Algirdas Kaupinis

Highlights of the Latest Advances in Research on CDK Inhibitors

Uncontrolled proliferation is the hallmark of cancer and other proliferative disorders and abnormal cell cycle regulation is, therefore, common in these diseases. Cyclin-dependent kinases (CDKs) play a crucial role in the control of the cell cycle and proliferation. These kinases are frequently deregulated in various cancers, viral infections, neurodegenerative diseases, ischemia and some proliferative disorders. This led to a rigorous pursuit for small-molecule CDK inhibitors for therapeutic uses. Early efforts to block CDKs with nonselective CDK inhibitors led to little specificity and efficacy but apparent toxicity, but the recent advance of selective CDK inhibitors allowed the first successful efforts to target these kinases for the therapies of several diseases. Major ongoing efforts are to develop CDK inhibitors as monotherapies and rational combinations with chemotherapy and other targeted drugs.

Roscovitine in cancer and other diseases

Roscovitine [CY-202, (R)-Roscovitine, Seliciclib] is a small molecule that inhibits cyclin-dependent kinases (CDKs) through direct competition at the ATP-binding site. It is a broad-range purine inhibitor, which inhibits CDK1, CDK2, CDK5 and CDK7, but is a poor inhibitor for CDK4 and CDK6. Roscovitine is widely used as a biological tool in cell cycle, cancer, apoptosis and neurobiology studies. Moreover, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, inflammation, viral infections, polycystic kidney disease and glomerulonephritis. This review focuses on the use of roscovitine in the disease model as well as clinical model research.

Klebsiella Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family Myoviridae

At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family.

Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis

Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.

Belinostat, a potent HDACi, exerts antileukaemic effect in human acute promyelocytic leukaemia cells via chromatin remodelling

Epigenetic changes play a significant role in leukaemia pathogenesis, therefore histone deacetylases (HDACis) are widely accepted as an attractive strategy for acute promyelocytic leukaemia (APL) treatment. Belinostat (Bel, PXD101), a hydroxamate‐type HDACi, has proved to be a promising cure in clinical trials for solid tumours and haematological malignancies. However, insight into molecular effects of Bel on APL, is still lacking. In this study, we investigated the effect of Bel alone and in combination with differentiation inducer retinoic acid (RA) on human promyelocytic leukaemia NB4 and HL‐60 cells. We found that treatment with Bel, depending on the dosage used, inhibits cell proliferation, whereas in combination with RA enhances and accelerates granulocytic leukaemia cell differentiation. We also evaluated the effect of used treatments with Bel and RA on certain epigenetic modifiers (HDAC1, HDAC2, PCAF) as well as cell cycle regulators (p27) gene expression and protein level modulation. We showed that Bel in combination with RA up‐regulates basal histone H4 hyperacetylation level more strongly compared to Bel or RA alone. Furthermore, chromatin immunoprecipitation assay indicated that Bel induces the accumulation of hyperacetylated histone H4 at the p27 promoter region. Mass spectrometry analysis revealed that in control NB4 cells, hyperacetylated histone H4 is mainly found in association with proteins involved in DNA replication and transcription, whereas after Bel treatment it is found with proteins implicated in pro‐apoptotic processes, in defence against oxidative stress and tumour suppression. Summarizing, our study provides some novel insights into the molecular mechanisms of HDACi Bel action on APL cells.

Isolation and characterization of vB_ArS-ArV2 - first Arthrobacter sp. infecting bacteriophage with completely sequenced genome.

This is the first report on a complete genome sequence and biological characterization of the phage that infects Arthrobacter. A novel virus vB_ArS-ArV2 (ArV2) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. Based on transmission electron microscopy, ArV2 belongs to the family Siphoviridae and has an isometric head (∼63 nm in diameter) with a non-contractile flexible tail (∼194×10 nm) and six short tail fibers. ArV2 possesses a linear, double-stranded DNA genome (37,372 bp) with a G+C content of 62.73%. The genome contains 68 ORFs yet encodes no tRNA genes. A total of 28 ArV2 ORFs have no known functions and lack any reliable database matches. Proteomic analysis led to the experimental identification of 14 virion proteins, including 9 that were predicted by bioinformatics approaches. Comparative phylogenetic analysis, based on the amino acid sequence alignment of conserved proteins, set ArV2 apart from other siphoviruses. The data presented here will help to advance our understanding of Arthrobacter phage population and will extend our knowledge about the interaction between this particular host and its phages.

Quantitative proteomic analysis of anticancer drug RH1 resistance in liver carcinoma.

UNLABELLED Acquired resistance of tumor cells to the therapeutic treatment is a major challenge in virtually any chemotherapy. A novel anticancer agent 2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) is designed to be activated by NAD(P)H quinone oxidoreductase, an enzyme expressed at high levels in many types of tumors. Here we investigated the potential mechanisms of acquired RH1 drug resistance in cancer cells by applying high-throughput differential quantitative proteomic analysis of the newly established RH1-resistant hepatoma cell lines. Over 400 proteins display significantly altered levels between drug-sensitive and drug-resistant cell lines. Differentially expressed proteins were clustered into more than 14 groups according to their functional annotation and protein-protein interactions. Bioinformatic analysis highlights the biological processes that might be responsible for acquired resistance to RH1. The level of several xenobiotic metabolism enzymes (total n=17) involved in RH1 activation and detoxification is decreased (Nqo1, catalase, Gst, Gsr), corresponding with the decrease in their catalytic activity. The altered biological processes also include the decrease of cell cycle positive regulators (n=15) and the increase of DNA repair proteins (n=5) as well as annexin family members (n=5) in the RH1-resistant cells. Drug-resistant hepatoma cell proteomes are also distinguished by the altered level of proteins involved in energy production and metabolism (n=55). Our data provide the basis for in-depth study of molecular mechanisms of tumor cell resistance to the promising anticancer drug RH1 enabling the further validation of protein biomarkers for the drug insusceptibility and of potential secondary pharmacological targets of RH1 resistant cells.

Isoenzyme Systems and Genotypes in Vendace (Coregonus Albula (Linnaeus, 1758)) from Lithuanian Lakes

Isoenzyme systems of vendace Coregonus albula from 18 lakes of Lithuania were studied. Muscle tissue homogenate was used for electrophoretic investigations on polyacrylamide gel. Six isoenzyme systems G3P, LDH, MDH, SOD, ME, EST and non-specific protein (NSP) were analysed. Seven polymorphic loci (Est-2, Est-3, Est-4, Est-5, Me-3, Nsp-2 and Nsp-3) were selected for the genotype analysis of different vendace populations. Homozygotic genotypes were found to be dominant and a deficit of heterozygotic genotypes was detected in the majority of vendace populations. In addition, a significant difference between the observed and expected genotype frequencies at various loci was recorded. It has been thus concluded, that genetic instability is a characteristic of vendace populations in most of Lithuanian lakes.

Vendace (Coreganus Albula (L.)) Growth and Morphological Diversity in Lakes of Lithuania

Growth rate of vendace inhabiting Lithuanian lakes differs markedly, a number of intermediate growth forms existing. Maximum sizes of vendace in various lakes are different, too. Large-sized vendace of Lithuanian lakes not being distinguishable by morphological characters, it would be not correct to treat it as a separate form. In the same lake and in different years, body lengths of the same age group vendace may vary. Differences among vendace populations of the lakes investigated, as established by the univariate analysis of characters, are significant. This fact proves that vendace adapt to definite conditions with the resultant modification of morphological characters. The most significant morphological characters in distinguishing vendace populations were determined. The most distinguishable are vendace inhabiting lakes of southern Lithuania. Some morphometric characters correlate with morphometric indices of the lake. Changes in meristic characters are less conspicuous. Differences between lake ven...

Gene expression and activity analysis of the first thermophilic U32 peptidase

Peptidase family U32 is one of the few whose catalytic type and structure has not yet been described. It is generally accepted that U32 peptidases represent putative collagenases and contribute to the pathogenicity of some bacteria. Meanwhile, U32 peptidases are also found in nonpathogenic bacteria including thermophiles and hyperthermophiles. Here we report cloning of the U32.002 peptidase gene from thermophilic Geobacillus thermoleovorans DSM 15325 and demonstrate expression and characterization of the recombinant protein. It has been determined that U32.002 peptidase is constitutively expressed in the cells of thermophilic G. thermoleovorans DSM 15325. The recombinant oligomeric enzyme showed its activity only against heat-treated collagen. It was unable to degrade albumin, casein, elastin, gelatine and keratin. In contrast to this, the monomeric recombinant protein showed no activity at all. This paper is the first report about the thermophilic U32 peptidase. As the thermophilic bacteria are non-pathogenic, the role of constitutively expressed extracellular collagenolytic U32 peptidase in these bacteria is unclear.