A Crosstalk Between Dual-Specific Phosphatases and Dual-Specific Protein Kinases Can Be A Potential Therapeutic Target for Anti-cancer Therapy.

While protein tyrosine kinases (PTKs) play an initiative role in growth factor-mediated cellular processes, protein tyrosine phosphatases (PTPs) negatively regulates these processes, acting as tumor suppressors. Besides selective tyrosine dephosphorylation of PTKs via PTPs may affect oncogenic pathways during carcinogenesis. The PTP family contains a group of dual-specificity phosphatases (DUSPs) that regulate the activity of Mitogen-activated protein kinases (MAPKs), which are key effectors in the control of cell growth, proliferation and survival. Abnormal MAPK signaling is critical for initiation and progression stages of carcinogenesis. Since depletion of DUSP-MAPK phosphatases (MKPs) can reduce tumorigenicity, altering MAPK signaling by DUSP-MKP inhibitors could be a novel strategy in anti-cancer therapy. Moreover, Cdc25A is, a DUSP and a key regulator of the cell cycle, promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases (CDK). Cdc25A-CDK pathway is a novel mechanism in carcinogenesis. Besides the mammalian target of rapamycin (mTOR) kinase inhibitors or mammalian target of rapamycin complex 1 (mTORC1) inhibition in combination with the dual phosphatidylinositol 3 kinase (PI3K)/mTOR or AKT kinase inhibitors are more effective in inhibiting the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and cap-dependent translation. Dual targeting of the Akt and mTOR signaling pathways regulates cellular growth, proliferation and survival. Like the Cdc2-like kinases (CLK), dual-specific tyrosine phosphorylation-regulated kinases (DYRKs) are essential for the regulation of cell fate. The crosstalk between dual-specific phosphatases and dual- specific protein kinases is a novel drug target for anti-cancer therapy. Therefore, the focus of this chapter involves protein kinase modules, critical biochemical checkpoints of cancer therapy and the synergistic effects of protein kinases and anti-cancer molecules.

Regulation and Effect of Telomerase and Telomeric Length in Stem Cells.

Telomeres are the protective end caps of eukaryotic chromosomes and they determine the proliferative lifespan of somatic cells, as the protectors of cell replication. Telomere length in leucocytes reflects telomere length in other somatic cells. Leucocyte telomere length can be a biomarker of human ageing. The risk of diseases associated with reduced cell proliferation and tissue degeneration, including aging or aging-associated diseases, such as dyskeratosis congenita, cardiovascular diseases, pulmonary fibrosis and aplastic anemia, is correlated with an increase in the shortening of telomeres. On the other hand, the risk of diseases that are associated with increased proliferative growth, including major cancers, is correlated with long telomeres. In most of the cancers, a telomere maintenance mechanism during DNA replication is essential. The reactivation of the functional ribonucleoprotein holoenzyme complex (telomerase) starts the cascade from normal and premalignant somatic cells to advanced malignant cells. Telomerase is overexpressed during the development of cancer and embryonic stem cells, through controlling genome integrity, cancer formation and stemness. Cancer cells have mechanisms to maintain telomeres to avoid initiation of cellular senescence or apoptosis, and halting cell division by critically short telomeres. Modulation of the human telomerase reverse transcriptase is the rate-limiting step for the production of functional telomerase and telomere maintenance. The human telomerase reverse transcriptase promoter promotes its gene expression only in tumor cells, but not in normal cells. Some cancers activate an alternative expansion of telomeres maintenance mechanism via DNA recombination to reduce the shortening of their telomeres. Not only heritability but also oxidative stress, inflammation, environmental factors, and therapeutic interventions have an effect on telomere shortening, explaining the variability in telomere length across individuals. There have been a large number of publications, which correlate human diseases with progressive telomere shortening. Telomere length of an individual at birth is also important to follow up telomere shortening, and it can be used as a biomarker for healthy aging. On the other hand, understanding of cellular stress factors, which affect stem cell behavior, will be useful in regeneration or treatment of cancer and age-associated diseases. In this review, we will understand the connection between stem cell and telomere biology, cancer, and aging-associated diseases. This connection may be useful for discovering novel drug targets and improve outcomes for patients having cancer and aging-associated diseases.

HDAC Inhibition Overcomes Acute Resistance to MEK Inhibition in BRAF-Mutant Colorectal Cancer by Downregulation of c-FLIPL.

PURPOSE: Activating mutations in the BRAF oncogene are found in 8% to 15% of colorectal cancer patients and have been associated with poor survival. In contrast with BRAF-mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of BRAFMT colorectal cancer patients. Therefore, identification of novel therapies for BRAFMT colorectal cancer is urgently needed. EXPERIMENTAL DESIGN: BRAFMT and wild-type (WT) colorectal cancer models were assessed in vitro and in vivo. Small-molecule inhibitors of MEK1/2, MET, and HDAC were used, overexpression and siRNA approaches were applied, and cell death was assessed by flow cytometry, Western blotting, cell viability, and caspase activity assays. RESULTS: Increased c-MET-STAT3 signaling was identified as a novel adaptive resistance mechanism to MEK inhibitors (MEKi) in BRAFMT colorectal cancer models in vitro and in vivo. Moreover, MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8 inhibitor c-FLIPL in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity abrogated MEKi-induced c-FLIPL expression. In addition, treatment with c-FLIP-specific siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIPL expression and resulted in significant increases in MEKi-induced cell death in BRAFMT colorectal cancer cells. Notably, combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth in BRAFMT xenografts. CONCLUSIONS: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIPL expression is an important escape mechanism following MEKi treatment in BRAFMT colorectal cancer. Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (e.g., HDAC inhibitors) could be potential novel treatment strategies for BRAFMT colorectal cancer.

Four novel mutations in the ß-galactosidase gene identified in infantile type of GM1 gangliosidosis.

OBJECTIVES: The aim of this study is to find out mutations of Turkish GM1 gangliosidosis patients and to make genotype-phenotype correlations. DESIGN AND METHODS: ß-galactosidase activities were measured by using fluorometric substrate. Mutation screening of 16 exons of ß-galactosidase gene and mutation detection were done by PCR-SSCP and DNA sequencing, respectively. RESULTS: Four new mutations, c.188_189insT in exon 2, c.569_570insA in exon 6, p.K142Q in exon 4, p.G190D in exon 6, and one known mutation p.P549L in exon 15, were identified in the ß-galactosidase gene in 5 Turkish patients. Mutations in exons 4 and 6 are in the active site and mutation in exon is in the galactose-binding domain of the ß-galactosidase gene. CONCLUSION: This is the first mutational analysis performed in Turkish GM1 gangliosidosis patients and shows the molecular heterogeneity of the disease in Turkish population. All identified mutations result in severe enzyme deficiency and infantile phenotype.

Two novel alpha-galactosidase A mutations causing Fabry disease: A missense mutation M11V in a heterozygote woman and a nonsense mutation R190X in a hemizygote man.

OBJECTIVES: To evaluate the nature of the molecular lesions in the alpha-galactosidase A gene of two patients having Fabry disease. METHODS: Enzyme analyses were done using 4-methylumbellyferyl alpha-galactoside as substrate. Single stranded conformational polymorphism analysis and DNA sequencing were performed following PCR amplification of seven exons of alpha-galactosidase A gene. RESULTS: Two new mutations, M11V and R190X, were identified. The female patient with M11V mutation had rheumatologic symptoms, microalbuminuria. The male patient with R190X mutation had a classical phenotype. M11V mutation is in the signal sequence of the peptide and may affect the targeting of the ribosomes to ER. R190X mutation causes premature termination, and probably leads to degradation of the protein. CONCLUSION: This is the first study in our country investigating the molecular aspects of Fabry disease. It provides the molecular basis for understanding the underlying mechanism of Fabry disease, allows prenatal diagnosis and provides genotype/phenotype correlations.

Methotrexate-induced apoptosis is enhanced by altered expression of methylenetetrahydrofolate reductase.

Folates are essential for DNA synthesis and methylation reactions. The antifolate methotrexate (MTX) is a widely used chemotherapeutic drug which inhibits DNA synthesis and induces apoptosis. Changes in activity of a critical folate-metabolizing enzyme, methylenetetrahydrofolate reductase (MTHFR), might alter the chemosensitivity to MTX, as the MTHFR substrate is required for nucleotide synthesis and its product is used in homocysteine remethylation to methionine. Mild MTHFR deficiency is common in many populations due to a polymorphism at bp 677. We previously showed that altered expression of MTHFR enhanced MTX-induced myelosuppression in mice. To determine the cause of the impaired hematopoietic profile in mice with decreased or increased MTHFR expression, we evaluated MTX-induced apoptosis in the major hemolytic organ, spleen, using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining and caspase-3/7 activity assays, in MTHFR-deficient mice and in MTHFR-overexpressing mice after MTX administration. Decreased or increased expression of MTHFR in mice significantly increased TUNEL-positive cells and caspase-3/7 activities in MTX-treated spleen, compared with that of wild-type littermates. Plasma homocysteine levels correlated with apoptotic index in MTX-treated MTHFR-deficient mice and dUTP/dTTP ratios correlated with apoptotic index in MTX-treated MTHFR-overexpressing mice. The increased apoptosis may therefore relate to hyperhomocysteinemia and deoxyribonucleotide pool imbalances, respectively. Our results suggest that MTHFR underexpression and overexpression enhances MTX-induced apoptosis and myelosuppression, and that genotyping for the MTHFR polymorphism may have therapeutic implications.

Protective role of 27bp repeat polymorphism in intron 4 of eNOS gene in lacunar infarction.

Association of the three potential endothelial nitric oxide synthase gene (eNOS) polymorphisms (T-786C in promoter region, G894T in exon 7 and tandem 27-bp repeats in intron 4) with an increased risk of lacunar infarction (LI) were investigated. Genotypes of 70 patients and 81 healthy controls were determined through PCR with or without RFLP. Flow-mediated dilatation (FMD) was performed to assess endothelial-dependent vasodilatation, whereas the endothelial-independent vasodilatation was assessed with nitroglycerin (NTG). Genotype distribution was significantly different between LI patients and controls for intron 4aa (alleles for four repeats), genotype frequency being 1.4% and 16.0%, respectively (odds ratio for additive effect, 0.47; 95% CI, 0.28-0.81; p=0.006). Haplotypes with the intron 4aa polymorphism were significantly higher in controls when compared with the LI group (p=0.001). Diminished FMD but normal NTG response confirmed that patients with LI have generalized endothelial dysfunction. Intron 4aa genotype of eNOS gene seems to be protective for isolated LI and the effect was potentiated by the absence of 786C polymorphism in any allele of the promoter region.

Altered expression of methylenetetrahydrofolate reductase modifies response to methotrexate in mice.

OBJECTIVE: Folates provide one-carbon units for nucleotide synthesis and methylation reactions. A common polymorphism (677C-->T) in methylenetetrahydrofolate reductase (MTHFR) encodes an enzyme with reduced activity. Response to the antifolate methotrexate (MTX) may be modified in 677TT individuals because MTHFR converts nonmethylated folates, used for thymidine and purine synthesis, to 5-methyltetrahydrofolate, used in homocysteine remethylation to methionine. To study potential interactions between MTHFR activity and MTX, we examined the impact of decreased and increased MTHFR expression on MTX response in mice. METHODS: Mthfr-deficient (Mthfr and Mthfr) and wild-type (Mthfr) mice were injected with MTX or saline and assessed for hematological parameters (hematocrit, hemoglobin, red, and white blood cell numbers), plasma homocysteine, nephrotoxicity, hepatotoxicity, and splenic 2'-deoxyuridine 5'-triphosphate/2'-deoxythymidine 5'-triphosphate ratios. MTHFR-overexpressing transgenic mice (MTHFR-Tg) were generated, metabolites and folate distributions were measured, and response to MTX was assessed. RESULTS: MTX-treated Mthfr and Mthfr mice displayed hyperhomocysteinemia and decreased hematocrit, hemoglobin, and red blood cell numbers compared with wild-type animals. Mthfr mice also showed increased nephrotoxicity and hepatotoxicity. MTHFR-Tg mice were generated and confirmed to have increased levels of MTHFR with altered distributions of folate and thiols in a tissue-specific manner. After MTX treatment, MTHFR-Tg mice exhibited the same decreases in hematological parameters as Mthfr-deficient mice, and significantly decreased thymidine synthesis (higher 2'-deoxyuridine 5'-triphosphate/2'-deoxythymidine 5'-triphosphate ratios) compared with wild-type mice, but they were protected from MTX-induced hyperhomocysteinemia. CONCLUSION: Underexpression and overexpression of Mthfr/MTHFR increase MTX-induced myelosuppression but have distinct effects on plasma homocysteine and nephrotoxicity. Pharmacogenetic analysis of polymorphisms in folate-dependent enzymes may be useful in optimization of MTX therapy.

Increased hexosaminidase activity in antipsychotic-induced extrapyramidal side effects: possible association with higher occurrence in bipolar disorder patients.

Dystonic movements and Parkinsonism are frequently seen in gangliosidoses and these conditions have been reported to modify dopaminergic plasticity. We investigated whether the activity of hexosaminidase, a type-two ganglioside (GM2) degrading enzyme, correlates with drug-induced extrapyramidal system (EPS) side effects in psychiatric patients. We compared hexosaminidase activity in the lymphocytes of 29 EPS-positive patients, 13 EPS-negative patients, and 30 healthy volunteers. The activities of A and B isoforms of hexosaminidase were higher in EPS-positive patients than EPS-negative patients and healthy controls. Multivariate analysis suggested an interaction with increased B isoform activity and EPS side effects in female bipolar disorder patients. Higher levels of hexosaminidase enzyme activity may explain the frequent occurrence of antipsychotic-induced extrapyramidal side effects in mood disorder patients.