Ehrlich Ascites Tumor Cell Line Research Articles

Enhanced antimicrobial, antioxidant, in vivo antitumor and in vitro anticancer effects against breast cancer cell line by green synthesized un-doped SnO2 and Co-doped SnO2 nanoparticles from Clerodendrum inerme

A novel approach was employed for the synthesis of un-doped tinoxide and Cobalt-doped tinoxide (Co-doped SnO2) nanoparticles (NAPs) by using aqueous extract of Clerodendrum inerme with the help of eco-friendly superficial solution combustion method. Synthesized NAPs were characterized by different spectroscopic techniques and results from XRD, TEM, SEM, EDX and UV–Vis examines confirmed the successful synthesis, crystalline nature and spherical structure of un-doped SnO2 and Co-doped SnO2 NAPs with the average grain size of 30 and 40 nm; and band gap energy of 3.68 and 2.76 eV respectively. Antimicrobial propensity of the synthesized NAPs was determined by agar well assay, SEM, TEM and confocal laser scanning microscopic analysis against various bacterial and fungal strains. Synthesized Co-doped SnO2 NAPs were unveiled the extraordinary antibacterial and antifungal activities against E. coli, B. subtilis, A. niger, A. flavus, and C. albicans with the zone of inhibitions of 30 ± 0.08 mm and 26 ± 0.06 mm, 17 ± 0.04 mm, 23 ± 0.08 mm and 26 ± 0.06 respectively which were also evidenced from SEM, TEM and confocal laser scanning microscopy. In addition, green synthesized Co-doped SnO2 NAPs were demonstrated the substantial antioxidant activity by scavenging DPPH, significant in vitro anticancer and in vivo antitumor activity on breast carcinoma cells (MCF-7) and Ehrlich ascites tumor cell lines respectively than standard. The hemolytic activity disclosed low cytotoxicity of fabricated NAPs (0.89 ± 0.05%) at 5 mg/mL, which was indicated their biocompatibility potential. Hence, the multi-purpose properties of synthesized NAPs presented in the current study can be further deliberated for pharmaceutical and nanomedicine applications.

Anti-proliferative and angio-suppressive effect of Stoechospermum marginatum (C. Agardh) Kutzing extract using various experimental models.

BACKGROUND/OBJECTIVESAbundant consumption of seaweeds in the diet is epidemiologically linked to the reduction in risk of developing cancer. In larger cases, however, identification of particular seaweeds that are accountable for these effects is still lacking, hindering the recognition of competent dietary-based chemo preventive approaches. The aim of this research was to establish the antiproliferative potency and angiosuppressive mode of action of Stoechospermum marginatum seaweed methanolic extract using various experimental models.MATERIALS/METHODSAmong the 15 seaweeds screened for antiproliferative activity against Ehrlich ascites tumor (EAT) cell line, Stoechospermum marginatum extract (SME) was found to be the most promising. Therefore, it was further investigated for its anti-proliferative activity in-vitro against choriocarcinoma (BeWo) and non-transformed Human embryonic kidney (HEK 293) cells, and for its anti-migratory/tube formation activity against HUVEC cells in-vitro. Subsequently, the angiosuppressive activity of S. marginatum was established by inhibition of angiogenesis in in-vivo (peritoneal angiogenesis and chorioallantoic membrane assay) and ex-vivo (rat cornea assay) models.RESULTSMost brown seaweed extracts inhibited the proliferation of EAT cells, while green and red seaweed extracts were much less effective. According to the results, SME selectively inhibited proliferation of BeWo cells in-vitro in a dose-dependent manner, but had a lesser effect on HEK 293 cells. SME also suppressed the migration and tube formation of HUVEC cells in-vitro. In addition, SME was able to suppress VEGF-induced angiogenesis in the chorio allantoic membrane, rat cornea, and tumor induced angiogenesis in the peritoneum of EAT bearing mice. A decrease in the microvessel density count and CD31 antigen staining of treated mice peritoneum provided further evidence of its angiosuppressive activity.CONCLUSIONSAltogether, the data underline that VEGF mediated angiogenesis is the target for the angiosuppressive action of SME and could potentially be useful in cancer prevention or treatment involving stimulated angiogenesis.

A pro-apoptotic 15-kDa protein from Bacopa monnieri activates caspase-3 and downregulates Bcl-2 gene expression in mouse mammary carcinoma cells

In diseases such as cancer, induction of apoptosis has been a new target for mechanism-based drug discovery. The central component of the process of apoptosis is a proteolytic system involving a family of proteases called caspases. Apoptosis involves characteristic morphological and biochemical events ultimately leading to cell demise. Apoptotic induction is evidently central to the mechanism of action of plant-derived anticancer drugs. Extract of the medicinal plant, Bacopa monnieri, inhibits tumor cell proliferation and accumulation of malignant ascites fluid. The crude sample when subjected to Soxhlet extraction yielded different solvent extracts of which the aqueous extract showed biological activity of apoptosis in Ehrlich ascites tumor cell lines (EAT). Bacopa monnieri water extract (BMWE) treatment of EAT cells produced apoptotic morphological characteristics and in-vivo DNA fragmentation, which is due to the activity of an endogenous endonuclease. The endonuclease responsible for DNA fragmentation acts downstream of caspase-3 activity and is also referred to as caspase-activated DNase (CAD). The CAD constitutively expressed in the cell cytoplasm is translocated into the nucleus upon BMWE treatment, as verified by Western blotting, leading to DNA fragmentation and to programmed cell death. The expression of the pro-apoptotic gene Bax was increased and the expression of the anti-apoptotic gene Bcl-2 was decreased by BMWE treatment. Considering the above results, BMWE was able induce apoptosis in EAT cells via Bax-related caspase-3 activation. This may provide experimental data for the further clinical use of BMWE in cancer.

MicroRNA Expression Profiles Associated with Development of Drug Resistance in Ehrlich Ascites Tumor Cells

Multidrug resistance (MDR) poses a major obstacle to successful chemotherapeutic treatment of cancer, and often involves multiple genes, which may be regulated post-transcriptionally by microRNAs (miRNAs). The purpose of the present study was therefore to identify any resistance-associated changes in miRNA expression in a sensitive and five increasingly drug-resistant Ehrlich ascites tumor (EAT) cell lines, representing different steps in the development of resistance. We used an LNA-enhanced microarray platform to study the global miRNA expression profiles in the six murine EAT cell lines, and identified growth-, hypoxia-, and resistance-specific miRNA patterns. Among the differentially expressed miRNAs, we found the two clusters miR-183∼miR-96∼miR-182 and miR-200b∼miR-200a∼miR-429 as well as miR-141 to be consistently upregulated in the MDR cell lines, while miR-125b-5p and the two clusters miR-30d∼miR-30b and miR-23b∼miR-27b∼miR-24-1 were downregulated in most of the resistant EAT cells. Several of the target genes for these miRNAs-including Zeb1/Zeb2 and members of the Fox gene family-could contribute to the drug-resistant phenotype, although we did not find that the degree of resistance was directly correlated to any specific changes in miRNA expression. Probably, the observed miRNA expression patterns reflect the underlying genomic instability of the tumor cells, and further studies are needed to explore how the highly complex regulatory miRNA networks contribute to the development of MDR.

Cytotoxic and antitumor effects of brucine on Ehrlich ascites tumor and human cancer cell line

Aims Brucine (BRU), a natural plant alkaloid is reported to possess cytotoxic and antiproliferative activities. In this study we aimed to investigate its in vitro and in vivo antitumor and antiangiogenic effects. Main methods Cell proliferation and viability was assessed using microculture tetrazolium tests (MTT). As predictive markers we determined intracellular levels of vascular endothelial growth factor (VEGF), Interleukin-12 (IL-12), tumor necrosis factor (TNF-α), caspase-3, -8 and -9 by ELISA and enzymatic activity assays. In addition, anti-VEGF neutralization effect was evaluated to assess whether it could result in augmented anticancer efficacy than the single agent. Antitumor activity was evaluated against Ehrlich ascites and solid tumor models. 15 × 10 6 EAC cells were implanted intraperitoneally (i.p., ascites tumor) and subcutaneous (s.c., solid tumor) in Swiss albino mice. Mice with established tumors received brucine i.p. at 12.5, 25, and 50 mg/kg for 14 days in ascites tumor and 50 mg/kg in solid tumor for 30 days. Tumor volume, cell viability, angiogenic, anti-angiogenic, anti-inflammatory factors and antioxidant parameters were determined. Immunohistochemistry analysis for VEGF and CD-31 was also performed. Key findings BRU produced time and dose-dependent inhibition of MCF-7 in vitro and EAC tumors in vivo. The anti-angiogenic effects were accompanied with decreased VEGF and TNF-α and increased IL-12 expression. BRU reduced peritoneal angiogenesis and microvessel density in vivo. Conclusion Our findings suggest that BRU possesses antitumor and anti-angiogenic activities in vitro and in vivo. The above results showed that BRU can be used as a potential anticancer agent as an antimetastatic and anti-angiogenic agent.

Development and characterization of hyaluronic acid decorated PLGA nanoparticles for delivery of 5-fluorouracil

The present investigation was aimed to develop and explore the prospective of engineered PLGA nanoparticles as vehicles for targeted delivery of 5-fluorouracil (5-FU). Nanoparticles of 5-FU-loaded hyaluronic acid-poly(ethylene glycol)-poly(lactide-co-glycolide) (HA-PEG-PLGA-FU) copolymer were prepared and characterized by FTIR, NMR, transmission electron microscopy, particle size analysis, DSC, and X-ray diffractometer measurement studies. The nanoparticulate formulation was evaluated for in vitro release, hemolytic toxicity, and hematological toxicity. Cytotoxicity studies were performed on Ehrlich ascites tumor (EAT) cell lines using MTT cell proliferation assay. Biodistribution studies of 99mTc labeled formulation were conducted on EAT-bearing mice. The in vivo tumor inhibition study was also performed after i.v. administration of HA-PEG-PLGA-FU nanoparticles. The HA conjugated formulation was found to be less hemolytic but more cytotoxic as compared to free drug. The hematological data suggested that HA-PEG-PLGA-FU formulation was less immunogenic compared to plain drug. The tissue distribution studies displayed that HA-PEG-PLGA-FU were able to deliver a higher concentration of 5-FU in the tumor mass. In addition, the HA-PEG-PLGA-FU nanoparticles reduced tumor volume significantly in comparison with 5-FU. Thus, it was concluded that the conjugation of HA imparts targetability to the formulation, and enhanced permeation and retention effect ruled out its access to the non-tumor tissues, at the same time favored selective entry in tumors, thereby reducing the side-effects both in vitro and in vivo.

Kidney uptake of 186/188Re(V)‐DMSA is significantly reduced when the reducing agent is changed from stannous ion to metabisulfite

AbstractWe describe the work carried out on the preparation of 186/188Re(V)‐DMSA, which showed lower kidney retention, formulated by using metabisulfite as the reducing agent. The complex was prepared by reducing 186/188ReO4− (100 μg, 0.54 μM, ∼150 MBq) in the presence of Na2S2O5 (30 mg, 0.15 mM) and reacting with DMSA (10 mg, 0.05 mM) in saline at pH 3.5 and at 80°C for 1 h. The complex was characterized by TLC using acetone and saline as two different solvent systems. Reverse phase HPLC carried out using isocratic system with 90:10 water/acetonitrile mobile phase showed the existence of four species. Biodistribution studies were carried out in albino mice with 186/188Re(V)‐DMSA prepared via metabisulfite reductant [186/188Re(V)‐DMSA (MBS)] as well as stannous chloride reductant [186/188Re(V)‐DMSA (SnCl2)]. The distribution patterns were similar except for kidney uptake. Kidney retention in case of 186/188Re(V)‐DMSA (MBS) was lower [0.68 (±0.06)%/g] than that observed in case of 186/188Re(V)‐DMSA (SnCl2) [2.93(±0.93)%/g] after 24 h p.i. Though bone uptake was initially similar with both the preparations, there was substantial decrease in bone activity after 24 h p.i. with 186/188Re(V)‐DMSA (MBS). In vitro cell uptake studies were carried out with 186/188Re(V)‐DMSA (MBS), 186/188Re(V)‐DMSA (SnCl2) and 99mTc(V)‐DMSA using Ehrlich Ascites Tumor Cell Lines. Approximately 15% cell uptake was observed with 186/188Re(V)‐DMSA (MBS) as well as with 186/188Re(V)‐DMSA (SnCl2) and was comparable with that of 99mTc(V)‐DMSA (19%). Our studies indicate that 186/188Re(V)‐DMSA prepared by using metabisulfite as a reducing agent has potential for use in targeted radiotherapy of medullary thyroid carcinoma. Copyright © 2002 John Wiley & Sons, Ltd.

Ehrlich ascites tumor cells expressing anti-sense glutaminase mRNA lose their capacity to evade the mouse immune system.

Glutaminase (EC 3.5.1.2) is a key enzyme in rapidly proliferating cells. Using anti-sense technology, an Ehrlich ascites tumor cell line (0.28AS-2) with reduced glutaminase activity has been obtained. We investigated the in vivo growth characteristics of the 0.28AS-2 cells. When injected i.p. into normal Swiss albino mice, the 0.28AS-2 cells were unable to grow. On the contrary, when injected into nude mice, they developed into solid tumors. Mice inoculated with 0.28AS-2 cells kept immunologic memory and rejected a second inoculation with parental Ehrlich ascites tumor cells. Expression of both polymorphic epithelial mucin-1 (MUC-1) and the enzyme N-acetyl-alpha-D-galactosaminidase, proteins implicated in host immune system escape, were markedly diminished in 0.28AS-2 cells. Study of the immune system response in mice inoculated with 0.28AS-2 cells revealed an increase in splenic CD18 cells and the presence of a large number of activated F4/80+ macrophages in the ascites cavity. These features, not observed in mice inoculated with parental Ehrlich ascites tumor cells, indicate that a distinctive, strong immune response occurred in animals inoculated with 0.28AS-2 cells. Our results suggest that inhibition of glutaminase expression using anti-sense technology induces phenotypic changes in Ehrlich ascites tumor cells that allow the development of an effective anti-tumor immune response, which makes the cells unable to develop in vivo tumors.

Ehrlich ascites tumor cells expressing anti-sense glutaminase mRNA lose their capacity to evade the mouse immune system

Glutaminase (EC 3.5.1.2) is a key enzyme in rapidly proliferating cells. Using anti-sense technology, an Ehrlich ascites tumor cell line (0.28AS-2) with reduced glutaminase activity has been obtained. We investigated the in vivo growth characteristics of the 0.28AS-2 cells. When injected i.p. into normal Swiss albino mice, the 0.28AS-2 cells were unable to grow. On the contrary, when injected into nude mice, they developed into solid tumors. Mice inoculated with 0.28AS-2 cells kept immunologic memory and rejected a second inoculation with parental Ehrlich ascites tumor cells. Expression of both polymorphic epithelial mucin-1 (MUC-1) and the enzyme N-acetyl-α-D-galactosaminidase, proteins implicated in host immune system escape, were markedly diminished in 0.28AS-2 cells. Study of the immune system response in mice inoculated with 0.28AS-2 cells revealed an increase in splenic CD18 cells and the presence of a large number of activated F4/80+ macrophages in the ascites cavity. These features, not observed in mice inoculated with parental Ehrlich ascites tumor cells, indicate that a distinctive, strong immune response occurred in animals inoculated with 0.28AS-2 cells. Our results suggest that inhibition of glutaminase expression using anti-sense technology induces phenotypic changes in Ehrlich ascites tumor cells that allow the development of an effective anti-tumor immune response, which makes the cells unable to develop in vivo tumors. © 2001 Wiley-Liss, Inc.

Kinetics of anthracycline accumulation in multidrug-resistant tumor cells: relationship to drug lipophilicity and serum albumin binding

A multidrug-resistant Ehrlich ascites tumor cell line (EHR2/DNR+) was used to examine the membrane transport kinetics of lipophilic anthracycline derivatives in the presence of serum albumin. We present a model for theoretical data analysis with consideration of drug–albumin complex formation. For a set of five derivatives (doxorubicin, daunorubicin, 4-demethoxydaunorubicin, 4′-deoxy-4′-iododoxorubicin, and 13-dihydro-4′-deoxy-4′-iododoxorubicin), data were given on the rates of diffusional drug uptake, and membrane permeability coefficients of the noncharged molecules were estimated. Both the initial rates and the steady-state levels of drug uptake were found to decrease by addition of BSA at concentrations ranging from 5 to 75 mg/mL. For each drug, this effect of serum albumin could be accounted for by the altered distribution between free and protein-bound drug molecules in the bulk aqueous medium. A good fit of theoretical accumulation curves to the experimental data was obtained. It was concluded that a mathematical simulation method makes it possible to predict the uptake characteristics of lipophilic anthracycline compounds into tumor cells under serum conditions.

ATPase activity of P-glycoprotein related to emergence of drug resistance in Ehrlich ascites tumor cell lines

We have characterized the ATPase activity of a sensitive and five progressively daunorubicin resistant Ehrlich ascites tumor cell lines passaged in mice. For the nine different modulators of drug resistance that we have studied, the ATPase activity first rose with the modulator concentration and then declined. We analyzed the ATPase activity profiles in terms of an activation constant and an inhibition constant for each of the nine drugs and six cell lines. In this series of cell lines, the drug-stimulatable ATPase activity was directly proportional to the amount of P-glycoprotein. Pumping of daunorubicin was also correlated with the amount of P-glycoprotein, except that, for a highly passaged line more daunorubicin was pumped than could be accounted for by the content of P-glycoprotein. Between the 12th and the 36th passage an additional source of resistance emerged, which was not correlated with P-glycoprotein. Pumping of daunorubicin was negatively correlated with the cell volume for the different lines.

Structure-cytotoxicity relationships of some helenanolide-type sesquiterpene lactones.

This study deals with the cytotoxicity of helenanolide-type (10 alpha-methylpseudoguaianolide) sesquiterpene lactones. We determined the influence of substitution patterns on the toxicity of 21 helenanolides to a cloned Ehrlich ascites tumor cell line, EN2. Within a series of helenalin esters, the acetate (2) and isobutyrate (3) were more toxic than helenalin itself (1). Esters with larger acyl groups (tiglate 4 and isovalerate 5) exhibited a decreased toxicity compared with the parent alcohol (1). Similar relationships were observed between the 6,8-diastereomer of helenalin, mexicanin I (6) and its acetate (7) and isovalerate (8). In contrast, cytotoxicity within a series of 11 alpha, 13-dihydrohelenalin esters (9-12) was shown to be directly related to the size and lipophilicity of the ester side chain, dihydrohelenalin (9) being the least toxic compound in this group. Investigation of several 2,3-dihydrohelenalin derivatives (13-21) with 2 alpha-hydroxy-4-oxo- and 2 alpha,4 alpha-dihydroxy- or -O-acyl-substituted cyclopentane rings (arnifolins and chamissonolides, respectively), for which no pharmacological data have been reported so far, revealed further interesting influences of the substitution pattern on cytotoxicity. The results may be interpreted in terms of lipophilicity and steric effects on the accessibility of the reactive sites considered responsible for biological activity.

Kinetics of daunorubicin transport in Ehrlich ascites tumor cells with different expression of P-glycoprotein: Influence of verapamil

The classical multidrug resistance (MDR) phenotype is characterized by a decrease in the intracellular drug concentration in resistant cells as compared to sensitive cells. P-glycoprotein (P-gp) is thought to be responsible for an active efflux of lipophilic drugs. Four Ehrlich ascites tumor cell lines selected in vivo for resistance to daunorubicin (DNR) and their sensitive counterpart were investigated. The resistant sublines EHR2/0.1, EHR2/0.2, EHR2/0.4, and EHR2/0.8 were developed by treatment of tumor bearing mice with DNR 0.1, 0.2, 0.4, and 0.8 mg/kg × 4 weekly, respectively. One passage from EHR2/0.1, EHR2/0.2, and EHR2/0.4 and two passages from EHR2/0.8 were investigated. Western blot analysis showed significantly different amounts of P-gp (a 6-fold variation). Efflux of DNR in a drug free medium was investigated with and without presence of verapamil (VER). Efflux from sensitive and resistant cells was described by mono- and bi-exponential kinetics, respectively. In all cases but one, a correlation between resistance, expression of P-gp, P-gp mediated efflux capacity and effect of VER was established. In passage No. 12 of EHR2/0.8, however, a high expression of P-gp was found in spite of a low degree of resistance and a low efflux capacity. In this subline the effect of VER did not correlate to the expression of P-gp. Active efflux seemed to be saturable and was suggested to constitute the major route of efflux in MDR cells. A dose-response relationship was established for the effect of VER on efflux. In conclusion, the results support that P-gp acts as a drug efflux pump. No simple correlation, however, could be established between P-gp and drug transport in all the investigated cell lines. Other factors which might influence transmembranous transportation of DNR are suggested. The active efflux capacity of the cell lines seemed to determine the degree of resistance and the sensitivity to circumvention by VER.

The effect of 9,11-secosterol, a newly discovered compound from the soft coral Gersemia fruticosa, on the growth and cell cycle progression of various tumor cells in culture

A new 9,11-secosterol, 24-nor-9,11-seco-11-acetoxy-3β,6α-dihydroxycholest-7,22(E)-dien-9-one, was found to exhibit growth inhibitory (IC 50 below 10 μM) and cytotoxic activities against human leukemia K562, human cervical cancer HeLa, and Ehrlich ascites tumor cells in vitro. The cytostatic concentrations of the compound generally caused the G 2/M block in the cell cycle progression, but differences between the three tumor cell lines in the events leading to cell death were remarkable. While inhibiting cell proliferation, 9,11-secosterol caused accumulation of HeLa and K562 cells in the metaphase of mitosis. So, abnormal mitosis can play an important role in the cytotoxicity of 9,11-secosterol in these cell lines. In the Ehrlich ascites tumor cell line the increasing concentrations of the drug (up to 40 μM) did not cause an immediate cell killing. Instead, due to continued DNA synthesis without entry into mitosis, cells with high DNA ploidy were produced. It was shown that the cytoskeletal systems such as microtubules and microfilaments were not damaged by the action of 9,11-secosterol. Further studies are necessary to elucidate the mechanism of the cytotoxic effect of 9,11-secosterol. (Steroids 59: 274–281, 1994)

Differences in cell surface carbohydrates, and in laminin and fibronectin synthesis, between adherent and non-adherent Ehrlich ascites tumor cells.

Differences in cell surface carbohydrates and in laminin and fibronectin synthesis between 2 Ehrlich ascites tumor (EAT) cell lines, the adherent and non-adherent EAT cells, have been studied. The adherent EAT (a-EAT) cells grow in monolayer in vitro in the presence of fetal bovine serum. The classical, or non-adherent EAT (na-EAT), cells grow in suspension in ascites form in the peritoneal cavity of mice, and they do not adhere when cultured in vitro. Both EAT cell lines express surface glycoproteins reactive with Maackia amurensis lectin (MAL) and Griffonia simplicifolia isolectin GS I-B4. However, only a-EAT cells react with elderberry (Sambucus nigra) bark lectin (SNA), suggesting that there are some differences in the sialylation of cell surface carbohydrate moieties between these 2 EAT cell lines. Removal of cell surface sialic acid by treating a-EAT cells with Vibrio cholerae neuraminidase did not abolish the ability of these cells to adhere to laminin- or fibronectin-coated plates, indicating that the sialic acid of the cell surface glycoproteins is not essential for their adhesion to these extracellular matrices. Therefore, the difference in sialylation of cell surface glycoproteins is not responsible for the difference in cell adhesion between these 2 lines of EAT cells. Both EAT cell lines express detectable amounts of laminin but not fibronectin on their surfaces; they both secrete fibronectin and entactin into the medium. The na-EAT cells (but not the a-EAT cells) also secrete laminin A chain into the culture medium; however, no B chain was detected in the culture medium of either cell line. The laminin isolated from the cell surface of na-EAT cells reacts with GS I and MAL lectins, but not with SNA, whereas the laminin isolated from a-EAT cells reacts with SNA, as well as GS I and MAL.

Subcellular Distribution of Adenine Nucleotides in Two Ehrlich Cell Lines Metabolizing Glucose

Adenine nucleotide compartmentation and cytochrome redox state were studied in two Ehrlich ascites tumor cell lines incubated in the presence of 5mM glucose to show differences between their energy metabolisms. Changes in the subcellular distribution of adenine nucleotides were very different in both cell lines. Glucose seemed to energize the less malignant cell line, as shown by the asymmetry of the ATP/ADP ratios between cytosolic and mitochondrial compartments, but this was not the case for the more malignant cell line. The cytochrome contents were similar in both cell lines, but the degree of cytochrome oxidation was higher in the less malignant cell line; this result is in agreement with a higher oxygen consumption in the less malignant cell line.

Analysis of X-ray induced aberrations in mammalian chromosomes by electrofusion induced premature chromosome condensation.

Premature chromosome condensation (PCC) was induced by electrofusion of metaphase cells of an Ehrlich ascites tumor cell line with interphase cells of a Muntjac cell line or of a Chinese Hamster subline. Electrofusion was performed by cell alignment in a weakly inhomogeneous a.c. field of 200 V/cm amplitude (peak-to-peak value) and of 1.7 MHz frequency, followed by the application of a series of breakdown (fusion) pulses of 5 kV/cm strength and 15 microseconds duration. Most of the PCC's were of the G2 type despite the large proportion of G1 and S cells in the suspension. The number of chromatid aberrations observed in electrofused cells which had not been subjected to irradiation was not significantly above the spontaneous level. This indicates that electrofusion, at least as used here, did not lead to lesions expressed as structural aberrations. When interphase cells were irradiated by X-ray doses below 3 Gy before electrofusion PCC analysis showed chromosome damage consisting mainly of breaks and gaps. The frequency of aberrations recorded by PCC was 6 to 40 fold larger than that seen in conventional metaphase analysis. This large increase probably arose because of an effective suppression of the G2 repair of chromosomal lesions by the fast condensation process which took place within about 30 min. This assumption was supported by PCC experiments in which the time between X-irradiation and fusion with subsequent chromosome condensation was varied. The results demonstrated that G2 repair of chromosomal lesions was not detectable until 20 min after fusion with a half-time of the repair kinetics of about 1.5 h. The selectivity of premature chromosome condensation in G2 cells is discussed in terms of the differences between electrofusion and chemically or virally induced fusion. It is assumed that the concentration and the transfer rate of the chromosome condensation factor from the metaphase to the interphase cell are the limiting factors in achieving PCC. This is because the localised permeabilisation of the membrane and the dominance of two-cell fusions are characteristic of electrofusion.

Detection of DNA cross-links in tumor cells with the ethidium bromide fluorescence assay.

Until now the fluorescence assay with ethidium bromide has only been used on pure DNA. This assay depends on the difference in fluorescence between single- and double-stranded DNA (dsDNA). Cross-links in DNA are measured by the return of fluorescence of dsDNA after heat denaturation at pH 12. Under these conditions denatured DNA gives very low fluorescence. In the present study this assay was applied to tumor cells. The mouse Ehrlich ascites tumor cell line (EAT) and a human small-cell carcinoma line (GLC4) were incubated for 4 hr at 37 degrees C, with the cross-linking agent cis-diamino-dichloro platinum (cDDP). The samples of whole cells were thereafter resuspended in potassium phosphate buffer with 10 mM EDTA, 4M NaCl, 0.1% Sarkosyl pH 7.2, for 16 hr at 37 degrees C. Measurements were performed with a spectrofluorometer with excitation wavelength 525 nm, emission wavelength 580 nm. There was a linear relationship for cDDP concentrations of 0-150 microM and the extent of DNA cross-links in EAT (r = 0.958). In GLC4 there was a linear relationship at low cDDP concentrations of 0-50 microM (r = 0.968) while between 50 and 150 microM a plateau was reached. RNase added to the lysate of whole cells had no influence on the extent of cross-links. This assay was compared with the alkaline elution assay, and results were identical.

Cellular retinol-binding protein

1. 1. A protein which binds retinol in vitro with high affinity and specificity was detected by sucrose gradient centrifugation or by gel filtration after preincubating rat tissue cytosols with all- trans-[ 3H]retinol. This protein sediments in the 2 S region of sucrose gradients. Molecular size determination by gel filtration indicates a molecular weight of 16 000. 2. 2. Competition studies revealed that only all- trans-retinol, not retinal or retinoic acid, competes for binding. The binding of radioactive retinol is reversible. 3. 3. This protein was detected in cytosols of rat liver, lung, spleen, brain, testis, ovaries, uterus and intestinal mucosa whereas heart or gastrocnemius muscle seem to lack this protein. 4. 4. The cellular retinol binding protein was found in fetuses as early as day 12 of the gestation period and possessed the same specifity for the ligand as the one in adult tissues. 5. 5. This binding component was not detected in cytosols prepared from Novikoff hepatoma, ascites hepatoma AS-30D, mouse Ehrlich ascites tumor and mouse pituitary tumor cell line AtT 20. 6. 6. The cellular retinol binding protein seems to be different from that described to be present in the serum as suggested by difference in size and by the inability of the antisera against the serum retinol binding protein to remove the cellular binding protein from the cytosol preparations.

The response of glycolytic and non-glycolytic mammalian cells to the inhibitory action of fluoropyruvate

A study on the effect of fluoropyruvate on the respiration of various mammalian tissues is reported. 1. 1. The endogenous respiration of chorioallantoic membranes, freshly prepared chick embryo cells, chick fibroblasts grown in tissue culture, Ehrlich tumor and a rat sarcoma cell line (PF line) are inhibited by small amounts of fluoropyruvate. 2. 2. In the presence of glucose, fructose or mannose the inhibition is abolished in cells which manifest a high aerobic glycolysis (chick fibroblasts, Ehrlich ascites tumor and PF cell line). Cells of low glycolytic activity (chorioallantoic membrane and chick embryo cells) are equally inhibited in the presence and in the absence of these sugars. 3. 3. The protective action of the sugars against inhibition by fluoropyruvate is tentatively explained by a rapid reduction of the inhibitor to fluorolactate by the glycolytic system of the cells. 4. 4. The use of fluoropyruvate for the selection of cell lines with high glycolytic activity is suggested.