Characterization of Cyclin E Expression in Multiple Myeloma and Its Functional Role in Seliciclib-Induced Apoptotic Cell Death

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator – cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator - cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion–mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy

Molecular recognition in selected biological systems

The focus of this work is the analysis of molecular recognition in selected biological systems. Geometrical parameters and graph-set analysis of molecular interactions were evaluated from molecular structure of proteins and small molecules determined by X-ray diffraction methods. The energetic aspects of the studied systems we evaluated using ab initio molecular orbital calculations. Hydrogen bonding between a donor, D-H, and an acceptor, A, were inspected in a variety of systems. One system involves the acceptor atoms 0 and/or N, in protein-protein, protein-nucleic acid, or basepair hydrogen bonding interactions, and the second involves the acceptor atom fluorine, common in drug-enzyme binding recognition. Molecular interactions involving the metal ions lead and copper were also analyzed; electronic configurations and effects on metalligand interactions, as well as geometrical consequences of relativistic effects on Pb(II) complexes, and the Jahn Teller effect on Cu(II) complexes, were studied. In addition, the secondary and quaternary structure of the enzyme porphobilinogen synthase were predicted based on preliminary X-ray results. Graph-set analysis showed that the bidentate hydrogen bonding pattern in protein-protein and base-pair recognition is R2 2(8) while that in protein DNA recognition is R2 2(9). The D-H...F interaction, although weaker than D-H ...O(N), is found to contribute to molecular alignment in the crystalline state, especially when several such interactions are present. It was also found that the fluorine atom tends to form contacts with C-H protons, where as O and/or N atoms tend to bind to the more acidic protons (O-H, N-H). The binding recognition of Pb(II) was shown to be either spherical or hemi-spherical; depending on the stereoactivity of the lone pair of electrons, the coordination number and the liganding atoms. The results have shown that geometrical differences between corresponding Cu(I) and Cu(II) complexes depends on specific ligand combinations, and can affect the functionality of the copper site. Type I copper enzymes were found to have similar geometrical parameters in the corresponding Cu(I) and Cu(II) centers. Porphobilinogen synthase was predicted to have a TIM barrel folding pattern. The proposed enzyme packing mode involved a non-crystallographic rotation axis of the two molecules in the asymmetric unit, and is consistent with earlier studies

Molecular recognition in selected biological systems

The focus of this work is the analysis of molecular recognition in selected biological systems. Geometrical parameters and graph-set analysis of molecular interactions were evaluated from molecular structure of proteins and small molecules determined by X-ray diffraction methods. The energetic aspects of the studied systems we evaluated using ab initio molecular orbital calculations. Hydrogen bonding between a donor, D-H, and an acceptor, A, were inspected in a variety of systems. One system involves the acceptor atoms 0 and/or N, in protein-protein, protein-nucleic acid, or basepair hydrogen bonding interactions, and the second involves the acceptor atom fluorine, common in drug-enzyme binding recognition. Molecular interactions involving the metal ions lead and copper were also analyzed; electronic configurations and effects on metalligand interactions, as well as geometrical consequences of relativistic effects on Pb(II) complexes, and the Jahn Teller effect on Cu(II) complexes, were studied. In addition, the secondary and quaternary structure of the enzyme porphobilinogen synthase were predicted based on preliminary X-ray results. Graph-set analysis showed that the bidentate hydrogen bonding pattern in protein-protein and base-pair recognition is R2 2(8) while that in protein DNA recognition is R2 2(9). The D-H...F interaction, although weaker than D-H ...O(N), is found to contribute to molecular alignment in the crystalline state, especially when several such interactions are present. It was also found that the fluorine atom tends to form contacts with C-H protons, where as O and/or N atoms tend to bind to the more acidic protons (O-H, N-H). The binding recognition of Pb(II) was shown to be either spherical or hemi-spherical; depending on the stereoactivity of the lone pair of electrons, the coordination number and the liganding atoms. The results have shown that geometrical differences between corresponding Cu(I) and Cu(II) complexes depends on specific ligand combinations, and can affect the functionality of the copper site. Type I copper enzymes were found to have similar geometrical parameters in the corresponding Cu(I) and Cu(II) centers. Porphobilinogen synthase was predicted to have a TIM barrel folding pattern. The proposed enzyme packing mode involved a non-crystallographic rotation axis of the two molecules in the asymmetric unit, and is consistent with earlier studies

Molecular and crystal structure of 1-(8-carboxyoctyl)-1,3,5,7-tetraazaadamantan-1-ium bromide and 1-(6-bromohexyl)-1,3,5,7-tetraazaadamantan-1-ium bromide

The molecular and crystal structures of a pair of quarternised bromide derivatives of hexamethylenetetramine have been determined. 1-(8-Carboxyoctyl)-1,3,5,7-tetraazaadamantan-1-ium bromide, 1, C<SUB>14</SUB>H<SUB>27</SUB>N<SUB>4</SUB>O<SUB>2</SUB>Br, M<SUB>r</SUB> = 363.31, triclinic, P , a = 9.759(5), b = 12.699(7), c = 14.125(6) &#197;, &#945; = 96.45(3), &#946; = 99.17(3), &#947; = 104.74(3)&#176; , V = 1649.7(14) &#197;<SUP>3</SUP>, Z = 4, D<SUB>c</SUB> = 1.463 g cm<SUP>-3</SUP>, &#955; (MoK&#945; ) = 0.7107 &#197; &#956; = 2.503 cm<SUP>-1</SUP>, F(000) = 760, R (on F) = 0.058, R<SUB>W</SUB> (on I) = 0.162 for 3625 unique reflections with [I -&#963; (I)]. There are two symmetry independent sets of ions, each set being linked with strong O&#8230;Br interactions to form linear arrays. The symmetry indenpendent arrays are in turn connected with C---H&#8230;O interactions to form a layer. 1-(6-Bromohexyl)-1,3,5,7-tetraazaadamantan-1-ium bromide, 2, C<SUB>12</SUB>H<SUB>29</SUB>N<SUB>4</SUB>Br<SUB>2</SUB>, M<SUB>r</SUB> = 384.17, monoclinic, P2<SUB>1</SUB>/n, a = 8.976(4), b = 15.743(8), c = 11.329(6) &#197;, &#946; = 103.70(2)&#176; , V = 1555.3(13) &#197;<SUP>3</SUP>, Z = 4, D<SUB>c</SUB> = 1.641 g cm<SUP>-3</SUP>, &#955; (MoK&#945; ) = 0.7107 &#197; &#956; = 5.203 cm<SUP>-1</SUP>, F(000) = 776, R (on F) = 0.066, R<SUB>W</SUB> (on I) = 0.168 for 1310 unique non-zero reflections with [I >>2&#963; (I)]. Two Br<SUP>-</SUP> anions are located between two cations resulting in the formation of a cyclic centrosymmetric dimer. Each dimer is surrounded by six similar dimers and is connected to them with C---H&#8230;N hydrogen bonds to form corrugated molecular sheets. The symmetrical bond lengths of the hexamethylenetetramine skeleton are distorted by quarternisation in both 1 and 2

3-(3',5'-dinitrophenyl)-4-(2',5'-dimethoxyphenyl)cyclobutane-1,2-dicarboxylic acid: engineered topochemical synthesis and molecular and supramolecular properties

This article does not have an abstract

Seliciclib downregulates MCL1 expression in hMMCLs.

<p>(A) The indicated multiple myeloma cell lines in logarithmic growth phase were extracted and subjected to immunoblotting, utilizing MCL1 antibodies. In all experiments CDK2 expression serves as an internal loading control. Experiments were performed at least 3 times and one representative result is presented. (B–C) MCL1 downregulation by seliciclib. Cells were extracted and subjected to immunoblotting. (B) Various hMMCLs were incubated in the presence of seliciclib 50 µM or DMSO for 6 hours and the level of MCL1 expression was analyzed. (C) RPMI8226, CAG and NCI H929 cells were incubated in the absence or presence of seliciclib 50 µM for the indicated time points, or in the presence of increasing concentrations of seliciclib for 8 hours. (D) Reduction in MCL1 phosphorylation by seliciclib. Cells were treated with 50 µM or DMSO for 8 hours and the levels of total and phosphorylated MCL1 were analyzed by immunoblotting using specific antibodies. β-actin was used to confirm equal protein loading. (E) CAG cells were incubated in the absence or presence of seliciclib or MG132 (10 µM) exclusively or combined. MCL1 level of expression was verified by immunoblotting. β-actin was used to confirm equal protein loading.</p