Aberrant expression of placental-like alkaline phosphatase in chronic myeloid leukemia cells in vitro and its modulation by vitamin E.

Placental-like alkaline phosphatase (PLAP) is expressed by many tumors and can be detected in sera of patients with various cancers. Its aberrant expression has been considered to be potentially useful as tumor marker. However, the biological background of the role of this aberrant alkaline phosphatase (AP) in cancer is still unclear. The expression of various forms of AP in cells of chronic myeloid leukemia (CML) has not yet been studied. AIM: To analyze the expression patterns of various AP forms in cells originated from CML patients in blast crisis and to modify their expression by vitamin E. MATERIALS AND METHODS: RNA extracted from leukemic cells was converted to cDNA and real-time reverse transcription polymerase chain reaction was performed using SYBR Green protocol with primers to tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase and CCAAT-enhancer-binding proteins alpha (C/EBPα). To analyze the modulation of expression of APs and C/EBPα, CML cells were incubated with 100 µM vitamin E. RESULTS: We have observed the aberrant expression of mRNA intestinal alkaline phosphatase in CML cells that upon sequencing demonstrated the significant alignment with PLAP sequence while no gene homology with tissue placental alkaline phosphatase (PAP) was revealed. Vitamin E decreases mRNA PLAP expression and increases mRNA TNAP expression. Moreover, along with down-regulation of aberrant PLAP and up-regulation of TNAP, vitamin E increases C/EBPα mRNA expression. CONCLUSION: The loss of TNAP in CML may contribute to pathogenesis of this disease. PLAP may be considered as a putative target in differentiation therapies in myeloid neoplasms. Our findings suggest the potential role of vitamin E as the inducer of differentiation potential of leukemic cells in CML.

Vitamin Е activates expression of С/EBP alpha transcription factor and G-CSF receptor in leukemic K562 cells.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the activity of BCR-ABL fusion oncogene. Tyrosine kinase inhibitors are the current treatment of CML, but secondary mutations finally contribute to therapy resistance and blast crisis of the disease. The search for the novel compounds for the effective control of CML is now in the spotlight. The progression of CML to blast crisis is correlated with down-modulation of C/EBP alpha. Therefore, C/EBP alpha may be considered as a putative target in differentiation therapies in myeloid leukemias. The aim of the study was to assess the potential of vitamin E as the possible inducer of C/EBP alpha expression in BCR-ABL-positive CML K562 cells. MATERIALS AND METHODS: RNA extracted from K562 cells cultured with valproic acid or vitamin E was converted to cDNA, RT-PCR reactions were carried out using HotStarTaq DNA polymerase with primers for C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR). RESULTS: We have not found detectable expression of C/EBP alpha in K562 cells. Upon 48-h culture with vitamin E at a dose of 100 µM, K562 cells expressed both C/EBP alpha and G-CSFR. CONCLUSION: Vitamin E restored the expression of C/EBP alpha mRNA in chronic myelogenous leukemia K562 cells. In this setting, G-CSFR expression in vitamin E treated K562 cells seems to suggest the activation to granulocytic differentiation. It should be further elucidated whether such effects of vitamin E on C/EBP alpha transcription factor are direct or mediated indirectly due to antioxidant properties of vitamin E.

DNA hypomethylation as Achilles' heel of tumorigenesis: a working hypothesis.

There are at least two findings that show DNA hypomethylation plays a key role in carcinogenesis. The first major evidence is that DNA hypomethylation induces target chromosomal and genomic instability with cancer manifestations. The second reason that cancer progression is associated with deepening DNA hypomethylation. Nevertheless, the evolution of this crucial epigenomic alteration in the somatic cellular malignant transformation remains unclear. From some of the experimental data to be present, a key role of DNA hypomethylation in early development of epigenetic somatic cancer biology is proposed. We have observed the significant increasing of genome ploidy at the level of peripheral blood lymphocytes taken from the patients with different solid carcinomas. Similarly, 5-azacytidine demethylating DNA treatment of cultured healthy lymphocytes induces increased nuclear DNA content. We argue that somatic lymphocyte ploidy induced by genomic DNA hypomethylation during carcinogenesis is related to global demethylation and decondensation of mitotic constitutive pericentromeric heterochromatin. This results in disturbances of pericentromeric heterochromatin that are expressed in nuclear heterochromatinization on the basis of extrachromosomal chromomerization. On the basis of literature searches and experimental findings, it is proposed that DNA hypomethylation plays the role of an initiator in epigenetic somatic cancer biology.

Alterations of constitutive pericentromeric heterochromatin in lymphocytes of cancer patients and lymphocytes exposed to 5-azacytidine is associated with DNA-hypomethylation.

BACKGROUND: DNA hypomethylation plays a key role in carcinogenesis. The malignant transformation of cells as well as tumor progression is accompanied with increasing DNA hypomethylation in cancer cells. Nevertheless, the evolution of dis-epigenetic genomic alteration in the somatic cellular malignant transformation has not yet been clear. AIM: To study the relationship between the pattern of genomic DNA hypomethylation and DNA hyperploidy. METHODS: The model of 5-azacytidine demethylating DNA treatment of the mitogen-stimulated lymphocytes in parallel with patients with solid cancer has been explored. DNA content was measured by quantitative DAPI and Hoechst 33258 fluorescence in situ hybridization on interphase nuclei. The conventional mitogen-stimulated blood lymphocyte culture development was performed in metaphase chromosomes and interphase nuclei assay. The light and fluorescent cytomorphological microscopy was performed. RESULTS: The model 5-azacytidine induced DNA demethylation results in increased DNA hyperploidy accompanying major pericentromeric heterochromatin (Alu) DNA repeats amplification similar to those during DNA hypomethylation-associated cancer events, and both contributed to nuclear heteropycnosis development. The constitutive pericentromeric heterochromatin consequent morphological disturbance to the latent polytene chromomerization and heteropycnosis development both in cancer patients and in model 5-azacytidine exposured lymphocytes are associated with DNA hypomethylation. CONCLUSION: We have observed that the induced global DNA hypomethylation triggers dis-epigenetic morphological reprogramming of constitutive pericentromeric heterochromatin on the extrachromosomal organization pathway as seen during the heterochromatin latent polytene features development, which is of importance as one of the mechanism involving DNA hypomethylation in initiation and progression of cancer.

[Study on secondary structure and properties of alpha- and gamma-forms of human thrombin].

Secondary structure and enzymatic properties of human a-thrombin and its gamma-form (obtaining during autolysis of the native enzyme) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). According to DSC-data both alpha-thrombin and gamma-thrombin contained only one thermal transition peak at 58.5 and 53.3 degrees C, respectively. A comparison of these values suggested that gamma-form is less stable than initial a-thrombin. In contrast to that the thermogram of DIP-a-thrombin had two peaks (57.5 and 64.5 degrees C). CD spectra showed that conversion a- to gamma-thrombin influenced the secondary structure of the enzyme slightly. The study of the inhibitory effect of such polyanions as ATP and dextran sulfate (DS) upon thrombin-catalyzed cleavages of fibrinogen has shown that the growth of the negative charge of the polyanion molecule resulted in the increase of its inhibitory activity. The catalytically non-active DIP-alpha-thrombin, which retained the native anion-binding exosite 1, was shown to decrease the inhibitory power of the dextran sulfate. It was explained by competition of DS with the exosite 1 of both alpha- and DIP-alpha -thrombin. In contrast to that DIP-gamma-thrombin having exosite 1 destroyed neither competed nor influenced the anticoagulant capacity of dextran sulfate toward the native alpha-thrombin. In accordance with our data thrombin consists of two rather strong interacting domains. It was shown further that its anion-binding exosite 1 may play a significant role in the interaction of the enzyme with dextran sulfate.

[Catalytic effect of gamma-thrombin on synthetic low molecular weight peptide substrates]. / Osobennosti kataliticheskogo deistviia gamma-trombina na sinteticheskie nizkomolekuliarnye peptidnye substraty.

Hydrolysis and respective catalytic parameters of hydrolysis of ester peptide substrates that contain residues of hydrophobic and nonpolar amino acids in P2, P3 subsites have been studied. It is shown that efficiency of hydrolysis by thrombin is determined by the length of polypeptide chains and by the nature of the amino acids in P2, P3 subsites in the substrate. In spite of the fact that gamma-thrombin retains the conformation activity of the catalytic centre the local conformation changes of the second binding region of the enzyme have been discovered.

[Affinity chromatography of human gamma-thrombin on fibrin-agarose]. / Afinnaia khromatografiia gamma-trombina cheloveka na fibrin-agaroze.

gamma-Thrombin was produced during autolysis or limited proteolysis of coagulant gamma-thrombin. This thrombin form loses its ability to coagulate fibrinogen but preserves the esterase and amidase activity on the low-molecular-weight synthetic substrates. This evidences for the integrity of the active site of gamma-thrombin and for the integrity break of the enzyme molecule region responsible for the binding with fibrinogen. gamma-Thrombin with the minimal coagulating activity, possessing high esterase and amidase activity is obtained. Fibrin-agarose possessing affinity to gamma-thrombin and specifically not binding gamma-thrombin was used to remove admixtures of the coagulant gamma-thrombin from the preparations of gamma-thrombin obtained during the enzyme autolysis.

[Effect of isopropanol on the enzymatic activity and stability of thrombin]. / Vliianie izopropanola na fermentativnuiu aktivnost' i stabil'nost' trombina.

Isopropanol is shown to affect considerably the thrombin activity. Its low concentrations (5-15%) activate the hydrolysis reaction of benzoyl-1-arginine ethyl ester (BAEE) by thrombin, whereas incomplete uncompetitive inhibition of the enzyme is observed in the presence of 20% isopropanol. Alcohol in the concentration of 25% results in complete reversible inhibition of the clothing and esterase activity of the enzyme. Isopropanol in the concentration of 25% is able to suppress the thrombin autolysis and therefore it may be used as a reagent which stabilizes thrombin during its storage in the aquatic-salt solutions.

[Isolation of human thrombin using affinity chromatography on gramicidin C-silochrome C 80]. / Poluchenie trombina cheloveka s pomoshch'iu affinnoi khromatografii na gramitsidin C-silokhrome C 80.

Thrombin purification is conducted by biospecific chromatography on gramicidin C-silochrome C 80. Preparations possessing the fibrinogen-coagulating activity of 2500-3200 NIH units per 1 mg of protein and containing 98% of active sites are obtained. Data obtained from electrophoresis in PAAG with the presence of DS-Na show the alpha-thrombin content to be 96%; the admixture of beta-thrombin possessing no coagulating activity does not exceed 4%. The kinetic constants are presented for thrombin hydrolysis of tosyl-L-arginine methyl ester (TAME), benzoyl-L-arginine ethyl ester (BAEE) and chromogenic substrate S-2238. The addition of isopropanol increases sharply the stability of thrombin when storing it in the aqueous-salt solutions.