Does transduced p27 induce apoptosis in human tumor cell lines?

p27 is a cyclin-dependent kinase inhibitor involved in the negative regulation of G1 progression in response to a number of antiproliferative signals. In this study, we examined the transduction of full-length Tat-p27, pt-mutated Tat-p27, and N'- Tat-p27 (truncated p27 on the C-terminal end) fusion proteins into human tumor cell lines and whether these transduced proteins induced apoptosis in the cells. Protein transduction can be described as the direct uptake by the cell of exogenous proteins/peptides as a result of a specific property of the protein/peptide component. The basic domain of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) protein possesses the ability to traverse biological membranes efficiently in a process termed protein transduction. Although the mechanism is unknown, transduction occurs in receptor/transporter-independent manner that appears to target the lipid bilayer directly. Thus, HIV-1 Tat proteins have tremendous potential to deliver large-sized compounds into the cells. Transduction of TAT-fusion proteins affected the proliferation of human tumor cell lines, depending on the type of protein and cell line. By Western blot analysis it was shown that some cell cycle regulatory proteins were affected, and that some proteins were responsible for the induction of apoptosis.

Synthesis, X-ray crystal structure study, and cytostatic and antiviral evaluation of the novel cycloalkyl-N-aryl-hydroxamic acids.

In vitro evaluation of the novel cycloalkyl-N-(4-chlorophenyl)-hydroxamic acids (2a-g) demonstrated that 2b,d,e exhibited rather marked inhibitory activity (IC50 = 7-10 microM) against pancreatic carcinoma, 2b-d against colon carcinoma, 2d against laryngeal carcinoma, and 2b,d against breast carcinoma. 2e showed the most pronounced anti-cytomegalovirus activity (EC50 = 1.5 and 0.8 microg mL(-1)) only at > or = 5-fold lower than the cytotoxic concentration. 2d and 2f showed modest, albeit selective, activity against cytomegalovirus (2d, EC50 = 7.3-8.9 microg mL(-1), selectivity index 7-10; 2f, EC50 = 7-13 microg mL(-1), selectivity index 10).

Sensitivity of B-cell chronic lymphocytic leukemia to Rituximab and Campath-1H and correlation with the expression of cell cycle regulatory proteins.

AIM: To assess the effect of monoclonal antibodies anti-CD20 (Rituximab) and anti-CD52 (Campath-1H) on the viability of B cells from patients with B cell chronic lymphocytic leukemia (B-CLL) in comparison with a cytotoxic drug fludarabine (Fluda), and to determine the influence of these agents on the expression of cell cycle regulatory proteins in vitro. METHODS: B-CLL cells were incubated in vitro in the presence of Rituximab, Campath-1H, and Fluda. The viability of the cells was measured by MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyltetrazolium bromide). Gel electrophoresis and Western blotting were used to determine the effect of these agents on the expression of cell cycle regulatory proteins in vitro. RESULTS: Both monoclonal antibodies, Rituximab and Campath-1H, were less toxic than Fluda to B-CLL cells. Combination of Campath-1H or Rituximab with Fluda did not have a stronger effect on the cells than Fluda alone. Both antibodies decreased the expression of p27 protein and increased the expression of p23; Fluda had a similar effect. The extent of cyclin D3 and cyclin E expression did not change significantly. The expression of cyclin D2 was slightly increased in the presence of Campath-1H, but in the presence of Rituximab it either decreased slightly or remained the same. Treatment of B-CLL cells with Fluda alone induced significant decrease in cyclin D2 expression. CONCLUSION: These results demonstrated that monoclonal antibodies Campath-1H and Rituximab antibodies, as well as a cytotoxic drug fludarabine, had cytotoxic effects on B-CLL cells. They most likely induce apoptosis of B-CLL cells, but their activity is mediated through different pathways.

Spirobipyridopyrans, spirobinaphthopyrans, indolinospiropyridopyrans, indolinospironaphthopyrans and indolinospironaphtho-1,4-oxazines: synthesis, study of X-ray crystal structure, antitumoral and antiviral evaluation.

The novel racemic indolinospirobenzopyrans (5-7), indolinospironaphthopyrans (11-14) and indolinospironaphtho-1,4-oxazine (17) were synthesized by an aldol type of condensation of 1',3',3'-trimethyl-2 '-methyleneindoline and its 5-substituted derivatives with an appropriately substituted hydroxybenzaldehyde, hydroxynaphthaldehyde or nitrosonaphthol. An unequivocal proof of the stereostructures of 9 and 17 was obtained by the single-crystal X-ray diffraction method. A substituted indoline ring and the benzopyran ring in 9 and the naphtho-1,4-oxazine moiety in 17 are interconnected via the common chiral atom and positioned almost perpendicularly to each other. The five-membered 2,3-dihydropyrrolo moiety of the indoline ring adopts an envelope conformation in both structures. Of all the compounds of this series, spirobipyridopyran (1) inhibited specifically the growth of human melanoma (HBL) (IC(50): 0.9 microM) cells but not the growth of normal fibroblasts (WI38). Indolinospirobenzopyrans (8-10) showed significant cytostatic activities against all tumor cell lines. However, these compounds also exhibited a cytotoxic effect on normal human fibroblasts. The indolinospirobenzopyrans 4, 6-8, 10 and the indolinospironaphtho-1,4-oxazine 16 showed, albeit modest, selectivity as antiviral agents against varicella-zoster virus (VZV) and/or cytomegalovirus (CMV) (EC(50) within the concentration range of 1.0-12.6 microM).

Synthesis and biological evaluation of iodinated and fluorinated 9-(2-hydroxypropyl) and 9-(2-hydroxyethoxy)methyl purine nucleoside analogues.

The novel fluorinated and iodinated purine derivatives containing 9-(2-hydroxypropyl) (1a-7a and 9a-13a) and 9-(2-hydroxyethoxymethyl) (1b-3b, 5b, and 7b-12c) side chains were synthesized by a multistep synthetic route involving Baltz-Schiemann's fluorination and diazotation/iodination as key reactions. An unequivocal proof for the stereostructure of 5b was obtained by X-ray structure analysis. New compounds were evaluated for their cytostatic activity against murine leukemia (L1210); mammary carcinoma (FM3A); and human T-lymphocytes (Molt4/C8 and CEM), melanoma (HBL), cervical carcinoma (HeLa), colon carcinoma (HT29 and SW620), laryngeal carcinoma (Hep2), and pancreatic carcinoma (MiaPaCa2) as well as diploid fibroblasts (WI38). Of all the compounds, the 2-aminopurin-6-thione derivative 9a showed the most pronounced inhibitory activity against human SW620 cells. The 2-aminopurin-6-thione derivative 9b exhibited the most selective inhibitory activity against human HeLa, Hep2, SW620, and murine L1210 cell proliferation as compared to normal fibroblast (WI38) cell proliferation. None of the compounds showed inhibitory activities against HIV-1, HIV-2, HSV-1, and HSV-2, vaccinia, vesicular stomatitis, parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, or respiratory syncytial virus. The new purine derivatives, and particularly 9a and 9b, appear to demonstrate sufficient cytostatic potency and selectivity to justify further evaluation of their potential.

Novel derivatives of benzo[b]thieno[2,3-c]quinolones: synthesis, photochemical synthesis, and antitumor evaluation.

Novel derivatives of benzo[b]thieno[2,3-c]quinolones 3a-j were synthesized in a multistep synthesis starting from substituted benzo[b]thiophene-2-carbonyl chlorides, to their corresponding benzo[b]thiophene-2-carboxamides, which were photochemically dehydrohalogenated to their corresponding substituted benzo[b]thieno[2,3-c]quinolones. Compound 4 was prepared from 3i by alkylation with 3-dimethylaminopropyl chloride in the presence of NaH. Compounds 7a,b were prepared from 3g in the multistep synthesis from compounds 5 and 6. Compounds 3b, 3c-f, 3h, 7a, and 7b were found to exert cytostatic activity against malignant cell lines: pancreatic carcinoma (MiaPaCa2), breast carcinoma (MCF7), cervical carcinoma (HeLa), laryngeal carcinoma (Hep2), colon carcinoma (CaCo-2), melanoma (HBL), human fibroblast cell lines (WI-38). The compounds that bear a 3-dimethylaminopropyl substituent on the quinolone nitrogen (3b, 3c-f, 3h) showed higher antitumor activity than compounds bearing the same substituent on the amidic nitrogen (7a and 7b). The compound 3h, which has a 3-dimethylaminopropyl substituent on the quinolone nitrogen and a methoxycarbonyl substituent at position 9, had marked antitumor activity. Because of strong cytotoxic effect of compound 4 on melanoma cells (HBL, ME 67.3, and ME 67.1), a potential mechanism of action was examined. Analysis of DNA and Annexin-V-FLUOS staining indicated that compound 4 causes cell death by apoptosis.

Synthesis, structural studies, and biological evaluation of some purine substituted 1-aminocyclopropane-1-carboxylic acids and 1-amino-1-hydroxymethylcyclopropanes.

The novel purine derivatives of 1-aminocyclopropane-1-carboxylic acid (8 and 9) and 1-amino-1-hydroxymethylcyclopropane (12 and 13) with methylene spacer between the base and the cyclopropane ring were prepared by multistep synthetic route involving alkylation of adenine and 6-(N-pyrrolyl)purine with 2-hydroxy-methyl-1-aminocyclopropane-1-carboxylic acid derivative 3 as a key reaction. All novel compounds were racemic. The N-9 substitution of the purine ring and the Z-configuration of the cyclopropane ring in 4-13 were deduced from their 1H and 13C NMR spectra by analyses of chemical shifts, H-H coupling constants and connectivities in two-dimensional homo- and heteronuclear correlation spectra. An unequivocal proof of the stereostructure of 1, 4 and 5 was obtained by their X-ray structure analysis. The novel compounds were evaluated on cytostatic and antiviral activities in several cell lines. The 6-(N-pyrrolyl)purine derivative of 1,2-aminocyclopropane alcohol 12 exhibited a more pronounced inhibitory activity against the proliferation of cervical carcinoma (HeLa) and human fibroblast (WI-38) cells than other types of tumor cell lines. None of the compounds showed inhibitory activities against cytomegalovirus, varicella-zoster virus or other viruses.

Influence of CD40 ligation on survival and apoptosis of B-CLL cells in vitro.

Modulation of signal transduction pathways represents a promising approach for altering the biological behavior of hematopoetic malignancies. The cells of chronic lymphocytic leukemia were treated in vitro with CD40-ligand or IL-4 to explore their effects on survival and sensitivity to apoptosis induced by Fluda. The expression of G1 cell cycle regulatory proteins was also measured. Stimulation via CD40-CD40L resulted in increased viability, as did stimulation with IL-4. A combination of the two stimulators (CD40L plus IL-4) induced increased expression of cyclins D3 and E, pRb phosphorylation and downregulated p27. Cdk2 and Cdk4 activities were not detected. It seems that this combination induced also some progression in the cell cycle. Furthermore, Fluda-induced apoptosis was not prevented by CD40L, IL-4, or a combination of both agents, although a delay in the onset of apoptosis was observed. Taken together, these results support the view that CD40L and IL-4 sustain B-cell chronic lymphocytic leukemia (B-CLL) survival by different pathways and their synergistic action might induce cell cycle progression in B-CLL. The exposure of B-CLL to CD40L, IL-4 or both did not impair the sensitivity of B-CLL to Fluda. CD40L and IL-4 postponed apoptosis induced by Fluda, depending on a fashion of administration.

Molecular and biochemical events during differentiation of the HD3 chicken erythroblastic cell line.

The chicken erythroblast cell line HD3 is transformed by a temperature-sensitive mutant of avian erythroleukemia virus. Upon shift to the non-permissive temperature in the presence of inducers (hemin and butyric acid), HD3 cells differentiate to an erythrocyte phenotype and provide a model system for analyzing events associated with this process. Expression of some cell surface proteins undergoes drastic changes as cells mature to the erythrocyte stage with a selective loss of membrane proteins that appears to be species-specific. Specific changes also occur in the expression and activities of cytosolic enzymes reflecting alterations of metabolism. HD3 differentiation is characterized by increased transferrin receptor (TFR) expression and increased hemoglobin (Hb) synthesis, a marker for the erythrocyte. In parallel, there is a decrease in glucose transport and an increase in nucleoside transport signifying a switch from glycolytic hexose metabolism to metabolism of pentose from nucleoside. Likewise the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAD) declines while glucose-6-phosphate dehydrogenase (G6PDH) activity remains constant. Commitment to the erythrocyte lineage alters expression of specific genes: TFR mRNA level increases while expression decreases for GLUT1 and GLUT3 glucose transporter mRNAs and GAD mRNA. However, the relationship between GAD activity and GAD mRNA was complex indicating modulation of GAD mRNA and protein half-lives. Serine/threonine and tyrosine phosphorylation and cAMP levels were shown to regulate the level of these messages. In this review, we describe how HD3 differentiation involves changes in plasma membrane composition, metabolism and gene expression that are orchestrated at different levels of control by multiple signaling modalities.