the use of the drug in combination with other cancer therapies.

A role for MAP kinase in the antitumor activity of a nucleoside analog.

Nucleoside analogs (NAs) have been used extensively in both antitumor and antiviral therapies. Their general mechanism of action has been postulated to result from incorporation into DNA, leading to disruption of DNA synthesis and DNA polymerase inhibition. To further explore the antitumor mechanisms of NAs we have evaluated ganciclovir (GCV), an NA antiviral agent, in herpes simplex virus thymidine kinase (HSV-TK) gene-modified tumor cells. This system allows specific evaluation of the antitumor effects of NAs because the antitumor effect is directly related to the phosphorylation of the prodrug GCV by the HSV-TK enzyme in the gene-modified tumor cells. We demonstrated that GCV incorporates into DNA and inhibits DNA polymerase, as has been observed in HSV-infected cells and with other antitumor NAs in tumor cells. A novel observation is that GCV activates MAP kinase within 1 hour of GCV exposure. This activation directly correlates with cytotoxicity, because inhibition of the MAP kinase extracellular regulated kinase (Erk) by PD98059, reversed GCV-mediated cytotoxicity. This effect appears to be specific to the Erk pathway, because inhibition of the p38 kinase with SB203580 had no effect on cytotoxicity. Further, GCV does not act as a DNA-damaging agent or activate general DNA-repair mechanisms, but does produce a number of metabolic disruptions, including a reversible decrease in NAD levels. These effects appear to be downstream of the earlier activation of Erk in this system, which may be a novel mechanism of action for GCV cytotoxicity in HSV-TK gene-modified tumor cells, and thus, needs to be further evaluated as the mechanism of tumor cell killing by other antitumor NAs.

Analysis of a murine anti-ganglioside GD2 monoclonal antibody expressing both IgG2a and IgG3 isotypes: monoclonality, apoptosis triggering, and activation of cellular cytotoxicity on human melanoma cells.

In this study we have documented a hybridoma secreting an unusual MAb, which expresses both IgG3 and IgG2a subclasses with a lambda-light chain. How this dual expression of isotypes was exactly brought about is not clear. To resolve this problem, it will have to wait the complete sequence analysis the heavy chain gene of MAb 9C4. Although the expression of IgG2a was about 50% that of IgG3, antibody titration studies showed the major binding affinity of MAb 9C4 to GD3-positive cells being mostly contributed by the IgG3 rather than IgG2a part of the antibody. This antibody could induce apoptosis in melanoma cells in 10-15% of cells in vitro, but the generality of this phenomenon is yet to be confirmed by the use of different cell targets and different anti-GD2 MAbs other than 9C4. Aside from the demonstrated indirect killing mechanisms of many anti-GD2 MAbs through CDC and ADCC, MAb 9C4 induction of apoptosis represents an alternative mechanism of tumor cell killing, by which direct killing of anti-GD2 antibody takes its effect. This apoptotic effect was demonstrated for the first time with an anti-ganglioside monoclonal antibody. From the therapeutic point of view, the cytolytic activity of MAb 9C4-targeted ADCC/LAK killing against GD2-positive tumor cells to be more effective than that of LAK alone and a possibility for dendritic cells to effectively acquire antigen through pulsing with MAb-induced apoptotic cells are both of great clinical importance. Further studies are warranted aiming at elucidating the molecular basis of bi-isotypic specificity and aberrant isotype switching, molecular pathway of anti-GD2 antibody-induced apoptosis, and ways to improve clinical utility of this unusual hybridoma/MAb 9C4.

Norcantharidin-Induced Post-G2/M Apoptosis Is Dependent on Wild-Type p53 Gene

Norcantharidin (NCTD), a synthetic analogue of phosphatase type 2A inhibitors, cantharidin, was shown to have limited effects in treating human and animal tumors. The tumor cell killing mechanisms by norcantharidin, however, remain unclear. In this report, we wished to investigate the mechanisms of norcantharidin-mediated cytotoxicity. Effort was made to investigate whether norcantharidin exerted its cytotoxicity through a p53-dependent or -independent mechanism. RT-2 (wtp53) and U251 (mutant p53) glioblastoma cell lines were exposed to norcantharidin at different dosages. Time-course fluorescent-activated cell sorting (FACS) analysis showed that high doses of norcantharidin arrested the cells at the G2/M phase and subsequent post-G2/M apoptosis in RT-2 cell line. In comparison, the U251 cell line was found resistant to norcantharidin-induced cytotoxicity. Restoring wild-type p53 gene function in the U251 cell line after adenoviral infections induced tumor cell cytotoxicity after exposure to norcantharidin. These results showed that norcantharidin kills tumor cells efficiently corresponding to their endogenous p53 gene status. The results also showed the feasibility of using adenoviral p53 gene therapy to enhance chemosensitivity of tumor cells to norcantharidin.

Overexpression of Fas-Ligand by neuroblastoma: A novel mechanism of tumor-cell killing

Background/Purpose: Binding of Fas ligand (Fas-L) to the membrane-bound Fas receptor incites a series of intracellular events that results in programmed cell death or apoptosis. Although this apoptotic phenomenon plays a key role in down-regulating cytotoxic T cells, the authors have shown previously that pancreatic beta cells (bTC) overexpressing Fas-L paradoxically undergo accelerated rejection that is dependent on a Fas/Fas-L interaction. This study evaluates whether a neuroblastoma (NB) cell line manipulated to overexpress Fas-L undergoes similar destruction and whether tumor-specific protective immunity can be produced.Methods: The authors transfected NB cells (SK-N-MC) with either mFas-L cloned into a pcDNA3.1/Zeo plasmid vector (NB/Fas-L) or with the vector alone (NB/control). Successful transfection of Fas-L was characterized by reverse transcription polymerase chain reaction (RTPCR) and the ability of transfectants to induce apoptosis of Fas-sensitive T cells (Jurkat). Expression of Fas and Fas-L in untransfected NB clones was characterized by immunohistochemistry and RNase protection assay (RPA). Apoptosis was measured by FACScan analysis using an Annexin V assay. A total of 3 × 106 NB/control and NB/Fas-L cells were implanted subcutaneously into the hind leg of Balb/C SCID mice. Tumor-specific protective immunity was also tested in this model by inoculating mice with NB/Fas-L before implanting NB/control cells.Results: Zeocin resistance and RT-PCR confirmed successful transfection of Fas-L into NB cells. Fas Ligand transfectants induced apoptosis in 17.6% ± 2.9% of Fas-sensitive T cells, whereas controls induced apoptosis in only 2.8% ± 1.2% (P= .01, n = 3). Although Fas appears to be constitutively expressed by NB in low amounts, introduction of Fas-L into NB cells did not induce suicide or affect tumor cell growth in vitro. In vivo, NB cells expressing Fas-L failed to grow in SCID mice (n = 3), whereas controls grew rapidly in all animals until death (n = 3). NB/control cells implanted into the opposite leg of mice that rejected initial NB/Fas-L transfectants also grew rapidly (n = 3) implying no protective immunity.Conclusions: Overexpression of Fas-L in NB clones targets such cells for rapid destruction even in immune compromised hosts, suggesting potential utility of Fas-L in combating NB. In this SCID mouse model, the observed effect is probably neutrophil mediated and does not provide tumor-specific protective immunity.

The role of radiation-induced apoptosis as a determinant of tumor responses to radiation therapy.

Ionizing radiation is an effective means of killing tumor cells. Approximately 50% of all American cancer patients are treated with radiotherapy at some time during the course of their disease, making radiation one of the most widely used cytotoxic therapies. Currently, much effort is focused on understanding the molecular pathways that regulate tumor cell survival following radiotherapy, with the long term goal of developing novel therapeutic strategies for specifically sensitizing tumors to radiation. At present, there is particular interest in the role of tumor cell apoptotic potential as a regulator of both intrinsic and extrinsic determinants of the response of tumors to radiation therapy. Here we review what is currently known about the role of apoptosis as a mechanism of tumor cell killing by ionizing radiation and the relative contribution of apoptosis to cellular radiosensitivity and the ability to control human cancers using radiotherapy. The following topics will be discussed: (1) radiation-induced apoptosis in normal and malignant cells, (2) clinical findings with respect to apoptosis in human cancers treated with radiotherapy, (3) the contribution of apoptosis to intrinsic radiosensitivity in vitro, (4) the relevance of apoptosis to treatment outcome in experimental tumor models in vivo and (5) the potential of exploiting apoptosis as a means to improve the therapeutic efficacy of radiotherapy.

135 Genetic radiotherapy using an adenoviral vector inhibits tumor growth by direct and indirect mechanisms of tumor cell killing

135 Genetic radiotherapy using an adenoviral vector inhibits tumor growth by direct and indirect mechanisms of tumor cell killing

Membrane-associated lymphotoxin expression and functional analysis of lymphokine-activated killer cells derived from tumor-infiltrating lymphocytes.

The expression of a membrane‐associated lymphotoxin molecule (mLT) on lymphokine‐activated killer (LAK) cells obtained from 18 patients with malignant tumors and its role in the tumor cell killing mechanisms were investigated. LAK cells from tumor‐infiltrating lymphocytes (TIL‐LAK cells) were mainly composed of CD3‐positive cells, whereas LAK cells from peripheral blood lymphocytes (PBL‐LAK cells) were mainly composed of CD16‐ and CD56‐positive cells. However, mLT was found to be expressed on TIL‐LAK cells as well as PBL‐LAK cells. The degree of mLT expression correlated with the killing activity of LAK cells towards L929 cells (r=0.806, P<0.01, n = 15), but not with that towards Daudi or K562 cells. Although the degree of mLT expression correlated with the amount of secreted lymphotoxin (LT) in the supernatant of LAK cell culture, the secreted LT itself could not account for the tumor cell killing activity of LAK cells. Polyclonal rabbit anti‐LT antibody partially inhibited the killing activities of LAK cells towards L929 cells and this inhibition was found in the combination of autologous tumor cells and PBL‐LAK cells. These findings suggest the possibility that the mLT‐related cytotoxicity is involved in the tumor cell killing mechanisms of TIL‐LAK cells as well as PBL‐LAK cells.

Anticancer activities of adenine nucleotides in tumor bearing hosts

AbstractThe utilization of adenosine 5′‐triphosphate (ATP) infusions against solid refractory cancers is based on the preclinical findings that low levels of extracellular ATP significantly inhibit the growth of a variety of human and murine tumor cells. The mechanisms of tumor cell killing by extracellular ATP are attributed to effects mediated by its interaction with P2 purine receptors as well as non‐receptor‐mediated pore formation in the tumor cell membrane. The achievement of elevated extracellular blood plasma pools of ATP is accomplished by the administration of adenine nucleotides which yield elevated liver, red blood cell, and plasma compartment pools of ATP. Administration of AMP or ATP to tumor‐bearing murine hosts produced, in addition to the cytotoxic effects against the tumor, a variety of host mediated anticancer activities, including significant inhibition of host weight loss in cachectic tumor models. The inhibition of several adverse events Which are among the hallmarks of cancer cachexia is the result of prevention of the significant depletion of visceral energy stores in cachectic animals after the administration of adenine nucleotides, leading to the successful maintenance of hepatic functions, the resumption of normal protein synthesis in the liver, and inhibition of the synthesis of hepatic acute‐phase proteins. Thus, the administration of ATP as an anticancer agent in humans at levels (below 0.1 mg/kg min) which do not adversely affect systemic and cardiovascular functions is expected to lead to the following activities resulting from the generation of elevated hepatic, red blood cell, and blood plasma ATP pools: cytolytic effects against the tumor, inhibition of host weight loss, and other adverse effects of cachexia, anti‐pain effects, modulation of blood flow to the tumor, positive effects on motor functions and performance status due to increases in several other parameters which are also known to be affected by elevated red blood cell and blood plasma (extracellular) ATP levels. © 1993 Wiley‐Liss, Inc.

Mechanism of tumor cell killing by HO-221, a novel antitumor compound

The mechanism of tumor cell killing by HO-221, a novel benzoylphenylurea derivative that shows broad-spectrum antitumor activities, was studied. HO-221 strongly inhibited the activity of mammalian DNA polymerase alpha but not that of DNA polymerases beta or gamma. The inhibition was equivalent to that induced by aphidicolin and ara-CTP, which were selective inhibitors of the enzyme. Furthermore, the inhibition by HO-221 of DNA polymerase alpha was found to be non-competitive with respect to dCTP as a substrate, unlike that induced by aphidicolin and ara-CTP. The inhibition was reduced the addition of an excess of DNA polymerase alpha but not by excess amounts of activated DNA as a template primer. These results suggest that HO-221 inhibits the activity of DNA polymerase alpha by direct interaction with the enzyme in contrast to the impairment of template activity through intercalation into DNA induced by anthracycline compounds. On the other hand, HO-221 showed almost no effect on RNA polymerase activity, the reverse transcriptase activity of avian myeloblastosis virus or protein synthesis in a cell-free system. The flow-cytometry analysis revealed that HO-221 accumulated HL-60 cells in G1-S phases at a low concentration but increased the number of cells in the G1 phase at a higher concentration, stopping cell-cycle progression. The results suggest a correlation between cell-cycle progression and inhibition by HO-221 of DNA polymerase alpha, which plays a role in DNA replication during the S phase in living cells.