Nucleotide Excision Repair and Vitamin D--Relevance for Skin Cancer Therapy.

Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation.

Eukaryotic TLS polymerases.

TLS polymerases are able to replicate damaged DNA (called translesion DNA synthesis, TLS). Their presence prevents cell death as a result of violating the integrity of the genome. In vitro, they are mutator, but in vivo are recruited by specific types of DNA damage and usually replicate them in a correct manner. The best-known TLS polymerases belong to the Y family, such as Rev1, κ, η, ι, and polymerase ζ from the B family. There are two mechanisms of TLS polymerases action: polymerase-switching model and the gap-filling model. Selection of the mechanism primarily depends on the phase of the cell cycle. The regulation of these polymerases may take place at the transcriptional level and at level of recruitment to the sites of DNA damage. In the latter case post-translational modification of proteins - ubiquitination and sumoylation, and protein-protein interactions are crucial.

Role of RUNX2 in Breast Carcinogenesis.

RUNX2 is a transcription factor playing the major role in osteogenesis, but it can be involved in DNA damage response, which is crucial for cancer transformation. RUNX2 can interact with cell cycle regulators: cyclin-dependent kinases, pRB and p21Cip1 proteins, as well as the master regulator of the cell cycle, the p53 tumor suppressor. RUNX2 is involved in many signaling pathways, including those important for estrogen signaling, which, in turn, are significant for breast carcinogenesis. RUNX2 can promote breast cancer development through Wnt and Tgfß signaling pathways, especially in estrogen receptor (ER)-negative cases. ERα interacts directly with RUNX2 and regulates its activity. Moreover, the ERa gene has a RUNX2 binding site within its promoter. RUNX2 stimulates the expression of aromatase, an estrogen producing enzyme, increasing the level of estrogens, which in turn stimulate cell proliferation and replication errors, which can be turned into carcinogenic mutations. Exploring the role of RUNX2 in the pathogenesis of breast cancer can lead to revealing new therapeutic targets.

Association between polymorphism of the DNA repair SMUG1 and UNG genes and age-related macular degeneration.

PURPOSE: To investigate the association between the g.4235T>C (rs2337395) polymorphism of the UNG gene and the c.-31A>G (rs3087404) polymorphism of the SMUG1 gene and the risk of age-related macular degeneration (AMD), as well as modulation of this association by some environmental and lifestyle factors. METHODS: Overall, 272 AMD patients and 105 control subjects were enrolled in this study. Both polymorphisms were genotyped by restriction fragment length polymorphism-polymerase chain reaction (PCR-RFLP). RESULTS: The C/C genotype of the g.4235T>C polymorphism of the UNG gene was associated with an increased risk of dry AMD (odds ratio, 2.54), whereas the T/T genotype of this polymorphism decreased such risk (odds ratio, 0.41). The presence of the T allele of the g.4235T>C polymorphism and the A allele of the c.-31A>G polymorphism of the SMUG1 gene (odds ratio, 2.17 and 2.18, respectively) was associated with an increased risk of AMD severity, expressed by the comparison of the frequencies of genotypes in the group of patients with wet AMD versus those with dry AMD. Conversely, the C/C genotype of the g.4235T>C polymorphism, the G/G genotype of the c.-31A>G polymorphism, and the C/C-G/G combined genotype of both polymorphisms had a protective effect (odds ratio, 0.48, 0.46, and 0.18; respectively). CONCLUSION: The results obtained suggest the potential role of the g.4235T>C and the c.-31A>G polymorphisms in AMD pathogenesis.

Dexamethasone and 1,25-dihydroxyvitamin D3 reduce oxidative stress-related DNA damage in differentiating osteoblasts.

The process of osteoblast differentiation is regulated by several factors, including RUNX2. Recent reports suggest an involvement of RUNX2 in DNA damage response (DDR), which is important due to association of differentiation with oxidative stress. In the present work we explore the influence of two RUNX2 modifiers, dexamethasone (DEX) and 1,25-dihydroxyvitamin D3 (1,25-D3), in DDR in differentiating MC3T3-E1 preosteoblasts challenged by oxidative stress. The process of differentiation was associated with reactive oxygen species (ROS) production and tert-butyl hydroperoxide (TBH) reduced the rate of differentiation. The activity of alkaline phosphatase (ALP), a marker of the process of osteoblasts differentiation, increased in a time-dependent manner and TBH further increased this activity. This may indicate that additional oxidative stress, induced by TBH, may accelerate the differentiation process. The cells displayed changes in the sensitivity to TBH in the course of differentiation. DEX increased ALP activity, but 1,25-D3 had no effect on it. These results suggest that DEX might stimulate the process of preosteoblasts differentiation. Finally, we observed a protective effect of DEX and 1,25-D3 against DNA damage induced by TBH, except the day 24 of differentiation, when DEX increased the extent of TBH-induced DNA damage. We conclude that oxidative stress is associated with osteoblasts differentiation and induce DDR, which may be modulated by RUNX2-modifiers, DEX and 1,25-D3.

An association of transferrin gene polymorphism and serum transferrin levels with age-related macular degeneration.

Age-related macular degeneration (AMD) is a degenerative disease of the eye, triggered by the damage of the macular cells. In the Western world it is the most frequent cause of blindness in the elderly. Oxidative stress is proved to play a key role in AMD pathogenesis and since iron accumulation has been found in AMD maculas, it may accelerate the oxidative processes in this tissue. In the present work we investigated the association between four polymorphisms of the transferrin gene (rs8177178; rs8177179; rs4481157; rs1130459) and AMD in dependence on the transferrin protein and iron serum levels. We employed PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) for genotype determination, ELISA assay for serum transferrin evaluation and colorimetric assay for measurement of iron concentration in the serum. We found that advanced age and AMD family history may be independent risk factors for AMD (1.02, p < 0.05 and 8.88, p < 0.001, respectively). At the rs4481157 site The GG genotype of the rs4481157 polymorphism decreased the risk of dry AMD (OR 0.50; p < 0.05), while the GA increased this risk (OR 1.07; p < 0.05). Moreover, the GA genotype of this polymorphism decreased the risk of progression to the wet form (OR 0.63; p < 0.05). The analysis of the gene-environment interactions showed that the rs4481157 polymorphism modulates the AMD risk among obese (BMI above 30) individuals. In the former smokers group we observed a moderate association between rs4481157 polymorphism and AMD risk while this association in current smokers was stronger. We found also that the serum level of transferrin was higher in the AMD group (p < 0.001) than in the control, but the total serum iron levels did not differ between both groups. We found that the serum transferrin was associated with the rs8177178 (p < 0.001) and rs4481157 (p < 0.01) polymorphisms, and the common variant (GG) of both sites was related to a lower level of transferrin. Presented data may contribute to the involvement of iron homeostasis in AMD risk.

An association between environmental factors and the IVS4+44C>A polymorphism of the DMT1 gene in age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is an ocular disease affecting macula - the central part of the retina, resulting in the degeneration of photoreceptors and retinal epithelium and causing severe central vision impairment. The pathophysiology of the disease is not completely known, but a significant role is attributed to genetic factors. The contribution of oxidative stress in AMD as a trigger of the degenerative process is well-established. Iron ions may act as a source of reactive oxygen species; therefore, maintaining iron homeostasis is important for redox balance in the organism. Diversity in iron homeostasis genes may counterpart in unbalanced redox state, and thus be involved in AMD pathophysiology. METHODS: In this work, we searched for an association between some single nucleotide polymorphisms in the divalent metal transporter 1 (DMT1) gene intronic IVS4+44C>A (rs224589) and 3'-UTR c.2044T>C (rs2285230) and environmental factors and AMD. Genotyping was performed using the PCR-RFLP method. DNA was obtained from 436 AMD patients and 168 controls. RESULTS: We did not find any association between the genotypes of the two polymorphisms and AMD occurrence. However, we observed that AMD patients living in a rural environment and having the CC genotype of the IVS4+44C>A polymorphism had an increased risk of AMD, while individuals with the CA genotype or the A allele had a decreased risk of the disease. Moreover, in male AMD patients the C allele increased the risk of the disease, while the AA genotype decreased it. CONCLUSIONS: These results suggest that the VS4+44C>A polymorphism of the DMT1 gene may interact with place of living and gender to modulate the risk of AMD.

[Perspectives of RNA interference application in the therapy of diseases associated with defects in alternative RNA splicing]. / Perspektywy wykorzystania interferencji RNA w terapii chorób zwiazanych z zaburzeniami alternatywnego skladania RNA.

The primary transcript of an eukaryotic gene (pre-mRNA) is composed of coding regions--exons intervened by non-coding introns--which are removed in the RNA splicing process, leading to the formation of mature, intron-free mRNA. Alternative splicing of pre-mRNA is responsible for high complexity of the cellular proteome and expresses effective use of genetic information contained in genomic DNA. Alternative splicing plays important roles in the organism, including apoptosis regulation or development and plasticity of the nervous system. The main role of alternative splicing is differential, dependent on conditions and the cell type, splicing of mRNA, generating diverse transcripts from one gene, and, after the translation, different isoforms of a particular protein. Because of the high complexity of this mechanism, alternative splicing is particularly prone to errors. The perturbations resulting from mutations in the key sequences for splicing regulations are especially harmful. The pathogenesis of numerous diseases results from disturbed alternative RNA splicing, and those include cancers and neurodegenerative disorders. The treatment of these conditions is problematic due to their genetic background and currently RNA interference, which is a common mechanism of eukaryotic gene regulation, is being studied. Initial successes in the attempts of silencing the expression of faulty protein isoforms support the idea of using RNA interference in targeting disease related to disturbances in alternative splicing of RNA.

Bisphenol A-glycidyl methacrylate induces a broad spectrum of DNA damage in human lymphocytes.

Bisphenol A-glycidyl methacrylate (BisGMA) is monomer of dental filling composites, which can be released from these materials and cause adverse biologic effects in human cells. In the present work, we investigated genotoxic effect of BisGMA on human lymphocytes and human acute lymphoblastic leukemia cell line (CCRF-CEM) cells. Our results indicate that BisGMA is genotoxic for human lymphocytes. The compound induced DNA damage evaluated by the alkaline, neutral, and pH 12.1 version of the comet assay. This damage included oxidative modifications of the DNA bases, as checked by DNA repair enzymes EndoIII and Fpg, alkali-labile sites and DNA double-strand breaks. BisGMA induced DNA-strand breaks in the isolated plasmid. Lymphocytes incubated with BisGMA at 1 mM were able to remove about 50% of DNA damage during 120-min repair incubation. The monomer at 1 mM evoked a delay of the cell cycle in the S phase in CCRF-CEM cells. The experiment with spin trap-DMPO demonstrated that BisGMA induced reactive oxygen species, which were able to damage DNA. BisGMA is able to induce a broad spectrum of DNA damage including severe DNA double-strand breaks, which can be responsible for a delay of the cell cycle in the S phase.

Lack of association between the c.544G>A polymorphism of the heme oxygenase-2 gene and age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a primary cause of blindness among the elderly in developed countries. The nature of AMD is complex and includes both environmental and hereditary factors. Oxidative stress is thought to be essential in AMD pathogenesis. Iron is suggested to be implicated in the pathogenesis of AMD through the catalysis of the production of reactive oxygen species, which can damage the retina. Heme oxygenase-2 is capable of degradation of heme producing free iron ions, thus, diversity in heme oxygenase-2 gene may contribute to AMD. In the present work we analyzed the association between the c.544G>A polymorphism of the heme oxygenase-2 gene (HMOX2) (rs1051308) and AMD. MATERIAL/METHODS: This study enrolled 276 AMD patients and 105 sex- and age-matched controls. Genotyping of the polymorphism was performed with restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) on DNA isolated from peripheral blood. RESULTS: We did not find any association between the genotypes of the c.544G>A polymorphism and the occurrence of AMD. This lack of association was independent of potential AMD risk factors: tobacco smoking, sex and age. Moreover, we did not find any association between AMD and smoking in our study population. CONCLUSIONS: The results suggest that the c.544G>A polymorphism of the heme oxygenase-2 gene is not associated with AMD in this Polish subpopulation.

The A Allele of the -576G>A polymorphism of the transferrin gene is associated with the increased risk of age-related macular degeneration in smokers.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries, and its pathogenesis is underlined by genetic and environmental factors. Oxidative stress is a major environmental risk factor of AMD; namely, AMD is associated with the increased level of reactive oxygen species, which may be produced in reactions catalyzed by iron present in the retina. Therefore, variability of the genes of iron metabolism may be important in the AMD risk. In the present study, we analyzed the association between AMD and the -576G>A polymorphism of the transferrin gene or the 1892C>T polymorphism of the transferrin receptor 2 (TFR2) gene in 278 patients with AMD and 105 controls. The former polymorphism is located in the promoter region of the transferrin gene and may affect the level of its transcription, while the latter is a synonymous mutation in the exon 16, which may affect the efficiency of translation of TFR2 mRNA. Transferrin and TFR2 are important in iron homeostasis. The A allele of the -576A>G polymorphism was significantly associated with the increased risk of AMD in tobacco smokers, whereas the 1892C>T polymorphism did not influence the risk of AMD related to smoking. Moreover, each polymorphism does not influence the risk of AMD associated with age, sex or the family history of the disease. In conclusion, the A allele of the -576A>G polymorphism of the transferrin gene may increase the risk of AMD in smokers.

[Involvement of nuclear domains in the cellular response to DNA damage]. / Udzial domen jadrowych w reakcji komórki na uszkodzenia DNA.

All cells of human organism are continuously damaged, and a damage of the genetic material can be especially dangerous. The reaction of the cell to DNA damage is a complex process, which includes damage signaling, repair, apoptosis or cell death. It is connected with serious changes in the cell nucleus, which are caused by posttranslational modifications and dynamic relocalizations of proteins as well as alterations in the expression of many genes. These changes are not limited to the sites of DNA damage, but involve whole cell nucleus, including its domains: PML bodies, nucleolus and Cajal bodies.

DNA damage and antioxidant properties of CORM-2 in normal and cancer cells.

In this study, we compared the effect of tricarbonyldichlororuthenium (II) dimer (CORM-2) and its CO-depleted molecule (iCORM-2) on human peripheral blood mononuclear cells (PBMCs) and human promyelocytic leukemia HL-60 cells. We determined cell viability, DNA damage and DNA repair kinetics. We also studied the effect of both compounds on DNA oxidative damage, free radical level and HO-1 gene expression. We showed that at low concentrations both CORM-2 and iCORM-2 stimulate PBMCs viability. After 24-h incubation, CORM-2 and iCORM-2, at the concentration of 100 µM, reduce the viability of both PBMCs and HL-60 cells. We also demonstrated that CORM-2 and iCORM-2, in the 0.01-100 µM concentration range, cause DNA damage such as strand breaks and alkaline labile sites. DNA damage was repaired efficiently only in HL-60 cells. CORM-2 significantly reduces oxidative stress induced by 1 mM H2O2 in normal and cancer cells. On the contrary, iCORM-2 in HL-60 cells increases the level of free radicals in the presence of 1 and 5 mM H2O2. We also revealed that both CORM-2 and iCORM-2 induce HO-1 gene expression. However, CORM-2 induces this gene to a greater extent than iCORM-2, especially in HL-60 cells at 100 µM. Finally, we showed that CORM-2 and iCORM-2 reduce H2O2-induced DNA oxidative damage. Furthermore, CORM-2 proved to be a compound with stronger antioxidant properties than iCORM-2. Our results suggest that both active CORM-2 and inactive iCORM-2 exert biological effects such as cyto- and genotoxicity, antioxidant properties and the ability to induce the HO-1 gene. The released CO as well as iCORM-2 can be responsible for these effects.

DNA damage and methylation induced by organophosphate flame retardants: Tris(2-chloroethyl) phosphate and tris(1-chloro-2-propyl) phosphate in human peripheral blood mononuclear cells.

Phosphorus flame retardants are a group of chemicals that are used to slow or prevent the spread of fire. These compounds have been detected in different environments including human organism. In the present study, we have investigated DNA-damaging potential and effect on DNA methylation of tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP) in human peripheral blood mononuclear cells (PBMCs). In order to determine DNA damage and repair, the alkaline and neutral versions of the comet assay were used. The level of DNA methylation was determined with specific antibodies against methylated DNA. PBMCs were exposed to TCEP and TCPP at the concentrations in the range of 1-1000 µM for 24 h. We have observed that TCEP and TCPP induced DNA damage-DNA breaks and alkali-labile sites. All DNA damages were effectively repaired during 120-min repair incubation. The results have also shown that TCEP and TCPP decreased the level of DNA methylation in PBMCs. In the case of TCEP, this effect was observed at a very low concentration of 1 µM.

Kaempferol derivatives isolated from Lens culinaris Medik. reduce DNA damage induced by etoposide in peripheral blood mononuclear cells.

Bioactive compounds isolated from plants are considered to be attractive candidates for cancer therapy. In this study, we examined the effect of kaempferol, its derivatives, the polyphenol fraction (PF) and an extract (EX) isolated from the aerial parts of Lens culinaris Medik. on DNA damage induced by etoposide in human cells. We also studied the effect of these compounds and their combinations on cell viability. The studies were conducted on HL-60 cells and human peripheral blood mononuclear cells (PBMCs). We used the comet assay in the alkaline version to evaluate DNA damage. To examine cell viability we applied the trypan blue exclusion assay. We demonstrated that kaempferol glycoside derivatives isolated from the aerial parts of Lens culinaris Medik. reduce DNA damage induced by etoposide in PBMCs, but do not have an impact on DNA damage in HL-60 cells. We also showed that kaempferol induces DNA damage in HL-60 cells and leads to an increase of DNA damage provoked by etoposide. Our data suggest that kaempferol derivatives can be further explored as a potential agent protecting normal cells against DNA damage induced by etoposide. Moreover, kaempferol's ability to induce DNA damage in cancer cells and to increase DNA damage caused by etoposide may be useful in designing and improving anticancer therapies.

Photoactive CO-releasing complexes containing iron - genotoxicity and ability in HO-1 gene induction in HL-60 cells.

This paper presents the results of research on the biological properties of two photoactive CO-releasing molecules containing iron, i.e. (η5-C5H5)Fe(CO)2(η1-N-maleimidato) (complex A) and (η5-C5H5)Fe(CO)2(η1-N-succinimidato) (complex B). We studied their cytotoxicity, genotoxicity and the ability of inducing the HO-1 gene in HL-60 cells. We also investigated the kinetics of DNA damage repair induced by complexes A and B. We demonstrated that complex B was not toxic to HL-60 cells in high doses (above 100 µM). The ability to induce DNA damage was higher for complex A. Importantly, there was no difference in irradiated and non-irradiated cells for both complexes. DNA damage induced by complex B was repaired efficiently, while the repair of DNA damage induced by complex A was disturbed. Complex B had a minor effect on HO-1 gene expression (less than 2-fold induction), while complex A had induced HO-1 gene expression to a great extent (over 17-fold for 10 µM) - similarly in irradiated and non-irradiated HL-60 cells. The results of our research indicate that the ability of both complexes to damage DNA and to upregulate HO-1 gene expression is not related to the release of CO. Further research is needed to test whether these compounds can be considered as potential CO carriers in humans.

RUNX2: A Master Bone Growth Regulator That May Be Involved in the DNA Damage Response.

RUNX2 is a member of the RUNX family of transcription factors, also containing the RUNX1 and RUNX3 proteins. These factors control the expression of genes essential for proper development in many cell lineages. RUNX2 plays a crucial role in the proliferation and differentiation of osteoblasts, required for bone formation. The cellular level of RUNX2 oscillates in a cell phase-specific manner, reaching a maximum at G2/M in some cells and overexpression of RUNX2 in osteoblasts blocked G1 to S phase progression. Recent studies have shown that RUNX2 may interact with p53 and change the activity of a histone deacetylase. Moreover, RUNX2 may act as an oncogene in cancer transformation, inevitably associated with genomic instability evoked by increased occurrence of DNA damage. We showed that some RUNX2 modifiers changed the sensitivity of differentiating preosteoblasts to DNA damage induced by oxidative stress. All these data suggest the involvement of RUNX2 in cellular DNA damage response (DDR), which is particularly important in osteogenesis as the process of osteoblast differentiation is associated with increasing oxidative stress. However, the mechanism underlying DDR involvement of RUNX2 is unknown. The basic question, whether RUNX2 plays a positive or destructive role in DDR in differentiating cells is still open.

Transferrin receptor levels and polymorphism of its gene in age-related macular degeneration.

The aim of the present study was to investigate the association of age related macular degeneration (AMD) risk with some aspects of iron homeostasis: iron concentration in serum, level of soluble transferrin receptor (sTfR), and transferrin receptor (TFRC) genetic variability. Four hundred and ninety one AMD patients and 171 controls were enrolled in the study. Restriction fragment length polymorphism PCR was employed to genotype polymorphisms of the TFRC gene, and colorimetric assays were used to determine the level of iron and sTfR. Multiple logistic regression was applied for all genotype/allele-related analyses and the ANOVA test for iron and sTfR serum level comparison. We found that the genotypes and alleles of the c.-253G > A polymorphism of the TFRC gene were associated with AMD risk and this association was modulated by smoking status, AMD family history, living environment (rural/urban), body mass index and age. The levels of sTfR was higher in AMD patients than controls, whereas concentrations of iron did not differ in these two groups. No association was found between AMD occurrence and the p.Gly142Ser polymorphism of the TRFC gene. The results obtained suggest that transferrin receptor and variability of its gene may influence AMD risk.

Variability of the transferrin receptor 2 gene in AMD.

Oxidative stress is a major factor in the pathogenesis of age-related macular degeneration (AMD). Iron may catalyze the Fenton reaction resulting in overproduction of reactive oxygen species. Transferrin receptor 2 plays a critical role in iron homeostasis and variability in its gene may influence oxidative stress and AMD occurrence. To verify this hypothesis we assessed the association between polymorphisms of the TFR2 gene and AMD. A total of 493 AMD patients and 171 matched controls were genotyped for the two polymorphisms of the TFR2 gene: c.1892C>T (rs2075674) and c.-258+123T>C (rs4434553). We also assessed the modulation of some AMD risk factors by these polymorphisms. The CC and TT genotypes of the c.1892C>T were associated with AMD occurrence but the latter only in obese patients. The other polymorphism was not associated with AMD occurrence, but the CC genotype was correlated with an increasing AMD frequency in subjects with BMI < 26. The TT genotype and the T allele of this polymorphism decreased AMD occurrence in subjects above 72 years, whereas the TC genotype and the C allele increased occurrence of AMD in this group. The c.1892C>T and c.-258+123T>C polymorphisms of the TRF2 gene may be associated with AMD occurrence, either directly or by modulation of risk factors.