Enhanced sensitivity of human colon tumor cell lines in vitro in response to thermochemoimmunotherapy.

We have investigated the cytotoxic responses in vitro of three human colon tumor cell lines with epithelial-like morphology, DLD-1, HCT-15, and HT-29, to thermochemoimmunotherapy with hyperthermia (42 degrees C for 2 h), carboplatin, and recombinant human tumor necrosis factor (TNF). Dose ranges of carboplatin and recombinant human TNF were administered essentially simultaneously and were followed 1 h later by hyperthermia. A two-tiered approach was used to evaluate cytotoxicity. In the first tier, a 5-day microcytotoxicity assay using vital dye staining was done; the effect on surviving fraction of simultaneously varying carboplatin and recombinant human TNF doses was evaluated by response surface methodology. From this analysis doses were selected for use in the second-tier clonogenic survival assays. A similar treatment protocol was used in clonogenic assays. Both assays revealed significant interline treatment response heterogeneity. Only the HCT-15 cells were sensitive to TNF alone; carboplatin activity against all three tumor cell lines was enhanced by TNF. Hyperthermia had minimal effect as a sole agent but enhanced the effects of carboplatin and TNF in DLD-1 and HCT-15 cells. Triple modality treatment resulted in 3-4-log decreased survival and could reduce cytotoxic resistance expressed against single- or dual-modality treatments by some of these cells.

Liposomal delivery of oligodeoxynucleotides.

We have previously demonstrated that liposome-incorporated methylphosphonate antisense oligodeoxynucleotides (oligos) specific for BCR-ABL can selectively inhibit the expression of p210Bcr-Abl protein and the proliferation of chronic myelogenous leukemia cells in vitro. Here, we show that liposome-entrapment of phosphodiester and phosphorothioate oligos specific for BCR-ABL can also selectively inhibit the proliferation of chronic myelogenous leukemia cells. We have studied the intracellular localization of liposomes by fluorescent microscopy and found that liposomes are readily taken up by leukemic cells and are localized in the cytoplasm, allowing increased access of oligos to target cells intracellularly. Liposomal oligos are not toxic to peripheral blood mononuclear cells nor to bone marrow progenitors isolated from normal hematological donors. These studies strongly suggest that liposomal delivery of oligos may indeed circumvent the major limitations that preclude the clinical development of antisense oligos.

Experimental and computational approach to the rational monitoring of hydrogen-bonding interaction of 2-Imidazolidinethione with DNA and guanine.

The hydrogen-bonding interaction of 2-Imidazolidinethione with DNA and guanine has been investigated using UV/vis, circular dichroism, differential pulse voltammetry and ab initio chemical mechanic quantum procedures. The bonding constant of 2-Imidazolidinethione with DNA was measured by UV/vis spectroscopy and is estimated to be 1.4x10(3). In order to prove of hydrogen-bonding formation, the bonding constants of 2-Imidazolidinethione with guanine in Tris-HCl buffer and binary mixture of buffer-acetonitrile were measured. In addition, density functional theory calculations, using ab initio methodology at the 6-31+G(d,p) level and simulating the effect of solvents on interaction by mean of the Cosmo Polarizable Continuum Model (CPCM), were performed on the single and complex of 2-Imidazolidinethione and guanine molecules. However, we concluded that 2-Imidazolidinethione interacted with N(3) of guanine in minor groove of DNA.

Fabrication of a novel iron(III)-PVC membrane sensor based on a new 1,1'-(iminobis(methan-1-yl-1-ylidene))dinaphthalen-2-ol synthetic ionophore for direct and indirect determination of free iron species in some biological and non-biological samples.

In this novel, the iron(III)-PVC membrane sensor was investigated based on a new 1,1'-(iminobis(methan-1-yl-1-ylidene))dinaphthalen-2-ol (IBMYD) synthetic ionophore as a suitable carrier. The best performance was observed for the membrane composition including 33.0% PVC, 65.0% TEHP, 1.0% NaTPB and 1.0% ionophore. The electrode displayed a linear potential response over a wide concentration range from 1.0 x 10(-7) to 1.0 x 10(-1)mol L(-1), with a detection limit of 5.0 x 10(-8)mol L(-1) and a good Nernstian slope of 19.9+/-0.3 mV decade(-1). The sensor possessed some advantages such as short conditioning time, very fast response time (<12s) and especially good discriminating ability towards Fe(III) ions over a wide variety of alkali, alkaline earth, transition, and heavy metal ions. The potential response of the proposed sensor was independent of the pH of the test solution, in the pH working range from 3.0 to 6.3. The fabricated electrode was applied for at least 2 months, without any measurable divergence in the potential characteristics. The optimized sensor was used successfully for direct and indirect determination of free iron species in some different synthetic and real samples with satisfactory results.

MRP- and BCL-2-mediated drug resistance in human SCLC: effects of apoptotic sphingolipids in vitro.

Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

Hyperthermia engages the intrinsic apoptotic pathway by enhancing upstream caspase activation to overcome apoptotic resistance in MCF-7 breast adenocarcinoma cells.

Febrile hyperthermia enhanced TNF-stimulated apoptosis of MCF-7 cells and overcame resistance in a TNF-resistant, MCF-7 variant (3E9), increasing their TNF-sensitivity by 10- and 100-fold, respectively. In either cell line, the hyperthermic potentiation was attributable to increased apoptosis that was totally quenched by caspase inhibition. In MCF-7 cells, hyperthermic potentiation of apoptosis was associated with sustained activation of upstream caspases in response to TNF and more prominent engagement of the intrinsic apoptotic pathway. Apoptotic enhancement by hyperthermia was primarily mediated by caspase-8 activation, as the specific inhibitor, Z-IETD, blocked cell death, whereas direct engagement of the intrinsic apoptotic pathway (with doxorubicin) was not affected. In 3E9 cells, hyperthermia alone induced activation of caspase-8, and was further enhanced by TNF. In 3E9 cells, hyperthermia caused TNF-dependent loss of mitochondrial membrane potential and activation of capspase-9 that was initiated and dependent on upstream caspases. MCF-7 and 3E9 cells were equally sensitive to exogenous C(6)-ceramide, but mass spectroscopic analysis of ceramide species indicated that total ceramide content was not enhanced by TNF and/or hyperthermia treatment, and that the combination of TNF and hyperthermia caused only modest elevation of one species (dihydro-palmitoyl ceramide). We conclude that febrile hyperthermia potentiates apoptosis of MCF-7 cells and overcomes TNF-resistance by sustained activation of caspase-8 and engagement of the intrinsic pathway that is independent of ceramide flux. This report provides the first evidence for regulation of caspase-dependent apoptosis by febrile hyperthermia.

Hyperthermia enhances the cytotoxicity of National Institutes of Health 3T3 cells transfected with a noncleavable transmembrane pro-tumor necrosis factor deletion mutant.

Hyperthermia has been shown to potentiate the cytotoxicity of exogenously added tumor necrosis factor (TNF) against tumor cell targets. The mechanism for that interaction is not known, but among the possibilities are that heat enhances internalization of ligand-bound TNF or enhances processing of internalized TNF. In this study, we found that NIH 3T3 cells transfected with an expression vector containing the full-length human pro-TNF secreted TNF and that hyperthermic treatment of chromium-labeled L929 target cells at 43 degrees C for 1 h potentiated the cytotoxicity of these transfectants against the L929 cells in clonogenic survival and chromium-release assays. On the other hand, transfectants expressing a transmembrane, nonsecretable pro-TNF mutant that kills L929 cells by cell-to-cell contact without internalization also exhibited enhanced cytotoxicity against heated L929 cell targets. Thus, potentiation of the cytotoxicity of TNF by hyperthermia is not strictly dependent on enhanced internalization of ligand-bound TNF or enhanced processing of internalized TNF.

Murine cells transfected with human Hsp27 cDNA resist TNF-induced cytotoxicity.

Hyperthermia sensitizes tumor cells to killing by tumor necrosis factor-alpha (TNF). Sensitization is greater in cells exposed to TNF before heating begins than with the reverse sequence, and heat-shock proteins (hsp) have been suggested to protect cells from TNF cytotoxicity. Here we examined the role of Hsp27 in TNF resistance. Murine L929 cells were stably transfected with the vector pRc/CMV constitutively to express an inserted human hsp27 complementary DNA (cDNA) sequence. Parental cells produced no detectable murine homolog to human hsp27. Hsp27-sense clones expressed hsp27 messenger RNA (mRNA) and protein at 37 degrees C. Cells transfected with the cDNA in the anti-sense orientation produced anti-sense mRNA but no protein, and cells transfected with the vector alone produced neither product. Expression of hsp27 conferred significant resistance to TNF cytotoxicity in both neutral red cytotoxicity and clonogenic survival assays. Vector along and hsp27 anti-sense transfectants had a TNF response similar to that of parental L929 cells. Kinetic studies in L929 cells showed that hsp27-expressing clones exhibited resistance relative to parental cells beginning 6 h after TNF exposure, and this differential response increased by 12 and 24 h. Addition of actinomycin D to the TNF cytotoxicity assays accelerated the cytotoxicity development in parental and transfected cells, but the hsp27-sense clones were still more resistant. Hsp27-sense clones of L929 cells were also resistant to oxidative stress induced by menadione and released less arachidonic acid in response to TNF induction. These results show that hsp27 can negatively regulate the TNF cytotoxic mechanism.