In vitro determination of cytotoxic drug response in ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA)

The fluorometric microculture cytotoxicity assay (FMCA), a short-term in vitro assay based on the concept of total tumor cell kill, was used for testing the cytotoxic drug sensitivity of tumor cells from patients with ovarian carcinoma. A total of 125 fresh specimens was obtained, 98 (78%) of which were analyzed successfully. Data from 45 patients were available for clinical correlations. The FMCA appeared to yield clinically relevant cytotoxic drug sensitivity data for ovarian carcinoma as indicated by a comparison with tumor samples obtained from patients with non-Hodgkin's lymphoma or kidney carcinoma. Considering the most active single agent in vitro actually given in vivo, and using the median drug activity among all ovarian carcinoma samples as a cut-off, the sensitivity of the assay and its specificity were 75 and 52%, respectively. Cross-resistance in vitro was frequently observed between standard drugs but not between standard drugs and Taxol. Ten percent of the specimens showed an extreme resistance for at least 4 of 6 of the drugs investigated.     

In vitro effect of the cysteine metabolites homocysteic acid, homocysteine and cysteic acid upon human neuronal cell lines

Cysteine (CYS) is a non-essential amino acid which elicits excitotoxic properties via the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor. CYS levels are known to be elevated in association with neurological disease such as Alzheimers Disease (AD) and Parkinsons Disease (PD). We have previously reported studies investigating the toxicity of CYS and its major metabolite cysteinesulfinic acid (CSA) to human neuronal cell lines in vitro and in continuation of this we now report the toxicity of other compounds (Homocysteic Acid, HCA; Homocysteine, HCYS; and Cysteic Acid, CA) in the CYS metabolic pathway. Three cell lines, all of human origin and derived from separate discrete areas of the brain were used in the neurotoxicity assays. Lactate dehydrogenase (LDH) release was assayed as a measure of cell death. The cell lines investigated showed varying degrees of toxic responses which were the reverse of those seen when they were exposed to CYS or CSA. The SK.N.SH (Neuroblastoma) cell line, which exhibits a high toxic response to CYS and CSA, gave a low toxic response to HCA and CA while the TE 671 (Medulloblastoma) cell line, which exhibits a low toxic response to CYS and CSA, showed a high toxic response to HCYS, HCA and CA. However, the U-87 MG (Glioblastoma) cell line, which has a median toxic response to CYS and CSA, also has median response to HCYS, HCA and CA. These results show that toxic responses are cell-type specific for CYS and its metabolites and this may be reflected in the patterns of neurodegeneration observed in such diseases as AD and PD. HCYS is selectively toxic to medulloblastoma cells; this may explain why high HCYS levels result in neural tube defects in prenatal humans, where the same cell-type is involved.     

Differentially potentiating effects by dipyridamole on cytotoxicity of 5-fluorouracil against three human maxillary cancer cell lines derived from a single tumor

Twenty specimens of six species of Nile fishes were examined for the presence of fungi. Of which 2 were from Alestes nurse; 3 from Bagrus docmac; 4 from Barbus bynni; 6 from Chrysichthys auratus; 4 from Lates niloticus and 1 from Malapterurus electricus. Forty-three fungal species in addition to 1 variety appertaining to fifteen genera were recovered from skin (15 genera and 34 species + 1 variety); gills, kidney (12 genera and 30 species + 1 variety, each); liver (11 genera and 30 species + 1 variety) and intestine (13 genera and 30 species + 1 variety) of all specimens, using glucose Czapek-Dox medium at 28 degrees C. The most common genera were Aspergillus, Penicillium and Trichoderma.     

Effects of diheptyldiselenide (DDS) on human tumor cell lines and on peripheral blood mononuclear cells

In vitro effects of graded concentrations of diheptyldiselenide (DDS) on human tumor cell proliferation, and on the proliferative responses and immunological functions of peripheral blood mononuclear cells (MNC) were investigated. The agent significantly decreased tumor cell proliferation in a dose and time dependent manner. Proliferative responses of MNC to phytohemagglutinin (PHA) and interleukin-2 (IL-2) were also significantly depressed when MNCs were exposed to DDS (250 microM for 18 h) led to a significant increase in NK activity only in MNC samples showing very limited baseline NK function. On the other hand, generation of LAK cells was significantly inhibited by DDS. However, when the agent was added to the effector and target cell mixture during the 4 h 51Cr release cytotoxicity assay, no influence was found on NK and LAK-mediated target cell lysis. These studies show that high concentrations of DDS inhibit tumor cell proliferation and could also impair certain proliferative-dependent immune functions, without directly affecting cell-mediated cytolytic activity of effector cells.     

Potentiation of tumour necrosis factor-mediated cell killing by VP16 on human ovarian cancer cell lines. In vitro results and clinical implications

Synergism between recombinant human tumour necrosis factor (rHuTNF) and DNA topoisomerase II inhibitor VP16 during the killing of cells has been studied in six human ovarian cancer cell lines (A2774, A2780, SW626, IGROV-1, SKOV3, Pa1) and a cervical carcinoma cell line (Me180). Studies were performed using an assay of colony formation inhibition (drug treatment for 1 h) and a growth inhibition assay (continuous exposure for 20 h). Concomitant treatment of cells with VP16+rHuTNF enhanced cell killing in all the cell lines tested--an effect observed in both short- and long-term cytotoxicity assays. This study suggests that the activity of VP16 in ovarian cancer cell lines might be enhanced by rHuTNF in in vitro models.     

In the search for new anticancer drugs. 29. A study on the correlation of lipophilicities, ionization constants and anticancer activities of aminoxyl labeled TEPA congeners

Ionization constants for a series of sterically hindered pyrrolidine, pyrroline and piperidine derivatives were determined by potentiometric titrations. The pKa values for the secondary amines as a group ranged from about 7.7 to 11.7, whereby the ring size had no decisive effect on the values. The corresponding hydroxylamine derivatives as a group had distinctly lower pKa values than the amine derivatives ranging between about 4.0 and 6.3. It was shown using the Henderson-Hasselbalch equation that at physiological pH, arbitrarily chosen 6, 7 and 8, the amine derivatives would exist mainly in the protonated form, whereas the hydroxylamine derivatives would be expected to be mainly in the unprotonated form. In contrast, the 4-hydroxy-2,2,6,6-tetramethylpiperdin-1-oxyl radical, under analogous conditions, was a neutral species, i.e. it could not be titrated in aqueous media. On the basis of these results, it was hypothesized that the alkylating anticancer drugs of TEPA (N,N:N'N':N",N"-tri-1,2-ethanediylphosphorictriamide) type, containing sterically hindered carrier moieties, would be expected to permeate across cell membranes, and, consequently, exhibit anticancer activities according to the following sequence: spin-labeled drugs containing no titratable components > hydroxylamine derivatives > secondary amine congeners. This assumption is confirmed by good correlations of anticancer activities of these drugs with their pKa values, and the partition coefficients. The conclusion was reiterated that, in the quest for a rational design of anticancer drugs, the aim should be to construct agents with partition coefficients approaching the logarithm of zero, either from the negative or positive side, and pKa values as low as practically possible and applicable.     

Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells. I. Comparative in vitro cytotoxicity of 27 drugs

Combined drug toxicity to HeLa cells was studied in vitro with use of the microtitre MIT-24 test system. Whether drug toxicity to HeLa cells is representative of drug toxicity to other cultivated cells was investigated by a comparison of the MIT-24 toxicity of 27 drugs to HeLa cells with their toxicity to various permanent cell lines and more differentiated primary cell cultures as reported in the literature, together with original recordings of the MIT-24 toxicity of 9 of the drugs to human fetal kidney cells. A similarity of drug toxicity to all cell types was found. Thus the MIT-24 recordings may be representative of a basal drug cytotoxicity, probably corresponding to local drug irritation and to causal systemic drug toxicity.     

Expression and cleavage of tumor necrosis factor-alpha and tumor necrosis factor receptors by human monocytic cell lines upon direct contact with stimulated T cells

Tumor necrosis factor-alpha (TNF-alpha) is a potent cytokine in inflammatory processes. A variety of mechanisms that modulate its activity have been described, one being its binding to soluble receptors (sTNFR). In this study, we demonstrate that human monocytic cells such as THP-1 respond to direct contact with a membrane preparation of stimulated HUT-78 cells by producing TNF-alpha and by releasing sTNFR-p75, but not sTNFR-p55, with different kinetics. TNF-alpha concentration peaked after 12 h of contact and then decreased, whereas sTNFR-p75 production increased progressively upon cell/cell contact. The decrease in TNF-alpha concentration is not due to trapping of TNF-alpha by its soluble receptors or other soluble or cell-associated molecules, but rather to a proteolytic activity associated to THP-1 cells. On the other hand, the increase in sTNFR-p75 release does not result from an increase in the cleavage of pre-existing cell-associated sTNFR-p75 but from an increase in TNFR-p75 expression, immediately followed by the cleavage of its extracellular domain. Phenylmethylsulfonylfluoride, a serine protease inhibitor, has a negative effect on both TNF-alpha degradation and sTNFR-p75 release by THP-1 cells. Thus, there may be an enzymatic activity associated to THP-1 cells that plays an important role in the neutralization of TNF-alpha activity both by degrading the molecule and by cleaving its receptors at the cell surface.     

In vitro and in vivo evaluation of the enhancing activity of glycyrrhizin on the intestinal absorption of drugs

PURPOSE: To describe a surgical technique to visualize the depth of corneal incisions and lamellar stromal dissections during surgery. METHODS: In porcine cadaver eyes, the aqueous was exchanged by air. Thus an air-to-endothelium interface (i.e., a useful optical surface) was created at the posterior corneal surface. The air-to-endothelium interface was used as a reference plane to visualize the corneal thickness and the relative depth of corneal incisions and dissections. Freehand peripheral corneal incisions, tangential keratotomy incisions, and lamellar stromal dissections were made at an intended corneal depth of 60, 80, and 99%. Light microscopy was used to measure the relative depth of the incisions and dissections. RESULTS: Achieved depth for peripheral corneal incisions averaged 65.2+/-5.3%, 78.8+/-5.1%, and 93.4+/-6.0%, respectively (p<0.05); and for tangential keratotomy incisions, 68.2+/-7.3%, 83.2+/-4.4%, and 95.8+/-3.6%, respectively (p<0.05). Achieved depth for lamellar stromal dissections averaged 58.3+/-9.4%, 81.1+/-3.4%, and 94.4+/-1.5%, respectively (p<0.05). Microperforations occurred with three incisions made at 99% intended depth. CONCLUSION: During surgery, the depth of incisions and lamellar dissections relative to the corneal thickness can be visualized by filling the anterior chamber with air (i.e., by creating an optical interface at the posterior corneal surface).     

In vitro invasiveness of human epithelioid-sarcoma cell lines: association with cell motility and inverse correlation with the expression of tissue inhibitor of metalloproteinases

Epithelioid sarcoma (ES) is a very aggressive soft-tissue tumor in vivo, but no experimental data on its invasive and metastatic behavior have been reported. In the present study, 3 different clonal sub-populations (GRU-1A, GRU-1B and GRU-1C), derived from the same human ES cell line, GRU-1, were investigated for in vitro invasiveness in relation to migration, adhesion and the expression of different invasion- and metastasis-related genes. Tumor spheroids of GRU-1A were markedly more invasive in the chick-heart invasion assay (CHIA) than spheroids of GRU-1B and GRU-1C. These results were paralleled by a significantly higher cell motility of GRU-1A than GRU-1B and GRU-1C (p < 0.05) on distinct substrates, suggesting that the observed differences in invasion result at least in part from differences in motility. When invasion was assayed with suspended tumor cells in the Matrigel assay, differences between the 3 cell lines were much more pronounced than in the CHIA, where cell-cell contacts are established. These results indicate that interclonal differences in ES invasion result mainly from differences in motility, but also partly depend on differences in cell-cell adhesion. On the molecular level, low invasive potential was associated with over-expression of distinct tissue inhibitor of metalloproteinases (TIMPs) relative to matrix metalloproteinase-2 and -9. However, no association was found between invasion and the expression of CD44 splicing variants or nm23 isoforms. Our results suggest that differences in invasion between GRU-1A, GRU-1B and GRU-1C are caused mainly by interclonal differences in migration, and might result from differences in the expression of distinct TIMPs.     

In vitro schedule-dependent interaction between paclitaxel and SN-38 (the active metabolite of irinotecan) in human carcinoma cell lines

METHODS: From 1983 to 1995, 72 patients with necrotizing pancreatitis were treated with a general approach involving planned reoperative necrosectomies and interval abdominal wound closure using a zipper. RESULTS: Hospital mortality was 25%. Multiple organ failure without sepsis caused early mortality in 3 of 4 patients and sepsis caused late mortality in 11 of the remaining 14. The mean number of reoperative necrosectomies/debridements was 2 (0 to 7). Fistulae developed in 25 patients (35%); 64% were treated conservatively. Recurrent intraabdominal abscesses developed in 9 patients (13%) but were drained percutaneously in 5. Hemorrhage required intervention in 13 patients (18%). Prognostic factors included APACHE-II score on admission < 13 (P = 0.005), absence of postoperative hemorrhage (P = 0.01), and peripancreatic tissue necrosis alone (P < 0.05). CONCLUSIONS: The zipper approach effectively maximizes the necrosectomy and decreases the incidence of recurrent intraabdominal infection requiring reoperation. APACHE-II score > or = 13, extensive parenchymal necrosis, and postoperative hemorrhage signify worse outcome.     

The effect of dacarbazine and vincristine sulfate on human melanoma cell lines. In vitro analysis of interaction on DNA synthesis, RNA synthesis and protein synthesis

The effect of anticancer agents, dacarbazine [DTIC (dimethyl-triazeno-imidazole-carboxamide)] and vincristine sulfate (VCR) on two human melanoma cell lines (G 361 and MeWo) was investigated. First we estimated the growth curve of two cell lines by using an isotope labelling technique with DNA, RNA and protein precursors to find the most appropriate conditions, and then, DTIC and VCR were added at various concentrations. When the drug was added alone, DNA, RNA and protein synthesis were suppressed in a dose-dependent fashion and the pattern of responses was similar between the two. When the two agents were administered in combination, responses were variable depending on the combinations of the concentrations of these agents. It was also observed that the response on DNA synthesis is not similar to that on RNA or protein synthesis, and that unbalanced growth can happen when two anticancer agents, whose mechanisms of action against tumour cells, are different, are administered at the same time. These observations also differed between the two cell lines. It suggests that these diverse responses of melanoma cells against chemotherapeutic agents hinder the establishment of a sensitive combination chemotherapeutic regimen.     

Effects of feeder cells (human cancer cell lines) on the development of mouse embryos by co-culture

The participation of apical membranes of uterine epithelial cells in the process of blastocyst adhesion makes them an interesting object in the study of changes occurring during early pregnancy. In the study of these changes alkaline phosphatase (AIP), a typical brush border enzyme, was chosen for demonstration with the scanning electron microscope (SEM) by means of a backscatter detector. Thus the temporal and spatial pattern of enzyme activity on the uterine luminal surface was made visible with lead salt procedures. AIP activity was shown to be located on apical membranes and microvilli of endometrial epithelial cells with high activity on day 2 of pregnancy decreasing to virtually no activity on day 5. This decrease in overall AIP activity was shown to be asymmetrical with respect to the uterine cavity. It begins on the antimesometrial half of the uterine lining on day 2. A distribution pattern demarcating a presumptive implantation site along the uterine horn was not found. However, on day 5 of pregnancy, a characteristic pattern of surface folds was found, dividing the uterine horn into 'implantation segments'. In addition, SEM investigation revealed a marked variation of AIP activity from one individual cell to the next on day 2 of pregnancy resulting in a mosaic-like pattern. This pattern is lost with the decrease of AIP activity on day 5. Thus heterogeneity of uterine epithelial cells in AIP activity is apparently a feature of nonreceptive epithelium in contrast to the homogeneous epithelium on day 5. It is proposed that epithelial cell homogeneity could be a marker for uterine receptivity.     

In vitro production of interleukin-1, interleukin-6 and tumor necrosis factor-alpha by different blood cells in patients on intermittent peritoneal dialysis

Stevens-Johnson syndrome (SJS) is an uncommon eruptive disorder of the skin and mucous membranes with systemic manifestation. It is extremely unusual for patients with a past history of SJS to present with indications for surgery necessitating thoracotomy. We describe herein the perioperative management of a patient with SJS who underwent surgery for a spontaneous pneumothorax.     

Conformationally restricted analogues of 1N,12N-bisethylspermine: synthesis and growth inhibitory effects on human tumor cell lines

Eight analogues of 1N,12N-bisethylspermine (BES) with restricted conformations were synthesized in the search for new spermine mimetics with cytotoxic activities. By replacing the central butane segment of BES with a 1,2-disubstituted cyclopropane ring, a pair of cis/trans-isomers was obtained that introduced a spatial constraint in the otherwise freely mobile butane chain. An analogous pair of isomers was obtained when the butane segment was replaced with a 1, 2-disubstituted cyclobutane ring or with a 2-butene residue. The six new BES analogues thus obtained (three pairs of cis/trans-isomers) were growth inhibitory at low-micromolar concentrations against four human tumor cell lines (A549, HT-29, U251MG, and DU145) but were less growth inhibitory against two other human tumor cell lines (PC-3 and MCF7). 1N,12N-Bisethylspermyne, where the central butane segment of BES was replaced by the rigid 2-butyne segment, was devoid of growth inhibitory activity against five of the six human cell lines studied (DU145 being the only exception), a clear indication of the importance of conformational mobility at the 4N, 9N-butane segment of BES for its biological activity. When the butane segment was replaced by a benzene-1,2-dimethyl residue, the resulting BES analogue was devoid of growth inhibitory activity despite its cisoid conformation. The cytotoxicity of the analogues does not seem to be directly related to their uptake by the cells or to their effects on cellular polyamine levels. BES analogues with restricted conformations but which contained the equivalent of a two-carbon unit, rather than the natural four-carbon unit, at the central segment, such as 1,2-diaminocyclopropyl or 1, 2-diaminocyclobutyl derivatives, were devoid of growth inhibitory effects at the concentrations studied. The development of conformationally restricted polyamine analogues appears to show promise in the further quest for polyamine-related therapeutic agents with specificity of action.     

The influence of gastrin and/or cholecystokinin antagonists on the proliferation of three human astrocytic tumor cell lines

We have investigated the potential role of gastrin in the regulation of cell growth in human astrocytic tumors. To this end we have used synthetic analogs of gastrin and cholecystokinin (CCK) which behave as gastrin and/or CCK antagonists, e.g. compounds JMV-97, JMV-209 and JMV-179. Their effects on astrocytic tumor cell proliferation was investigated by the use of the colorimetric MTT assay. The in vitro biological models used in the present study included the SW1088, U87 and U373 astrocytic tumor cell lines. The results demonstrated marked influence of gastrin and CCK antagonists in the regulation of astrocytic tumor growth. This suggests that gastrin and/or CCK antagonists might be tested in experimental glioblastoma.     

Human tumor cells grown in fetal calf serum and human serum: influences on the tests for lymphocyte cytotoxicity, serum blocking and serum arming effects

Peripheral blood lymphoid cells (PBL) from cancer patients and normal donors were tested against three melanoma cell lines grown in either 10% fetal calf serum (FCS) or 2.5-5% human AB serum in order to determine if the heterologous membrane (HM) antigen or other FCS antigens acquired from the bovine serum supplement could influence lymphoid cell-mediated cytotoxicity in vitro. FCS-grown melanoma cells were more susceptible than the AB serum-grown subline to lymphocyte cytotoxic effects. Arming effects by autologous sera on normal donor lymphocytes and to a lesser extent on lymphocytes of cancer patients were more pronounced on the FCS-grown M12 melanoma cells. This effect was abrogated when the cells were grown in human AB serum for at least 8 weeks. The non-HM tumor-associated antigen remained at the same original low level. Blocking effects were more evident on the AB-grown M14 melanoma line. These data suggest that the FCS antigens on the cell surface may have been responsible for the augmented PBL cytotoxicity. The anti-FCS antibody present in normal and cancer patients' blood induced an antibody-dependent cellular cytotoxicity (ADCC). Elimination of arming activity against HM or other FCS antigens from AB-grown cells may have made the serum blocking factors more apparent. However, cytotoxicity against tumor cells by PBL from normal donors was still apparent even on the human serum-grown cells, suggesting that a different antigen-antibody system was also responsible for this "non-specific" activity.     

In vitro and in vivo characteristics of human squamous cell carcinoma of the head and neck cells engineered to secrete interleukin-2

Two human squamous cell carcinoma of the head and neck (SCCHN) cell lines, PCI-13 and PCI-52, were transduced with the retroviral construct containing human interleukin-2 (IL-2) cDNA and selected for neomycin resistance in G418 medium. Stably transduced SCCHN cells produced and secreted IL-2, which was shown to have biologic activity in a bioassay, using an IL-2-dependent CTLL-2 cell line. By immunohistochemistry, IL-2 gene-transduced PCI-13 cells were strongly positive for IL-2, and by flow cytometry showed both cell surface and intracytoplasmic expression of IL-2 protein. Expression of IL-2 mRNA was measured by quantitative RT-PCR and found to be considerably increased in transduced SCCHN relative to that in parental cells. There was no difference in expression of IL-2R between the parental and IL-2 gene-transduced cells. In vitro proliferation of IL-2 gene-transduced tumor cells was consistently more rapid than that of parental cells. Sensitivity of the parental and IL-2 gene-transduced targets to lysis or apoptosis mediated by purified human natural killer (NK) cells or IL-2-activated NK (A-NK) cells was comparable as measured in 4-hour 51Cr-release and 1-hour [3H]thymidine-release assays, respectively. However, transduced cells were significantly more sensitive than parental cells to these effectors in 24-hour MTT assays, most likely due to IL-2 production by the transduced targets. PCI-52 cells selected for in vivo experiments formed large subcutaneous tumors in immunosuppressed nude mice. Tumors established by subcutaneous injections of 1 x 10(7) IL-2 gene-transduced cells regressed completely by day 25, while those formed by parental or LacZ gene-transduced tumor cells grew progressively. Tumor regression was mediated by numerous mononuclear cells, identified as murine NK cells and macrophages by immunohistochemistry, which accumulated around the IL-2-secreting, but not parental, tumors within 5-6 days after tumor cell injections. Thus, IL-2 gene-transduced SCCHN cells produce functional IL-2 in vivo in amounts sufficient to support the recruitment to the tumor site and antitumor activity of cytotoxic effector cells. IL-2-secreting SCCHN cells may be a useful component of vaccines designed to induce and sustain effector cell activation at the tumor site.