Murine Leukemia Cell Line L1210 Research Articles

Establishment of Transgenic Mouse Leukemia Cell Lines Expressing Human CD4/CCR5/CyclinT1 Infected with HIV-1.

Establishing a cross-species animal model of human immunodeficiency virus (HIV) infection is crucial for the study of HIV/acquired immunodeficiency syndrome (AIDS). However, due to the species-specific characteristics of HIV-1, the virus can only infect directly humans and a small number of non-human primates. It cannot directly infect mouse cells across species. A mouse leukemia cell line with high CD4 (clusters of differentiation 4)/CCR5 (CC-chemokine receptor 5)/CyclinT1 expression was constructed in this study. First, CD4/CCR5/CyclinT1 lentiviral vector was used to infect a murine leukemia cell line L1210 to express the receptor CD4, co-receptor CCR5 and tat protein driving factor CyclinT1, which are required to infect L1210 cells with HIV-1. The results of sequencing identification and fluorescence expression showed that the plasmid expressing CD4, CCR5, and CyclinT1 vector was successfully constructed and wrapped as the lentiviral vector. Moreover, it was observed that CD4, CCR5, and CyclinT1 proteins were highly expressed in mouse leukemia cells L1210 compared to empty lentiviral vector-transfected cells. Next, viral entry and replication were demonstrated when HIV-1 RNA was detected in body cells and cultured supernatants. Transgenic mice cells L1210 showed significantly greater content of HIV-1 RNA compared to control L1210 cells. Finally, CEMx174 was infected with cell culture supernatants to clarify that the progeny virus is an active virus with infection ability. HIV-1 RNA was highly expressed in CEMx174 cells. This study made the foundation for future studies evaluating HIV-1 cross-species infection in a murine animal model. The results provided new direction for studies investigating the development of vaccines, antiviral drugs screening, and HIV/AIDS pathogenesis.

In vitro evaluation of immunobiological activity of simple mannolipids

Immunomodulation, cytotoxicity and anti-cancer activity of selected amphiphilic non-ionic (thio)alkyl α-D-mannosides (with aglycone of C6-C12) were investigated in vitro in human cervix epitheloid carcinoma cell line HeLa, murine melanoma cancer cells B16, murine lymphocytic leukemia cell line L1210, murine fibroblast cell line NIH 3 T3 and murine macrophage cell line RAW 264.7. Toxicological studies revealed structure-dependent immunobiological effectivity based on a tight interaction with relevant cells. The results demonstrated diverse immunomodulation of macrophage cell-line RAW264.7 proliferation and production of Th1 and Th2 cytokines, and induction of pro-inflammatory interleukins IL-1α, TNFα, IL-6, IL-12 and IL-17 and anti-inflammatory IL-10 following (thio)alkyl α-D-mannosides 24 and 48 h exposure. Direct application of alkyl mannosides MOC10 and MOC12 and their thio analogues MSC10 and MSC12 in reconstructed human EpiDerm™ and MOC12 and MSC12 in EpiOcular™ model assays for dermal and ocular irritation together with quantification of human proinflammatory cytokines IL-1α, TNFα, IL-6 and IL-8 culture media release was used to ascertain toxicological safety.

Activity Trends in Desoxy Anthrapyrazoles: The Influence of Molar Volume, Polarizability and Lipophilicity of N<sub>2</sub> C<sub>5</sub> Side Chains on Their Anticancer Response

QSAR methodology was used to assess the effects of lipophilicity (logP), molar volume (MV) and polarizability (pl) of the side chains at N2 and C5 of 20 known desoxy anthrapyrazoles on their in vitro anticancer activity expressed as the negative logarithm of the inhibitory concentration of 50% of L1210 murine leukemia cell line (1/logIC50). The main data set shows poor correlations between biological response and the descriptors with exception of MV of the C5 side chain, where a moderate correlation was discerned ( =0.60, n = 18, two outliers). To extract more information regarding mechanism, the main data set was visually classified to three clusters depending on N2 side chain. Cluster 1 containing six 5-substituted 2-[(2-hydroxyethyl) amino] ethyl anthrapyrazoles; cluster 2 contains ten 5-subsitutes 2-(diethyl amino) ethyl anthrapyrazoles and cluster 3 contains four anthrapyrazoles with miscellaneous substituents at both N2 and C5. For cluster 1, MV and pl of C5 show high correlation with biological response (R2’s = 0.75 and 0.72 respectively) while logP gives a weak correlation (R2 = 0.44). For cluster 2, the correlations of logP and pl of C2side chain are higher (=0.66 and 0.62 respectively) compared with MV (=0.16). Cluster 3 shows very poor correlation with all descriptors (~0.3). This indicates mechanistic distinction between the three clusters. Derived descriptors which represent the difference between the descriptors of N2 and C5 side chains where used to explore the presence of interplay between these descriptors in affecting variability of the biological response.

Ma’edamines C and D, New Bromotyrosine Alkaloids Possessing a Unique Tetrasubstituted Pyridinium Moiety from an Okinawan Marine Sponge Suberea sp.

Two new bromotyrosine alkaloids, ma'edamines C and D, were isolated from an Okinawan marine sponge Suberea sp. The structures of ma'edamines C and D were elucidated on the basis of spectroscopic data. Ma'edamines C and D were the first natural products possessing a unique tetrasubstituted pyridinium moiety such as N-alkyl-3,5-diethyl-2-propylpyridinium and N-alkyl-3,5-diethyl-4-propylpyridinium, respectively. Ma'edamines C and D exhibited moderate cytotoxicity against murine leukemia cell line L1210 in vitro.

Characterization of the novel 4-thiazolidinone derivatives: luminescence spectra and potential antitumor activity

This study was aimed at measurement of the transmission, luminescence spectra, and luminescence excitation spectra of three novel anticancer 4-thiazolidinone derivatives (ID 3288, ID 3882, ID 3833) in order to characterize the photoinduced cytotoxicity of these compounds against murineleukemia L1210 cell line. The luminescence and luminescence excitation spectra of the ID 3288, ID 3882 and ID 3833 powders differed significantly from those measured in aqueous solution. Furthermore, the luminescence of the ID 3288 powder had lower intensity as compared to that of the ID 3882 and ID 3833 powders. The luminescence of the ID 3882 aqueous solution showed the lowest intensity, while the ID 3833 demonstrated the highest intensity. The potential of application of the obtained results in the photodynamic anticancer chemotherapy has been discussed.

Synthesis and anticancer evaluation of benzyloxyurea derivatives.

A series of novel benzyloxyurea derivatives was designed, synthesized by substituting different benzyls or phenyls on N,N'-positions of the hydroxyurea (HU). These target compounds were evaluated for their anticancer activity in vitro against human leukemia cell line K562 and murine leukemia cell line L1210 in comparison with HU by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Some of the compounds showed promising anticancer activity against the cells. Molecular docking experiments with Saccharomyces cerevisiae R1 domain indicated that 4a and 4f' have stronger affinity than 4m and 4n. Flow cytometry study showed that compound 4g exerted greater apoptotic activity against K562 cells line than HU.

Synthesis, spectroscopic characterization, in vitro cytotoxic and structure activity relationships of some mononuclear Ru(II) complexes

New mononuclear Ru(II) complexes [Ru(A)2(B)]2+, where A = 2,2′-bipyridine/1,10-phenanthroline and B = 3,4,5-tri-OCH3-DPC, 4-CH3-DPC, 4-N(CH3)2-DPC, 4-NO2-DPC, N-BITSZ, PTSZ and PINH, were prepared and characterized by spectroscopic methods. The in vitro cytotoxic activities of the complexes and their corresponding ligands were investigated against the human cancer T-lymphocyte cell lines molt 4/c8 and CEM and the murine tumor leukemia cell line L1210, human promyelocytic leukemia cells (HL-60) and Bel-7402 liver cancer cells by MTT assay. The complexes [Ru(A)2(B)]2+ (A = 1,10-phenanthroline, B = 3,4,5-tri-OCH3-DPC) exerts rather more potent activities against all of these cell lines, especially for CEM and L1210. Ru complexes and structure–activity relationships and anticancer mechanisms are also discussed.

Solvolysis and cytotoxicity of dinuclear ruthenium(II)-2,2′-bipyridine complexes with various bridged-ligands

In order to reveal the solvolysis reactions of dinuclear ruthenium(II)-2,2′-bipyridine complexes with various bridged-ligands (BL), [{RuCl(bpy)2}2(μ-BL)]2+ ([Ru2Cl2-BL]2+), we have studied the kinetics of these complexes in H2O and H2O–dmso (1:1 v/v). In H2O–dmso (1:1 v/v), time-course UV–Vis absorption and IR spectra indicate that the labile Cl− ligands of [Ru2Cl2-BL]2+ are successively substituted by H2O followed by dmso, [{Ru(dmso-S)(bpy)2}2(μ-BL)]4+. Cytotoxicity assays of dinuclear ruthenium(II) complexes were performed in L1210 murine leukemia cell line. [Ru2Cl2-BL]2+ complexes exhibit much lower cytotoxicity compare to cisplatin. However, [{Ru(dmso-S)(bpy)2}2(μ-BL)]4+ complexes produced in H2O–dmso are found to much more cytotoxic than [Ru2Cl2-BL]2+ and in particular the cytotoxicity of [{Ru(dmso-S)(bpy)2}2(μ-dado)]4+ (dado=1,12-diaminododecane) is comparable to that of cisplatin.

DNA damage induction and antiproliferative activity of vanadium(V) oxido monoperoxido complex containing two bidentate heteroligands

Several peroxidovanadium(V) complexes have been shown as a potent anticancer agents. The aim of this study was to investigate the interaction of monoperoxidovanadium(V) complex Pr4N[VO(O2)(ox)(phen)], (Vphen), [phen=1,10-phenantroline, ox=oxalate(2−) and Pr4N=tetra(n-propyl)ammonium(1+)] with DNA. UV–Vis spectrophotometry and the alkaline single-cell gel electrophoresis (SCGE, the comet assay) were used to examine the possibility of the vanadium(V) complex to induce changes in DNA. The interaction of Vphen with calf thymus DNA resulted in absorption hyperchromicity in DNA spectrum and shift of the absorption band of DNA to longer wavelengths for the [complex]/[DNA] concentration ratio equals to 4 and after 60min of incubation. The rise in DNA absorption (by 34%) and bathochromic shift (Δλmax=6nm) are indicative of the interaction between DNA and the complex molecules. DNA strand breaks in cellular DNA were investigated using the comet assay. The human lymphocytes were exposed to various concentrations of Vphen for 30min. The results revealed that Vphen contributed to the DNA damage expressed as DNA strand breaks in concentration dependent manner. The used concentrations of Vphen (ranging from 0.1 to 100μmol/L) caused higher DNA damage in lymphocytes compared to untreated cells (from 1.2 times for 0.1μmol/L to 1.8 times for 100μmol/L). Vphen was screened for its potential antitumor activity towards murine leukemia cell line L1210. Vphen exhibited significant antiproliferative activity depending on its concentration and time of exposure. The IC50 values were 0.247μg/mL (0.45μmol/L) for 24h, 0.671μg/mL (1.21μmol/L) for 48h and 0.627μg/mL (1.13μmol/L) for 72h.

Study of interrelations between resistance to cisplatin and composition of low molecular weight thiols in murine leukemia L1210 cell lines

Cis -dichlorodiammineplatinum (II), commonly known as cisplatin, is a widely used anticancer drug. However, the therapeutic efficacy of the drug is limited due to various dose-limiting side effects and development of acquired resistance. The resistance of malignant cells to cisplatin may be connected to elevated concentration of cellular thiols. To verify this hypotheses, we compared the concentrations of low weight cellular thiols, metallothionein (MT) and glutathione (reduced, GSH and oxidized, GSSG) and also the metal-binding properties of MT in the two cell lines, known to be resistant (L1210R) or sensitive (L1210S) to cisplatin. It was established that R-cells were characterised by hi­gher concentrations of MTs and GSH, higher ratio of copper in the composition of MTs in comparison to zinc than S-cells. The treatment by cisplatin provoked the decrease of the GSSG concentration only in the S-cells without the changes in GSH concentration. Hence, R-cells insensitivity to cisplatin can be related to higher levels of MTs and GSH. Reported for the first time peculiar metal composition of MTs in R-cells can also give an advance to these cells by particular oxidizing properties of copper-MT. These specific characteristics of thiols in malignant cells could be useful for the indication of cisplatin resistance. Keywords: cisplatin, resistanse, metallothionein, glutathione, copper.

Copper(II) and Palladium(II) Complexes with Cytotoxic and Antibacterial Activity

The synthesis of eight square pyramidal copper complexes with general structure [Cu(IL)(AL)H2O]2+, where IL represents various methylated 1,10-phenanthrolines, and AL represents either 1S,2S- or 1R,2R-diaminocyclohexane, is reported, with the complexes synthesised as both the perchlorate and chloride salts. The crystal structures of [Cu(1,10-phenanthroline)(1S,2S-diaminocyclohexane](ClO4)2·H2O and [Cu(5,6-dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane](ClO4)2·1.5H2O are reported. Four square planar palladium complexes with general structure [Pd(IL)(AL)]Cl2 have also been synthesised. These complexes were synthesised in order to investigate the structure–activity relationship against both cancer cell lines and bacterial cultures. The copper complexes display anticancer activity similar to cisplatin and 1,10-phenanthroline (phen) in the L1210 murine leukaemia cell line. Methylation of the phen increased the copper complex cytotoxicity by approximately four-fold, compared with the non-methylated complex. No significant difference in activity was observed by altering the chirality of the diaminocyclohexane ligand. The copper complexes demonstrated antibacterial activity against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli; however, high levels of toxicity (30–60 % of death) were observed in the nematode Caenorhabditis elegans. The copper complexes have also been shown to act as DNA nucleases, with the ability to cleave plasmid DNA in the presence of hydrogen peroxide. The palladium complexes all have half maximal inhibitory concentration (IC50) values of ~10 μM in the L1210 cell line, with no significant difference in the cytotoxicity of any of the compounds tested. Minimal antibacterial activity of the palladium complexes was observed.

ChemInform Abstract: Design, Synthesis, and Cytotoxic Activity Evaluation of New Linear Pyranoxanthone Aminoderivatives.

AbstractThe hydrochlorides of the compounds (IV), the free amines (VIII) and (XIIIa), and the difumarate of the compound (XIIIb) are screened for their in vitro cytotoxicity against the murine leukemia L1210 cell line, human colorectal adenocarcinoma (HT‐29), and human uterine sarcoma (MES‐SA and the multidrug resistant variant MES‐SA/Dx5) cell lines in comparison to the reference drugs mitoxantrone and doxorubicin.

Synthesis, nucleic acid binding and cytotoxicity of oligonuclear ruthenium complexes containing labile ligands

We report the synthesis, nucleic acid binding and cytotoxicity of the complexes [Ru(terpy)(Me2bpy)Cl]+, [Ru(terpy)(phen)Cl]+ and dinuclear [{Ru(terpy)Cl}2(μ-bbn)]2+ {where Me2bpy = 4,4′-dimethyl-2,2′-bipyridine; phen = 1,10-phenanthroline; and bbn = bis[4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane, with n = 7, 10, 12, 14}. The complexes were isolated from the reaction of the [Ru(terpy)Cl3] precursor with the respective bidentate and di-bidentate bridging ligands. The time-course UV–Visible spectroscopy of the reaction of the mono- and dinuclear complexes with guanosine 5-monophosphate (GMP) showed the movement of the metal-to-ligand charge transfer (MLCT) band to lower wavelengths, accompanied by a hypochromism effect. The formation of the aqua complex and phosphate-bound intermediates in the reaction were detected by the time-course 1H NMR and 31P NMR experiments, which also demonstrated that the complex bound to the N7 guanine was the major product. The UV–Visible and 1H NMR studies showed no evidence of the interaction of the complexes with both adenosine 5-monophosphate (AMP) and cytidine 5-monophosphate (CMP). Cytotoxicity studies of these complexes against a murine leukemia L1210 cell line revealed that the dinuclear [{Ru(terpy)Cl}2(μ-bbn)]2+ complexes were significantly more cytotoxic than mononuclear [Ru(terpy)(Me2bpy)Cl]+. The [{Ru(terpy)Cl}2(μ-bb14)]2+ complex appeared to be the most active (IC50 = 4.2 μM).

Pro-oxidant and antiproliferative effects of the 1,3,4-thiadiazole–based Schiff base and its metal complexes

Adverse biological activities of Schiff base (SB) derivatives are well known. In this study, the ligand and its metal complexes have been synthesized and characterized by IR, 1H-NMR spectra, elemental analyses, magnetic susceptibility, UV-Vis spectra, and thermogravimetry/differential thermal analysis. From the elemental analyses data, the complexes were proposed to have the general formula [Mn(L)2(H2O)2], [Co(L)2(H2O)2], and [Ni2(L)(H2O)4(Cl)3]. From the magnetic moment and UV-Vis spectra data, it was found that the geometrical structures of these complexes are octahedral. In the in vivo experiment, adult male rats were injected subcutaneously with a new SB (L) and its [Mn(L)2(H2O)2], [Co(L)2(H2O)2], and [Ni2(L)(H2O)4(Cl)3] complexes (25 mg/kg body weight) and were then sacrificed 16 days later. Effects of these compounds on serum antioxidant vitamins (i.e., vitamins A, E, and C) and malondialdehyde (MDA) levels were measured in blood serum, liver, and kidney tissues. In an in vitro experiment, antiproliferative effects of these compounds were assessed on the human breast carcinoma MCF-7 and murine leukemia L1210 cell lines. Serum MDA and vitamins A, E, and C levels did not change by the treatments. However, in the kidney and liver tissues, MDA levels were higher, whereas vitamin levels were lower in treatment groups, compared to the control group. All compounds inhibited cell proliferation of MCF-7 and L1210 cancer cell lines in a dose- and time-dependent manner. In conclusion, SB derivatives tested in the current study induced oxidative stress in vivo and exhibited anti-proliferative effects in an in vitro culture system.

Design, synthesis, and cytotoxic activity evaluation of new linear pyranoxanthone aminoderivatives

AbstractWith the aim of enlightening some structure‐activity correlation within the pyranoxanthenone series, we have designed and synthesized a number of new 5‐aminosubstituted pyrano[3,2‐b]xanthen‐6‐ones bearing various 12‐substituents. In vitro cytotoxic potencies of the new derivatives toward the murine leukemia L1210 cell line, human colorectal adenocarcinoma (HT‐29), and human uterine sarcoma (MES‐SA and its 100‐fold resistant to doxorubicin variant MES‐SA/Dx5) cell lines, are described and compared with that of reference drugs. Among the studied compounds, those possessing a second aminosubstituted side‐chain exhibit interesting cytotoxic activity against the solid tumor cell lines, and they retain activity against the multidrug resistant MES‐SA/Dx5 subline. Their selective effect on a phase of the cell cycle was evaluated using HT‐29 cells providing evidence that the compounds induce a G0/G1 arrest. J. Heterocyclic Chem., (2011).

Abstract 1720: Tyrosine phosphorylation of HSC70 located in the cell membrane may regulate methotrexate transportation in murine L1210 leukemia cells

Abstract Bhushan and co-workers previously described an alteration in an unidentified 66-68 kDa membrane-associated, tyrosine-phosphorylated, methotrexate (MTX)-binding protein in the L1210 murine leukemia cell line. This protein was absent, at least in the tyrosine phosphorylated form, in cisplatin-resistant, methotrexate cross-resistant L1210/DDP cells. The 66-68 kDa protein was isolated by affinity chromatography, first on MTX-agarose followed by purification using an anti-phosphotyrosine immunoaffinity column. Amino acid sequence analysis identified the protein as heat shock cognate protein 70 encoded by the HSPa8 gene. The HSPa8 gene was found to be transcribed in both sensitive and resistant cell lines by RT-PCR and the sequence of the HSPa8 gene was identical in both cell lines. Western blot analysis showed that HSC70 was also expressed in both cell lines. A binding pull-down assay with MTX-agarose beads, followed by western blotting for HSC70, showed HSC70 to be present in cell lysates and in the MTX-binding fraction. Fractionation of cell homogenates into membrane and cytosolic fractions showed that HSC70 was found in both fractions but was not detected in the MTX-binding assay with the membrane fraction from the L1210/DDP cells, suggesting HSC70 located in the membrane fraction may be controlling MTX transport in the sensitive cells. Comparing the tyrosine phosphorylation status between L1210/0 and L1210/DDP cells, a band around 70 kDa was observed in the membrane fraction of L1210/0 cell but was not present in the L1210/DDP cells. Additionally, immunoprecipitation with anti-phosphtyrosine (PY20) agarose beads and detection of HSC70 by western blot showed little to no PY-HSC70 in the membrane fraction from the L1210/0 cells. Thse results suggest that HSC70 is a putative MTX binding protein, and HSC70 associated with the cell membrane may regulate methotrexate uptake. Tyrosine phosphorylation may contribute to the function of HSC70 in the cross-resistance mechanism in the murine L1210 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1720. doi:10.1158/1538-7445.AM2011-1720

Mechanism of Cytotoxicity and Cellular Uptake of Lipophilic Inert Dinuclear Polypyridylruthenium(II) Complexes

The accumulation, uptake mechanism, cytotoxicity, cellular localisation of-and mode of cell death induced by-dinuclear ruthenium(II) complexes ΔΔ/ΛΛ-[{Ru(phen)(2) }(2) {μ-bb(n) }](4+) (Rubb(n)), where phen is 1,10-phenanthroline, bb(n) is bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n=2, 5, 7, 10, 12 or 16), and the corresponding mononuclear complexes containing the bb(n) ligands, were studied in L1210 murine leukaemia cells. Cytotoxicity increased with linker chain length, and the ΔΔ-Rubb(16) complex displayed the highest cytotoxicity of the series, with an IC(50) value of 5 μM, similar to that of carboplatin in the L1210 murine leukaemia cell line. Confocal microscopy and flow cytometry studies indicated that the complexes accumulate in the mitochondria of L1210 cells, with the magnitude of cellular uptake and accumulation increasing with linking chain length in the bb(n) bridge of the metal complex. ΔΔ-Rubb(16) entered the L1210 cells by passive diffusion (with a minor contribution from protein-mediated active transport), inducing cell death via apoptosis. Additionally, metal-complex uptake in leukaemia cells was approximately 16-times that observed in healthy B cells highlighting that the bb(n) series of complexes may have potential as selective anticancer drugs.

Synthesis, biological evaluation and docking studies of 4-amino-tetrahydroquinazolino[3,2- e]purine derivatives

The synthesis of new 4-amino-tetrahydroquinazolino[3,2- e]purine derivatives along with their activity in cell-free enzymatic assays on Src is reported. Some compounds emerged as moderately active inhibitors of the enzyme and showed antiproliferative effects on the murine leukemia L1210 cell line. Docking studies have been also performed to analyze the binding mode of compounds under study and to identify the structural determinants of their interaction. Therefore, this study provides a new promising scaffold with moderate enzymatic inhibitory activities for further development of new anticancer drugs targeting Src tyrosine kinase.

Purification and Characterization of a Laccase with Inhibitory Activity Toward HIV-1 Reverse Transcriptase and Tumor Cells from an Edible Mushroom (Pleurotus cornucopiae)

A 66-kDa laccase, with an N-terminal sequence different from those of other mushroom laccases, was purified from fresh fruiting bodies of the edible mushroom Pleurotus cornucopiae by using affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Mono Q and gel filtration on Superdex 75. The procedure resulted in a 16-fold purification and a specific enzyme activity of 17.3 U mg(-1). The optimum pH and temperature for the purified laccase were pH 4 and 40 degrees C, respectively. This laccase inhibited proliferation of murine leukemia cell line L1210 and human hepatoma cell line HepG2, and reduced the activity of HIV-1 reverse transcriptase with an IC50 of 22 microM. There was neither mitogenic activity toward mouse splenocytes, nor hemagglutinating/hemolytic activity toward rabbit erythrocytes. This study yielded information about the potentially exploitable activities of P. cornucopiae laccase.

Effect of berberine on proliferation, cell cycle and apoptosis in HeLa and L1210 cells.

Previous studies on the anticancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human tumour HeLa and murine leukemia L1210 cell lines. An attempt was also made to investigate the relationship between the cytotoxic activity of berberine and its molecular mechanism of action. Cytotoxicity was measured in-vitro using a primary biochemical screening according to Oyama and Eagle, and the growth inhibition assay. The in-vitro cytotoxic techniques were complemented by cell cycle analysis and determination of apoptotic DNA fragmentation in L1210 cells. Berberine acted cytotoxically on both tumour cell lines. The sensitivity of leukemia L1210 cells to the berberine was higher than that of HeLa cells. The IC(100) was below 100 microg mL(-1) for HeLa cells and approached a 10 microg mL(-1) limit for the leukemia L1210 cells. For both cell lines the IC(50) was found to be less than 4 microg mL(-1), a limit put forward by the National Cancer Institute (NCI) for classification of the compound as a potential anticancer drug. In L1210 cells treated with 10-50 microg mL(-1) berberine, G(0)/G(1) cell cycle arrest was observed. Furthermore, a concentration-dependent decrease of cells in S phase and increase in G(2)/M phase was detected. In addition, apoptosis detected as sub-G(0) cell population in cell cycle measurement was proved in 25-100 microg mL(-1) berberine-treated cells by monitoring the apoptotic DNA fragmentation (DNA ladder) using agarose gel electrophoresis.