Modified protein kinase activity and isozyme distribution in adenocarcinoma of the human colon

Protein kinase activity and histone kinase isozyme distribution have been determined in soluble extracts of adenocarcinoma of the human colon and compared to adjacent normal mucosa. The results show an enhancement in endogenous protein kinase activity and the presence of an additional isozyme (PKI) for histone kinase activity in the tumour tissue. PKI activity exhibited a peculiar behaviour in comparison to the isozyme. PKII present in both carcinoma and normal mucosa after dialysis of the soluble extracts. It is suggested that alteration of intracellular regulatory processes involved in PKI activity might be related to the maintenance of the proliferate state in human colon carcinoma.     

Histone specific proteinase and DNase activity of spleen lymphocyte nuclei during oncogenesis and under the effect of hydrobromide-5-(5',6' benzocoumaroyl-3')-methylaminouracil

The features of histone proteolysis, their carbonylation level and endonucleolysis intensity were studied in the spleen lymphocytes of rats with transplanted Guerin's carcinoma and under the conditions of hydrobromide-5-(5',6' benzocoumaroyl-3')-methylaminouracil (BCU) action. The intensification of oxidizing histone destruction and histone-specific protease activity during oncogenesis and under conditions of BCU action was shown. DNase I and DNase II enzymatic activities of spleen lymphocytes of rats with tumour were decreased on different stages of tumour development, however, they were increased under conditions of chemical compound administration.     

Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

The dual activity of pyruvate kinase type M2 from chromatin extracts of neoplastic cells

Pyruvate kinase type M(2) from Morris hepatoma 7777 tumour cell nuclei and cytosol, in contrast to types L and M(2) from nuclei and cytosol of normal rat liver, shows the histone H(1) kinase activity. Moreover, in the presence of L-cysteine and without ADP it converts 2-phosphoenolpyruvate (PEP) to pyruvate while in the presence of L-arginine or L-histidine does not. L-Cysteine markedly stimulates the activity of histone H(1) kinase transferring a phosphate group from PEP to, as results suggested, the epsilon -amino group of L-lysine of histone H(1). This, L-cysteine which is known to inhibit the activity of pyruvate kinase type M(2) from neoplastic cells transfering a phosphate from PEP to ADP, can act as a control factor champing the direction of enzymatic reaction in cancer cells.     

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.     

Activity of chromatin-bound protease from rat liver and Morris hepatomas.

1. The activity of serine protease was studied in chromatin and in histone preparations from rat liver and Morris hepatomas, lines 5123 D and 7777. 2. Electrophoretic patterns of histones and the amounts of acid-soluble peptides released during incubation of chromatin and histones showed that there was no significant correlation between protease activity and tumour differentiation and its growth rate.     

Valproic acid inhibits the growth of cervical cancer both in vitro and in vivo

Valproic acid (VPA), a well-known anti-convulsant, is currently under extensive evaluation as an anti-cancer agent. It is known to exert its anti-cancer effect mainly by inhibiting the enzyme histone deacetylase I. In our study, we investigated the effects of VPA on cervical cancer both in vitro and in vivo cancer models. We examined the effects of acute VPA (0, 1.2, 2.4, 5.0 mM) treatment on cell proliferation in cervical cancer cell lines HeLa, SiHa and Ca Ski and histone acetylation, p21 and p53 gene expression in HeLa cell line. We also investigated the effect of chronic VPA administration in tumour xenograft growth studies. Our results show that with acute treatment, VPA can increase the expression of net histone H3 acetylation and up-regulate p21 expression with no effect on p53 expression. Chronic administration of VPA had a net cytostatic effect that resulted in a statistically significant reduction of tumour growth and improved survival advantages in tumour xenografts studies. Furthermore, we also demonstrated that VPA has a direct anti-angiogenic effect in tumour studies and could potentially be a promising candidate for further cervical cancer trails.     

The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth

E7 is the main transforming protein of human papilloma virus type 16 (HPV16) which is implicated in the formation of cervical cancer. The transforming activity of E7 has been attributed to its interaction with the retinoblastoma (Rb) tumour suppressor. However, Rb binding is not sufficient for transformation by E7. Mutations within a zinc finger domain, which is dispensable for Rb binding, also abolish E7 transformation functions. Here we show that HPV16 E7 associates with histone deacetylase in vitro and in vivo, via its zinc finger domain. Using a genetic screen, we identify Mi2beta, a component of the recently identified NURD histone deacetylase complex, as a protein that binds directly to the E7 zinc finger. A zinc finger point mutant which is unable to bind Mi2beta and histone deacetylase but is still able to bind Rb fails to overcome cell cycle arrest in osteosarcoma cells. Our results suggest that the binding to a histone deacetylase complex is an important parameter for the growthpromoting activity of the human papilloma virus E7 protein. This provides the first indication that viral oncoproteins control cell proliferation by targeting deacetylation pathways.     

Anti-malarial effect of histone deacetylation inhibitors and mammalian tumour cytodifferentiating agents

The histones of Plasmodium falciparum represent a potential new target for anti-malarial compounds. A naturally occurring compound, apicidin, has recently been shown to inhibit the in vitro growth of P. falciparum. Apicidin was shown to hyperacetylate histones, suggesting that its mode of action is through histone deacetylase inhibition. We have tested the ability of known histone deacetylase inhibitors, mammalian tumour suppressor compounds, and cytodifferentiating agents to inhibit the in vitro growth of a drug sensitive and resistant strain of P. falciparum. Seven of the tested compounds had microM IC50 values, and trichostatin A, a histone deacetylation inhibitor and cytodifferentiating agent, was active at low nM concentrations. One compound, suberic acid bisdimethylamide, which selectively arrests tumour cells as opposed to normal mammalian cells, had an in vivo cytostatic effect against the acute murine malaria Plasmodium berghei, and one round of treatment with the compound failed to select for resistant mutations. These results suggest a promising role for histone deacetylase inhibitors and cytodifferentiating agents as antimalarial drug candidates.     

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.     

Synthesis of a novel series of thiazole-based histone acetyltransferase inhibitors

Acetylation, which targets a broad range of histone and non-histone proteins, is a reversible mechanism and plays a critical role in eukaryotic genes activation/deactivation. Acetyltransferases are very well conserved through evolution. This allows the use of a simple model organism, such as budding yeast, for the study of their related processes and to discover specific inhibitors. Following a simple yeast-based chemogenetic approach, we have identified a novel HAT (histone acetyltransferase) inhibitor active both in vitro and in vivo. This new synthetic compound, 1-(4-(4-chlorophenyl)thiazol-2-yl)-2-(propan-2-ylidene)hydrazine, named BF1, showed substrate selectivity for histone H3 acetylation and inhibitory activity in vitro on recombinant HAT Gcn5 and p300. Finally, we tested BF1 on human cells, HeLa as control and two aggressive cancer cell lines: a neuroblastoma from neuronal tissue and glioblastoma from brain tumour. Both global acetylation of histone H3 and specific acetylation at lysine 18 (H3AcK18) were lowered by BF1 treatment. Collectively, our results show the efficacy of this novel HAT inhibitor and propose the utilization of BF1 as a new, promising tool for future pharmacological studies.     

The methylation landscape of tumour metastasis

The metastatic cascade which leads to the death of cancer patients results from a multi‐step process of tumour progression caused by genetic and epigenetic alterations in key regulatory molecules. It is, therefore, crucial to improve our understanding of the regulation of genes controlling the metastatic process to identify predictive biomarkers and to develop more effective therapies to treat advanced disease. The study of epigenetic mechanisms of gene regulation offers a novel approach for innovative diagnosis and treatment of cancer patients. Recent discoveries provide compelling evidence that the methylation landscape (changes in both DNA methylation and histone post‐translational modifications) is profoundly altered in cancer cells and contributes to the altered expression of genes regulating tumour phenotypes. However, the impact of methylation events specifically on the advanced metastatic process is poorly understood compared with the initial oncogenic events. Moreover, the characterisation of a large number of histone‐modifying enzymes has revealed their active roles in cancer progression, via the regulation of specific target genes controlling different metastatic phenotypes. Here, we discuss two main methylating events (DNA methylation and histone‐tail methylation) involved in oncogenesis and metastasis formation. The potential reversibility of these molecular events makes them promising biomarkers of metastatic potential and potential therapeutic targets.