Tomasz Poplawski

Tyrosine Kinase Blockers: New Hope for Successful Cancer Therapy

Tyrosine kinases (TKs) are attractive targets for cancer therapy, as quite often their abnormal signaling has been linked with tumor development and growth. Constitutive activated TKs stimulate multiple signaling pathways responsible for DNA repair, apoptosis, and cell proliferation. During the last few years, thorough analysis of the mechanism underlying tyrosine kinases activity led to novel cancer therapy using TKs blockers. These drugs are remarkably effective in the treatment of various human tumors including head and neck, gastric, prostate and breast cancer and leukemias. The most successful example of kinase blockers is Imatinib (Imatinib mesylate, Gleevec, STI571), the inhibitor of Bcr/Abl oncoprotein, which has become a first-line therapy for chronic myelogenous leukemia. The introduction of STI571 for the treatment of leukemia in clinical oncology has had a dramatic impact on how this disease is currently managed. Others kinase inhibitors used recently in cancer therapy include Dasatinib (BMS-354825) specific for ABL non-receptor cytoplasmic kinase, Gefitinib (Iressa), Erlotinib (OSI-774, Tarceva) and Sunitinib (SU 11248, Sutent) specific for VEGF receptor kinase, AMN107 (Nilotinib) and INNO-406 (NS-187) specific for c-KIT kinase. The following TK blockers for treatment of various human tumors are in clinical development: Lapatinib (Lapatinib ditosylate, Tykerb, GW-572016), Canertinib (CI-1033), Zactima (ZD6474), Vatalanib (PTK787/ZK 222584), Sorafenib (Bay 43-9006, Nexavar), and Leflunomide (SU101, Arava). Herein, we discuss the chemistry, biological activity and clinical potential of new drugs with tyrosine kinase blockers for cancer treatment.

Genotoxicity and cytotoxicity of 2-hydroxyethyl methacrylate

Resin-based methacrylate materials are widely used in restorative dentistry. They are viscous substances that are converted into solid material via polymerization. This process, however, may be incomplete, leading to the release of monomers into the oral cavity and the pulp, which can be reached through the dentin micro-channels. This opens the opportunity for the monomers to reach the bloodstream. Monomers can reach concentrations in the millimolar range, high enough to cause cellular damage, so it is justified to study their potential toxic effects. In the present work we investigated the cytotoxicity and genotoxicity of 2-hydroxyethyl methacrylate (HEMA) in human peripheral blood lymphocytes and A549 lung-tumour cells. HEMA at concentrations up to 10mM neither affected the viability of the cells nor interacted with isolated plasmid DNA during a 1h exposure. However, HEMA induced concentration-dependent DNA damage in lymphocytes, as assessed by alkaline and pH 12.1 versions of the comet assay. HEMA did not cause double-strand breaks, as assessed by the neutral version of the comet assay and pulsed-field gel electrophoresis. The use of DNA repair enzymes, spin traps and vitamin C produced results suggesting that HEMA induced oxidative modifications to DNA bases. DNA damage caused by HEMA at 10mM was removed within 120min. HEMA induced apoptosis in a concentration-dependent manner and caused cell-cycle delay at the G0/G1-checkpoint. Methylglycol chitosan displayed a protective effect against the DNA-damaging action of HEMA. The results obtained in this study suggest that HEMA induces adverse biological effects, mainly via reactive oxygen species, which can lead to DNA damage, apoptosis and cell-cycle delay. Chitosan and its derivatives can be considered as additional components of dental restoration to decrease the harmful potency of HEMA.

BCR/ABL Stimulates WRN to Promote Survival and Genomic Instability

BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSB) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), nonhomologous end-joining (NHEJ), and single-strand annealing (SSA). Here, we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTK), such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC-mediated activation of transcription and Bcl-xL-dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability.

Cytotoxicity and genotoxicity of glycidyl methacrylate.

Methacrylates are used in the polymer form as composite restorative materials in dentistry. However, the polymers can release monomers and co-monomers into the oral cavity and pulp, from where they can migrate into the bloodstream reaching virtually all organs. The local concentration of the released monomers can be in the millimolar range, high enough to induce adverse biological effects. Genotoxicity of methacrylate monomers is of a special significance due to potential serious phenotypic consequences, including cancer, and long latency period. In the present work, we investigated cytotoxicity and genotoxicity of glycidyl methacrylate (GMA) in the human peripheral blood lymphocytes and the CCR-CM human cancer cells. GMA at concentrations up to 5mM evoked a concentration-dependent decrease in the viability of the lymphocytes up to about 80%, as assessed by flow cytometry. This agent did not induce strand breaks in the isolated plasmid DNA, but evoked concentration-dependent DNA damage in the human lymphocytes evaluated by the alkaline and neutral comet assay. This damage included oxidative modifications to the DNA bases, as checked by DNA repair enzymes Endo III and Fpg as well as single and double DNA strand breaks. The lymphocytes exposed to GMA at 2.5 microM were able to remove about 90% of damage to their DNA in 120 min. The ability of GMA to induce DNA double-strand breaks was confirmed by pulsed field gel electrophoresis. The drug evoked apoptosis and induced an increase in the G2/M cell population, accompanied by a decrease in the S cell population and an increase in G0/G1 cell population. Due to broad spectrum of GMA genotoxicity, including DNA double-strand breaks, and a potential long-lasting exposure to this compound, its use should be accompanied by precautions, reducing the chance of its release into blood stream and the possibility to induce adverse biological effects.

Therapeutic effect of melatonin in patients with functional dyspepsia

Background and Goal Melatonin may inhibit reactive oxygen species-related pathogenesis in the alimentary tract by neutralizing free radicals. In the present study we assessed the potential protective action of melatonin in ulcerlike dyspepsia. Study Sixty patients aged 19 to 39 years with the diagnosis of functional dyspepsia according to the Rome Criteria II and no Helicobacter pylori infection were involved in the study. Melatonin, at a dose of 5 mg (n=30), or placebo (n=30) were taken in the evening for a period of 12 weeks. At this time, patients were on an equivalent diet and were only to take an alkaline drug in case of the abdominal pain. Results After 12 weeks, the dyspeptic symptoms completely subsided in 17 patients in the melatonin-treatment group (56.6%). In other 9 individuals (30.0%) a partial improvement in health was achieved, especially in the frequency and intensity of nocturnal pain. After placebo, the majority of patients (93.3%) did not experience any improvement in symptoms. Multivariate analysis indicated that melatonin (odds ratio 95.86, 95% confidence interval 3.72-2469.37, P<0.01) correlated independently with significantly improved patients health. H. pylori past infection decreased positive effect of melatonin in ulcerlike dyspepsia. Conclusions Melatonin can be considered as an auxiliary drug in the treatment of ulcerlike dyspepsia.

Mutations in the PAX9 gene in sporadic oligodontia

OBJECTIVES Oligodontia, a congenital lack of six or more teeth, is often associated with mutations in the PAX9 gene; therefore, we searched for mutations in this gene. DESIGN In the present work, we sequenced fragments of the PAX9 gene in individuals with sporadic oligodontia. Next, we genotyped some mutations we found in patients with oligodontia and individuals without tooth agenesis. SETTING AND SAMPLE POPULATION DNA sequencing was performed in the material isolated from peripheral blood lymphocytes of six unrelated patients with sporadic, non-syndromic oligodontia. These patients were selected based upon explorative cluster analysis. Genotyping was performed in 38 patients with oligodontia and 100 control individuals. MATERIAL AND METHODS Direct sequencing and restriction fragment length polymorphism PCR were employed. RESULTS We detected two homozygotic substitutions, IVS2-109G>C and IVS2-54A>G, in intron 2 in three patients. Another homozygotic substitution in intron 2, IVS2-41A>G, was revealed in two patients. Two patients had an IVS3+40G>A homozygotic change in intron 3 and 4 patients displayed a 717C>T transition in exon 4 (silent mutation). One patient had a heterozygotic 718G>C transversion, resulting in a missense Ala240Pro substitution. We detected also several other intronic substitutions. Further genotyping of the IVS2-54A>G, IVS2-109G>C, and IVS2-41A>G mutations suggested that they can display polymorphic changes. CONCLUSION The IVS2-54A>G, IVS2-109G>C, and IVS2-41A>G mutations of the PAX9 gene may represent polymorphism associated with sporadic oligodontia.

Polymorphisms of the DNA Mismatch Repair Gene HMSH2 in Breast Cancer Occurence and Progression

SummaryThe response of the cell to DNA damage and its ability to maintain genomic stability by DNA repair are crucial in preventing cancer initiation and progression. Therefore, polymorphism of DNA repair genes may affect the process of carcinogenesis. The importance of genetic variability of the components of mismatch repair (MMR) genes is well documented in colorectal cancer, but little is known about its role in breast cancer. hMSH2 is one of the crucial proteins of MMR. We performed a case-control study to test the association between two polymorphisms in the hMSH2 gene: an A → G transition at 127 position producing an Asn → Ser substitution at codon 127 (the Asn127Ser polymorphism) and a G → A transition at 1032 position resulting in a Gly → Asp change at codon 322 (the Gly322Asp polymorphism) and breast cancer risk and cancer progression. Genotypes were determined in DNA from peripheral blood lymphocytes of 150 breast cancer patients and 150 age-matched women (controls) by restriction fragment length polymorphism and allele-specific PCR. We did not observe any correlation between studied polymorphisms and breast cancer progression evaluated by node-metastasis, tumor size and Bloom-Richardson grading. A strong association between breast cancer occurrence and the Gly/Gly phenotype of the Gly322Asp polymorphism (odds ratio 8.39; 95% confidence interval 1.44–48.8) was found. Therefore, MMR may play a role in the breast carcinogenesis and the Gly322Asp polymorphism of the hMSH2 gene may be considered as a potential marker in breast cancer.

Genotoxicity of urethane dimethacrylate, a tooth restoration component.

Urethane dimethacrylate (UDMA) is used in dental restorative materials in its polymeric form. However, the process of polymerization is usually incomplete and the monomers of UDMA can diffuse into the oral cavity and the pulp, reaching millimolar concentrations. In the present work we showed that UDMA at 0.1 and 1.0 mM decreased the viability of and induced DNA damage in lymphocytes in a concentration dependent manner, but it did not affect a plasmid DNA in vitro. UDMA at 1mM induced apoptosis in lymphocytes. The lymphocytes exposed to UDMA were able to repair their DNA within 60 min. Analysis with DNA repair enzymes Endo III and Fpg showed that UDMA induced mainly oxidative DNA lesions. Vitamin C and chitosan decreased genotoxic effect of UDMA. Our results show that monomers of UDMA may exert pronounced cyto- and genotoxic effects in human lymphocytes and chitosan can be considered as a protection against such effects.

DNA Damage/Repair and Polymorphism of the hOGG1 Gene in Lymphocytes of AMD Patients

Oxidative stress is thought to play a role in the pathogenesis of age-related macular degeneration (AMD). We determined the extent of oxidative DNA damage and the kinetics of its removal as well as the genotypes of the Ser326Cys polymorphism of the hOGG1 gene in lymphocytes of 30 wet AMD patients and 30 controls. Oxidative DNA damage induced by hydrogen peroxide and its repair were evaluated by the comet assay and DNA repair enzymes. We observed a higher extent of endogenous oxidative DNA damage and a lower efficacy of its repair in AMD patients as compared with the controls. We did not find any correlation between the extent of DNA damage and efficacy of DNA repair with genotypes of the Ser326Cys polymorphism. The results obtained suggest that oxidative DNA damage and inefficient DNA repair can be associated with AMD and the variability of the hOOG1 gene may not contribute to this association.

BCR/ABL downregulates DNA-PK CS -dependent and upregulates backup non-homologous end joining in leukemic cells

Non-homologous end joining (NHEJ) and homologous recombination repair (HRR) are the main mechanisms involved in the processing of DNA double strand breaks (DSBs) in humans. We showed previously that the oncogenic tyrosine kinase BCR/ABL stimulated DSBs repair by HRR. To evaluate the role of BCR/ABL in DSBs repair by NHEJ we examined the ability of leukemic BCR/ABL-expressing cell line BV173 to repair DNA damage induced by two DNA topoisomerase II inhibitors: etoposide and sobuzoxane. DNA lesions induced by sobuzoxane are repaired by a NHEJ pathway which is dependent on the catalytic subunit of protein kinase dependent on DNA (DNA-PKCS; D-NHEJ), whereas damage evoked by etoposide are repaired by two distinct NHEJ pathways, dependent on or independent of DNA-PKCS (backup NHEJ, B-NHEJ). Cells incubated with STI571, a highly specific inhibitor of BCR/ABL, displayed resistance to these agents associated with an accelerated kinetics of DSBs repair, as measured by the neutral comet assay and pulsed field gel electrophoresis. However, in a functional NHEJ assay, cells preincubated with STI571 repaired DSBs induced by a restriction enzyme with a lower efficacy than without the preincubation and addition of wortmannin, a specific inhibitor of DNA-PKCS, did not change efficacy of the NHEJ reaction. We suggest that BCR/ABL switch on B-NHEJ which is more error-prone then D-NHEJ and in such manner contribute to the increase of the genomic instability of leukemic cells.