Sensitivity to EBV superinfection and IUdR inducibility of hybrid cells formed between a sensitive and a relatively resistant Burkitt lymphoma cell line

The sensitivity of Burkitt lymphoma-derived cell lines to Epstein-Barr virus (EBV) superinfection and the inducibility of the latent EBV genome in these lines were studied by somatic cell hybridization. Two non-EBV producer lines; AG R3, an adherent, 8-azaguanine-resistant variant of the Rajiline; and Namalwa, a non-adherent, 8-azaguanine-sensitive line, were fused with the aid of β-propiolactone-inactivated Sendai virus. The resident EBV genome in Raji is inducible with 5-iododeoxyuridine and the line is also sensitive to EBV superinfection. Namalwa is relatively resistant to both super-infection and induction, and synthetizes surface-associated IgM-lambda. Cytological studies showed that hybrid cells appear to be much larger than either parent and attach to culture plates less firmly than the adherent Raji variant parent. Karyological analyses showed that hybrids contain the expected sum of the parental chromosome markers. Membrane immunofluorescence tests also showed that hybrids synthetize IgM. All the hybrid cells appear to be non-EBV producers, but they are sensitive to both EBV super-infection and induction of latent EBV. These findings suggest the following explanations: (1) there is no evidence of any complementation between the two non-EBV producer lines (Raji and Namalwa) to elicit spontaneous EBV production in hybrid cells; (2) Namalwa is deficient in some factors required for the synthesis of EBV-specified early antigens after EBV superinfection and after induction of latent EBV by IUdR; these factors are supplied by the Raji parent in the hybrids; or (3) Raji, Namalwa and hybrid cells or EBV all produce a substance which inhibits activation of a productive viral cycle, but whose action is antagonized in Raji and hybrid cells to allow the synthesis of EBV-specific early antigens.     

Epstein-Barr virus strain-specific differences in transformed cell lines demonstrated in growth characteristics, induction of viral antigens and ADCC susceptibility

Paired lymphoid cell lines were established by transformation with both B95-8 (B) and QIMR-WIL (Q) EBV strains, of cells either from cord blood or from peripheral blood or spleens of patients with leukemias or lymphomas. The morphologies of the transformed cell clumps differed consistently between B-transformed lines (B-lines) and Q-transformed lines (Q-lines) even after 1 year in culture. When the B- and Q-lines were compared for superinfection with P3HR-1 EBV, B-lines had a higher frequency of EA induction than did the Q-lines. The shape of dose-response curves for superinfection indicated that a much higher multiplicity of infection by P3HR-1 EBV was required for EA expression in Q-lines than in B-lines. This difference in superinfection was independent of the EBV receptor concentration on the two types of lines and reflected apparent control of the level of EA induction by the resident EBV genome of the transformed line. Transforming EBV could be rescued by P3HR-1 EBV superinfection of both B- and Q-lines originating from cells of patients with Hodgkin's disease and hairy cell leukemia but not from cord blood. The cord blood lines transformed with virus produced spontaneously from these B- or Q-lines, showed that the cell lines contained antigen-determining information of the resident genome in the original B- or Q-lines, respectively. Superinfected B-lines were more susceptible to ADCC with anti-EBV-positive sera than were superinfected Q-lines. These experiments demonstrate that distinct biology. differences exist in paired cell lines transformed by different EBV strains.     

T and B leukemic cell lines exhibit different requirements for cell death: correlation between caspase activation, DFF40/DFF45 expression, DNA fragmentation and apoptosis in T cell lines but not in Burkitt's lymphoma.

The execution phase of apoptosis occurs through the activation and function of caspases which cleave key substrates that orchestrate the death process. Here, we have compared the sensitivity of various T and B cell lines to death receptor or staurosporine-induced apoptosis. First, we found a lack of correlation between death receptor expression and sensitivity to Fas or Trail. By contrast, a correlation between caspase activation, DNA fragmentation and cell death in T cell lines was evidenced. Among T cells, CEM underwent apoptosis in response to CH11 but were resistant to Trail in agreement with the absence of Trail receptors (DR4 and DR5) on their surface. The B cell line SKW 6.4 was sensitive to CH11 and staurosporine but resistant to Trail. As B cell lines expressed significant levels of DR4 and DR5, resistance to Trail in SKW 6.4 is likely due to the expression of the decoy receptor DcR1. Burkitt's lymphoma such as RPMI 8866 and Raji did not exhibit DNA fragmentation in response to CH11, Trail or staurosporine but showed long-term caspase-dependent loss of viability upon effector treatment. The B cell lines used in this study express very weak or undetectable levels of DFF40 and relatively high levels of DFF45. Interestingly, cytosolic extracts from RPMI 88.66 but not other B lymphoma exhibit altered levels of cytochrome c-dependent caspase activation. Taken together, our results show that: (1) death receptor expression does not correlate with sensitivity to apoptosis; (2) the very low ratio of DFF40 vs. DFF45 is unlikely to explain by itself the lack of DNA fragmentation observed in certain B cell lines; and (3) a defective cytochrome c-dependent caspase activation might account at least in part for the insensitivity of certain Burkitt's lymphoma (RPMI 88.66) to apoptosis. Thus it seems that resistance of Burkitt's lymphoma to apoptosis is not governed by a general mechanism, but is rather multifactorial and differs from one cell line to another.     

Variable susceptibility to NK activity of cloned cell lines derived from a primary rat rhabdomyosarcoma: relationship to metastatic potential.

In vitro cloned lines derived from a primary nickel-induced rat rhabdomyosarcoma exhibited diverse levels of susceptibility to spontaneous NK activity. The presence of NK target structures was revealed by competition assays on all cloned cell lines, and the NK susceptibility of the tumour lines varied according to their osmotic fragility. Tumour cell lines derived from metastatic lung nodules presented similar NK susceptibilities to cells originating from the primary tumour. However, cloned cell lines differed in their capacity to form lung colonies after i.v. injection, and in their potential for invading lungs after s.c. primary tumour development. No correlation was found between lung colonization potential and NK resistance. Studies of the correlation between metastatic potential and NK sensitivity revealed that (1) all the NK resistant tumour cells were highly metastatic; (2) NK susceptible tumour cells could be either highly or weakly metastatic. Therefore, highly metastatic tumour cells could be either resistant or susceptible to NK lysis. We conclude that the property of resistance to NK contributes to a high metastatic potential. However, other properties could counterbalance and finally prevail over NK susceptibility thus enabling NK susceptible cell lines to be also highly metastatic.     

Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially triggers apoptosis in tumor cells versus normal cells, thus providing a therapeutic potential. In this study, we examined a large panel of human malignant glioma cell lines and primary cultures of normal human astrocytes for their sensitivity to TRAIL. Of 13 glioma cell lines, 3 were sensitive (80-100% death), 4 were partially resistant (30-79% death), and 6 were resistant (< 30% death). Normal astrocytes were also resistant. TRAIL-induced cell death was characterized by activation of caspase-8 and -3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation. Decoy receptor (DcR1 and DcR2) expression was limited in the glioma cell lines and did not correlate with TRAIL sensitivity. Both sensitive and resistant cell lines expressed TRAIL death receptor (DR5), adapter protein Fas-associated death domain (FADD), and caspase-8; but resistant cell lines expressed 2-fold higher levels of the apoptosis inhibitor phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15). In contrast, cellular FADD-like IL-1beta-converting enzyme-like inhibitory protein (cFLIP) expression was similar in sensitive and resistant cells. Transfection of sense PED/PEA-15 cDNA in sensitive cells resulted in cell resistance, whereas transfection of antisense in resistant cells rendered them sensitive. Inhibition of protein kinase C (PKC) activity restored TRAIL sensitivity in resistant cells, suggesting that PED/ PEA-15 function might be dependent on PKC-mediated phosphorylation. In summary, TRAIL induces apoptosis in > 50% of glioma cell lines, and this killing occurs through activation of the DR pathway. This caspase-8-induced apoptotic cascade is regulated by intracellular PED/PEA-15, but not by cFLIP or decoy receptors. This pathway may be exploitable for glioma and possibly for other cancer therapies.     

Differential inducibility of Epstein-Barr virus in cloned, non-producer Raji cells.

Cells of the human lymphoblastoid non-producer line Raji were cloned in soft agar. Individual colonies were isolated and analyzed for their inducibility of the Epstein-Barr virus-associated early antigen (EA). The induction of EA by the tumor promoter TPA varied among the different cell clones. Clones with very high and very low inducibility of the resident Epstein-Barr virus genome were further analyzed. Constant differences in the inducibility of EA were observed after activation by tumor promoters, 5-iododeoxyuridine or antibodies to human IgM. Induction of EA synthesis by superinfection with Epstein-Barr virus from the P3HR-1 line, however, did not vary among the clones tested. No differences in expression of the Epstein-Barr virus-associated nuclear antigen (EBNA) were noted in cells of clones with high or low susceptibility to EA induction. DNA reassociation kinetics demonstrated that Raji cells with high susceptibility to EA induction contained a significantly higher number of Epstein-Barr virus genome equivalents per cell than cells with low susceptibility. Treatment of Raji cells with the tumor promoter TPA did not change the ratio of Epstein-Barr virus-specific DNA to cellular DNA.     

胰腺癌细胞株Puta8988对培美曲塞产生获得性耐药的分子生物学机制初步研究

目的探索胰腺癌细胞株Puta8988对培美曲塞（pemetrexed,Alimate）产生获得性耐药的机制,分析这种耐药与培美曲塞调控通路上相关基因的关系。方法对胰腺癌细胞株Puta8988进行耐药诱导,使其对培美曲塞产生获得性耐药。然后检测正常Puta8988细胞和耐药Puta8988细胞中胸苷酸合酶（thymidylate synthase,TS）,叶酰聚谷氨酸合酶（folylpolyglutamate synthetase,VPGS）,还原型叶酸盐载体（reduced folate carrier,RFC）的mRNA表达水平变化。结果胰腺癌细胞株Pum8988对培美曲塞产生了耐药作用,培美曲塞长期作用后,Puta8988的50％抑制浓度（IC50）上升2．2倍（P〈0．05）。mRNA分析结果提示耐药细胞株偈的mRNA表达上调,FPGS变化不明显,RFC表达下调。结论化疗药物长期作用后,TS、FPGS、RFC的基因表达失衡,胰腺癌细胞产生了获得性耐药。对这些基因进行干预有可能提高胰腺癌细胞对培美曲塞的敏感性,从而提高疗效。     

Correlation of frequency of induced mutation and metastatic potential in tumor cell lines from a single mouse mammary tumor

Spontaneous mutation rates were determined in mouse mammary tumor subpopulation lines that differ in metastatic phenotype. Although there was almost a 9-fold difference in spontaneous rates to ouabain resistance among the three lines tested, the difference did not correlate with ability to metastasize. Similarly a 10-fold difference in spontaneous rates to 6-thioguanine resistance did not correlate with metastatic ability. In contrast, the frequency of ethyl methanesulfonate-induced mutations was associated with metastatic potential. Thus, ethyl methanesulfonate only induced significant numbers of 6-thioguanine resistant colonies in 66 and 410.4 cells, the only 2 of 5 lines tested that spontaneously metastasize at high frequency, and of ouabain resistant colonies in 66, 410.4, and 168 cells, the only lines tested that produce experimental lung metastases after i.v. injection. Differential sensitivity to induced mutation was not correlated with differences in plating efficiency, wild type sensitivity to ethyl methanesulfonate, 6-thioguanine, or ouabain toxicity, ploidy, cell shape, cell size, or ability to engage in metabolic cooperation.     

Induction of the EBV cycle in B-lymphocyte-derived lines is accompanied by increased natural killer (NK) sensitivity and the expression of EBV-related antigen(s) detected by the ADCC reaction

Human B-lymphocyte-derived lines were forced to enter the EBV-cycle by superinfection with the P3HR-l substrain of EBV or sodium butyrate treatment. The induced cells were used as targets for natural killing (NK) and EBV-specific, antibody-dependent cellular cytotoxicity (ADCC). Two Burkitt lymphoma lines, Raji and Daudi, and one normal adult derived lymphoblastoid cell line, NAD-7, were comparable in their ADCC-sensitivity after induction, but only the Burkitt lymphoma-derived lines showed a major increase in NK-sensitivity. The superinfection-induced membrane change, responsible for both NK and ADCC sensitivity, is an early function of the viral cycle, correlated with the appearance of early antigens (EA). Indirect evidence indicates that the NK and ADCC target sites are different but this problem requires further investigation. Sodium butyrate induced an increased NK sensitivity and EBV-related ADCC sensitivity in the Burkitt lymphoma-derived P3HR-l line. Lymphocyte effectors from different donors showed great differences in their NK and ADCC activity. Optimal ADCC could be demonstrated with effectors that were intermediate in their NK-activity.     

Mouse natural killer (NK) cell activity against human cell lines is not influenced by superinfection of the target cell with xenotropic murine C-type virus.

Mouse natural killer (NK) cells can lyse a variety of syngeneic, allogeneic and xenogeneic target cells in short-term 51Cr release assays. The target specificity of NK cells is not known, but endogenous C-type viral antigens have been suggested as possible target structures. To test this hypothesis, human lymphoid lines were superinfected with xenotropic mouse C-type virus either by repeated dosage through nude mice or by in vitro superinfection with the supernatants of nude-mouse-passaged lines. The appearance of surface-associated MuLV antigens after superinfection was confirmed in a complement-dependent cytotoxicity test. Subsequently, the NK sensitivity of each infected line was compared with its non-infected counterpart in direct cytolytic and competition assays. None of these two assay systems showed a consistent difference in NK sensitivity of infected and non-infected cell lines. These findings do not lend support to the concept tht murine C-type viral antigens are responsible for NK sensitivity.     

Disruption of Golgi processing by 2-phenyl benzimidazole analogs blocks cell proliferation and slows tumor growth

PURPOSE: Cancer chemotherapy continues to be challenged by the emergence of resistant tumors, and one organelle entwined in the development of drug resistance is the Golgi apparatus. Recently, we discovered a group of 2-(substituted phenyl)-benzimidazole (2-PB) compounds that displace resident Golgi proteins from the juxtanuclear region resulting in their degradation. These compounds are also potent anti-proliferative agents, which together with their action on the Golgi made a compelling case for testing them against cancer. METHODS: The anti-tumor activity of a group of 2-PB compounds was examined both in vitro and in vivo. The role of the Golgi in the anti-proliferative effect was assessed by comparing the proliferation of individual cell lines with the distribution and total cellular expression of selected resident Golgi proteins. RESULTS: The anti-proliferative activity of 2-PB compounds is partially reversible (time- and concentration-dependent), non-cell-cycle-specific, and translates to tumor growth inhibition in vivo. While 2-PB compounds displace resident Golgi proteins from the juxtanuclear region in all cells, those that are resistant to the anti-proliferative effects differ from sensitive cells in that they have the capacity to protect these Golgi proteins from degradation. CONCLUSIONS: These results illustrate the utility of targeting the Golgi for cancer drug development. They also reveal a cellular strategy for resisting 2-PB drug effects through protection of displaced Golgi proteins from degradation thus allowing their continued function.     

Resistance to cytolysis by tumor necrosis factor alpha in malignant gynecological cell lines is associated with the expression of protein(s) that prevent the activation of phospholipase A2 by tumor necrosis factor alpha.

Although there are a limited number of cell lines that are sensitive to cytolysis by tumor necrosis factor alpha (TNF alpha), the vast majority are resistant. The analysis of TNF alpha-sensitive cells has shown that phospholipase A2 is activated by TNF alpha in these cells and that the activity of phospholipase A2 is required for their cytolysis. Many cell lines that are resistant to TNF alpha-mediated cytolysis are dependent on the maintenance of protein synthesis for their resistance. We have recently shown that this is also true for TNF alpha-resistant cell lines derived from cervical (ME-180 and SiHa) and ovarian (SK-OV-3 and OVCAR-3) carcinomas, in that they are sensitive to cytolysis by TNF alpha only in the presence of protein synthesis inhibitors. Here we show that the TNF alpha-mediated cytolysis of these resistant cell lines in the presence of the protein synthesis inhibitor emetine is similar to that of sensitive cells, in that cytolysis is inhibited by the inhibitors of phospholipase A2. The measurement of the release of radiolabeled material from cervical and ovarian carcinoma cell lines prelabeled with [3H]arachidonic acid showed that not only was phospholipase A2 required for the cytolysis of these cells by TNF alpha in the presence of protein synthesis inhibitors, but more importantly, phospholipase A2 was not activated by TNF alpha unless protein synthesis was inhibited. These results indicate that a protein synthesis-dependent resistance mechanism expressed by these cell lines blocks TNF alpha-mediated cytolysis by preventing the activation of phospholipase A2 by TNF alpha.     

Characterization of Acquired Resistance to cis-Diamminedichloroplatinum(II) in Mouse Leukemia Cell Lines

We have established in vivo cisplatin-resistant mouse leukemia cell lines, L-1210/DDP and P388/ DDP, in order to elucidate the mechanism of acquired resistance to cisplatin. Resistance indices were 22 and 14, respectively, when the cells were exposed to cisplatin for 48 h. Uptake of cisplatin by both resistant lines was significantly reduced, compared with values for the respective parent lines (17% for L-1210/DDP and 27% for P388/DDP, at 100 μM for 1 h). While glutathione contents in the resistant cells were 1.7-1.9 times higher than those in the sensitive ones, their reduction by preincubation with buthionine sulfoximine did not influence the sensitivity of the cells to cisplatin. In addition, the resistant lines dill not show lower sensitivity to CdCl₂, than the respective sensitive ones, suggesting that intracellular SH groups might contribute little to the mechanism of cisplatin resistance in these cells. Postincubation with DNA repair inhibitors, caffeine and aphidicolon, did not selectively enhance the sensitivity of the resistant cells to cisplatin. These results suggested that reduced drug uptake would be a primary mechanism of cisplatin resistance in L-1210/DDP and P388/DDP. Cross-resistance patterns to platinum complexes were quite different between L-1210/DDP and P388/DDP. Colon 26/DDP, another cisplatin-resistant mouse tumor showed a different pattern from those observed with L-1210/DDP and P388/DDP. In the development of new platinum complexes we should use plural resistant lines for examining cross-resistance patterns to candidate platinum complexes.     

Genetic basis of drug sensitivity in human testis tumour cells

Testicular germ cell tumours (TGCT) are cured in over 80% of patients by using combination chemotherapy. However, the mechanism regulating this sensitivity has not been defined. Because cells derived from patients with DNA repair syndromes are similar to TCGT in their sensitivity to certain DNA-damaging agents and some of the genes involved have been cloned by functional complementation, the purpose of our study was to determine whether drug sensitivity in TGCT also has a genetic basis. Three testis tumour cell lines (cisplatin-sensitive) and 3 bladder cancer cell lines (cisplatin-resistant) were fused with a cisplatin-sensitive cell line (D98 or CI). The authenticity of the hybrids was confirmed by karyotyping and PCR analysis of locus-specific sites, and sensitivities to cisplatin were measured by colony forming assays. The hybrids between sensitive cell lines were more resistant to cisplatin than the parental cells, indicating that functional complementation had occurred. The hybrids between the cisplatin-resistant and sensitive cells were intermediate in their cisplatin sensitivity, indicating that resistance is incompletely dominant. We conclude that cisplatin sensitivity has a genetic basis in TGCT and that resistance to cisplatin can be conferred by somatic cell fusion. Our data indicate that gene(s) controlling sensitivity to chemotherapy in TGCT might be identified by expression cloning. © 1996 Wiley-Liss, Inc.     

Intrinsic resistance to the MEK1/2 inhibitor AZD6244 (ARRY‐142886) is associated with weak ERK1/2 signalling and/or strong PI3K signalling in colorectal cancer cell lines

Mutations in KRAS or BRAF frequently manifest in constitutive activation of the MEK1/2‐ERK1/2 signalling pathway. The MEK1/2‐selective inhibitor, AZD6244 (ARRY‐142886), blocks ERK1/2 activation and is currently undergoing clinical evaluation. Tumour cells can vary markedly in their response to MAPK or ERK kinase (MEK) inhibitors, and the presence of a BRAF mutation is thought to predict sensitivity, with the RAS mutations being associated with intrinsic resistance. We analysed cell proliferation in a panel of 19 colorectal cancer cell lines and found no simple correlation between BRAF or KRAS mutation and sensitivity to AZD6244, though cells that harbour neither mutation tended to be resistant. Cells that were sensitive arrested in G₁ and/or underwent apoptosis and the presence of BRAF or KRAS mutation was not sufficient to predict either fate. Cell lines that were resistant to AZD6244 exhibited low or no ERK1/2 activation or exhibited coincident activation of ERK1/2 and protein kinase B (PKB), the latter indicative of activation of the PI3K pathway. In cell lines with coincident ERK1/2 and PKB activation, sensitivity to AZD6244 could be re‐imposed by any of the 3 distinct PI3K/mTOR inhibitors. We conclude that AZD6244 is effective in colorectal cancer cell lines with BRAF or KRAS mutations. Sensitivity to MEK1/2 inhibition correlates with a biochemical signature; those cells with high ERK1/2 activity (whether mutant for BRAF or KRAS) evolve a dependency upon that pathway and tend to be sensitive to AZD6244 but this can be offset by high PI3K‐dependent signalling. This may have implications for the use of MEK inhibitors in combination with PI3K inhibitors. © 2009 UICC     

Heterogeneity in the sensitivities of the 13762NF rat mammary adenocarcinoma cell clones to cytolysis mediated by extra- and intratumoral macrophages

The susceptibility of cloned cell lines of the 13762NF rat mammary adenocarcinoma to macrophage-mediated cytolysis was investigated using both intra- and extratumoral macrophages. The percentage of Fc receptor-positive cells in tumors growing s.c. in syngeneic F344 rats ranged from 8 to 20%, but we could not demonstrate a significant correlation between the number of Fc receptor-positive cells within tumors and their spontaneous metastatic potentials. In macrophage-mediated cytolysis assays, cloned 13762NF cell lines of differing metastatic potential, established from tissue culture lines, fresh tumor explants, or short-term cultures (one passage in vitro), were used as targets. Effector cells were thioglycolate-elicited peritoneal macrophages (activated in vitro with bacterial lipopolysaccharide) or intratumoral macrophages (activated in vitro with lipopolysaccharide). When the effector cells were peritoneal macrophages, established cloned 13762NF cell lines showed little correlation in their susceptibility to macrophage-mediated cytolysis and metastatic potential, while this was not observed when fresh tumor explants were used. Highly metastatic MTLn3 cells were the least sensitive, less metastatic MTF7 and MTLn2 cells were more susceptible, and the low metastatic parental MTPa cells were the most sensitive in 72-h cytolysis assays. When the effector cells were intratumoral macrophages, all 13762NF cell lines showed less sensitivity in cytolysis assays than similar assays using thioglycolate-elicited peritoneal macrophages. With the exception of line MTLn2, short-term cultures (one passage in vitro) did not differ substantially in susceptibility to intratumoral macrophages compared to fresh explants. In this system, the sensitivity of 13762NF cells to macrophage-mediated cytolysis is a function of effector as well as target cell source.     

Resistance of human ovarian cancer cells to tumor necrosis factor and lymphokine-activated killer cells: correlation with expression of HER2/neu oncogenes

Since overexpression of HER2/neu oncogenes in breast cancer cells is associated with resistance to the cytotoxic effect of tumor necrosis factor (TNF), we investigated whether this correlation also existed for ovarian cancer targets. Nine continuously cultured human ovarian cancer lines were studied and compared to 3 breast cancer lines. Three of the ovarian and 1 breast cancer line demonstrated amplified HER2/neu genes by Southern analysis, increased HER2/neu RNA by Northern analysis, and marked immunoperoxidase staining for HER2/neu protein. The other 8 lines contained unamplified genes and undetectable RNA and protein. All 4 overexpressed lines were relatively resistant to the cytotoxic effects of TNF. Interestingly, they were also resistant to lymphokine-activated killer cells. In contrast, 7 of 8 nonexpressed lines showed sensitivity to TNF and all 8 were sensitive to lymphokine-activated killer cells. There was no difference in sensitivity to lysis by hydrogen peroxide or peptide defensins between over- and nonexpressed lines. These data indicate that expression of HER2/neu oncogenes may impart a proliferative advantage in tumor cells due to induction of resistance to several different cytotoxic mechanisms.     

The effect of adenovirus-IkappaBalpha transduction on the chemosensitivity of lung cancer cell line with resistance to cis-diamminedichloroplatinum(II)(cisplatin) and doxorubicin(adriamycin)

Resistance to chemotherapeutic agents is the major reason for treatment failure of cancer chemotherapy. Some chemotherapeutic drugs induce the activation of NF-kappaB in cancer cells that results in their resistance to anticancer drugs. But the role of NF-kappaB in acquired resistance has not been well investigated. In this study, we transferred the "super-repressor" form of the NF-kappaB inhibitor by adenoviral vector (ad-IkappaBalpha) to human lung cancer cell lines with resistant to cisplatin (PC-14-DDP) and adriamycin (PC-14-ADR), and observed the sensitivity change. Electrophoretic mobility shift assay showed that ad-IkappaBalpha blocked the activation of NF-kappaB induced by cisplatin and adriamycin. Transduction with ad-IkappaBalpha restored the sensitivity of cisplatin and adriamycin resistant lung cancer cell lines (PC-14-DDP and PC-14-ADR) to a level compatible to the parental cell lines. Annexin-V analysis suggested that the enhancement of chemosensitivity was probably a result of the induction of apoptosis. These data demonstrated that ad-IkappaBalpha blockade of chemotherapeutic induced NF-kappaB activation increased apoptosis induction and the chemosensitivity of lung cancer cell lines with acquired resistance to cisplatin and adriamycin. Therefore, gene transfer of IkappaBalpha-SR seems to represent a new therapeutic strategy for the solution of low sensitivity and lung cancer resistance to anticancer drugs.     

Cytolytic activity against tumor cells by macrophage cell lines and augmentation by macrophage stimulants

Previous studies have shown that macrophage cell lines retained the ability to phagocytize, to secrete lysosomal enzymes, and to function as effector cells in antibody-dependent cellular cytoxicity. In this paper, the cytolytic activity of murine macrophage cell lines against tumor target cells was assessed using an 18-h 51Cr release assay. Of the macrophage cell lines tested, RAW 264, PU5-1.8 and IC-21 had intermediate to high levels of spontaneous cytolytic activity, P388D, and J774 had low to intermediate levels, while /WEHI-3 showed little or no cytolytic activity against RBL-5, MBL-2 and TU-5 target cells. Tumor-cell killing by macrophage cell lines could be augmented by the addition of macrophage stimulants, such as bacterial lipopolysaccharide and poly I:C, indicating that the activation of macrophages by these stimulants does not require the participation of other cell types. Treatment with interferon also augmented the tumor-cell killing by macrophage cell lines. Although the mechanism by which these cell lines exert their spontaneous or boosted cytotoxic activity is not clear, it does not appear to be due to depletion of nutrients since cell lines with high metabolic and proliferative activities, such as WEHI-3 and RBL-5, showed little or no cytotoxicity and supernatants from the macrophage cell lines did not exert any cytotoxic effects in their essay. Thus, it appears that the different macrophage cell lines represent different levels of activation and/or differentiation and may be useful for studying the development of these processes as well as providing a useful tool for analyzing the mechanisms of macrophage-mediated cytolysis.     

Loss of caspase-8 expression in highly malignant human neuroblastoma cells correlates with resistance to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis

Human neuroblastoma (NB) is a highly heterogeneous childhood cancer that is aggressively malignant or can undergo spontaneous regression that may involve apoptosis. NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Noninvasive S-type cell lines (NB cell lines of substrate adherent phenotype) are highly sensitive to TRAIL, whereas invasive N-type cell lines (NB cell lines of neuronal phenotype) are resistant. Whereas both S- and N-type cell lines express TRAIL-R2, FADD, and caspase-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a malignant stage IV NB tumor when compared with a benign ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage NB tumors. Caspase-8 expression can be induced by demethylation with 5-aza-2'deoxycytidine, which enhances sensitivity to TRAIL. Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.