Anna Gozdzicka-Jozefiak

Epigenetic mechanisms in virus-induced tumorigenesis

About 15–20% of human cancers worldwide have viral etiology. Emerging data clearly indicate that several human DNA and RNA viruses, such as human papillomavirus, Epstein–Barr virus, Kaposi’s sarcoma-associated herpesvirus, hepatitis B virus, hepatitis C virus, and human T-cell lymphotropic virus, contribute to cancer development. Human tumor-associated viruses have evolved multiple molecular mechanisms to disrupt specific cellular pathways to facilitate aberrant replication. Although oncogenic viruses belong to different families, their strategies in human cancer development show many similarities and involve viral-encoded oncoproteins targeting the key cellular proteins that regulate cell growth. Recent studies show that virus and host interactions also occur at the epigenetic level. In this review, we summarize the published information related to the interactions between viral proteins and epigenetic machinery which lead to alterations in the epigenetic landscape of the cell contributing to carcinogenesis.

Estrogen and progesterone receptor expression in HPV-positive and HPV-negative cervical carcinomas.

Human papillomavirus (HPV) is widely accepted as the main cause of cervical cancer. However, the presence of HPV DNA does not inescapably lead to the development of the cancerous phenotype of the infected cell. Therefore, it is considered that the induction of full cancerous expression of HPV requires additional cofactors. The aim of this study was to assess the expression of estrogen receptor α (ERα) and progesterone receptor (PR) in archived tissue blocks of squamous cell carcinoma and adenocarcinoma of the uterine cervix and to ascertain whether expression of these receptors is associated with the presence of HPV DNA. The investigation was performed using formalin-fixed, paraffin-embedded cervical cancer specimens obtained from 250 women who underwent surgery for histologically confirmed neoplastic lesions. The control group consisted of normal cervical tissues obtained from 50 patients who underwent myomectomy. The results of this study revealed that the expression of ER and PR in planoepithelial cancers and adenocarcinomas of the cervix were decreased to undetectable levels. Only in singular cases in the pattern of staining the expression of ER and PR was noted. In stromal cells of the tested neoplasms, higher expression of both types of receptors was found. Comparison of the expression of ER and PR in the staining pattern and stroma of both squamous cell carcinoma and adenocarcioma of the cervix, showed statistically higher expression in the stromal cells. Strong expression (+1, +2, +3) of ER and PR was noted in the stromal cells irrespective of HPV infection, histopathological type of cancer, and clinical and histopathological grade.

Alterations in mtDNA: A qualitative and quantitative study associated with cervical cancer development

OBJECTIVE High-risk human papillomaviruses are the causative agent of cervical carcinogenesis. Additionally, a number of other unknown factors are also instrumental in the development of cancer. The aim of this present study was the analysis of the mutations in the D-loop region of mitochondrial DNA, and 4.997 bp deletion during cervical cancer development. Our research also extended to the relationship between mtDNA copy number, ROS (reactive oxygen species) production and the MnSOD (manganese superoxide dismutase) expression level. METHODS The study group consisted of postoperative tissues from patients diagnosed with L-SIL, H-SIL and squamous cell cervical carcinomas. A quantitative real-time polymerase chain reaction was used to determine the copy number of the mitochondrial DNA, and MnSOD mRNA expression levels. A PCR amplification and a sequencing of DNA were used for the identification of HPV DNA and mtDNA mutations. RESULTS A total of 62 point mutations in the D-loop region of mtDNA were found in study patients. The mitochondrial DNA copy number increased during cervical cancer development when compared to the corresponding tissues in the control samples. About 70% of the mtDNA copy number have a 4.997 bp deletion in L-SIL. We also observed an increase in ROS generation during cervical cancer development. CONCLUSION Alterations in mtDNA both qualitatively (by mutations) and quantitatively (by mtDNA copy number) are associated with cervical cancer developments. High levels of mtDNA copy with a 4.997 bp deletion in L-SIL cells can be associated with the susceptibility of cells to HPV persistent infection and cervical cancer development.

Insulin-like growth factor (IGF) axis in cancerogenesis

Determination of the role of insulin-like growth factor (IGF) family components in carcinogenesis of several human tumors is based on numerous epidemiological and pre-clinical studies, experiments in vivo and in vitro and on attempts at application of drugs affecting the IGF axis. Investigative hypotheses in original studies were based on biological functions manifested by the entire family of IGF (ligands, receptors, linking proteins, adaptor molecules). In the context of carcinogenesis the most important functions of IGF family involve intensification of proliferation and inhibition of cell apoptosis and effect on cell transformation through synthesis of several regulatory proteins. IGF axis controls survival and influences on metastases of cells. Interactions of IGF axis components may be of a direct or indirect nature. The direct effects are linked to activation of PI3K/Akt signaling pathway, in which the initiating role is first of all played by IGF-1 and IGF-1R. Activity of this signaling pathway leads to an increased mitogenesis, cell cycle progression, and protection against different apoptotic stresses. Indirect effects of the axis depend on interactions between IGF and other molecules important for cancer etiology (e.g. sex hormones, products of suppressor genes, viruses, and other GFs) and the style of life (nutrition, physical activity). From the clinical point of view, components of IGF system are first of all considered as diagnostic serous and/or tissue biomarkers of a given cancer, prognostic factors and attractive target of modern anti-tumor therapies. Several mechanisms in which IGF system components act in the process of carcinogenesis need to be clarified, mainly due to multifactorial etiology of the neoplasms. Pin-pointing of the role played in carcinogenesis by any single signaling pathway remains particularly difficult. The aim of this review is to summarize the current data of several epidemiological studies, experiments in vitro and on animal models, to increase our understanding of the complex role of IGF family components in the most common human cancers.

Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells

BackgroundCorydalis cava Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group Chelidonium majus L. (Greater Celandine), belong to the family Papaveraceae. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The Corydalis sp. tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in Corydalis cava tubers.MethodsNucleolytic proteins were isolated from the tubers of C. cava by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS).ResultsThe studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml) - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR) proteins.ConclusionsThe cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most probably, represents the first investigations on the effect of purified PR proteins from tuber extracts of a pharmacologically active plant on cell lines.

Analysis of coding and promoter sequences of the IGF-I gene in children with growth disorders presenting with normal level of growth hormone.

The insulin-like growth factor-I (IGF-I) gene was analyzed in a population of children with growth disorders presenting normal GH and low IGF-I. We thus tried to detect any mutation in the IGF-I gene that could be responsible for short stature in children, using PCR, single-strand conformation polymorphism (SSCP) analysis, followed by DNA cloning and sequencing. We demonstrated in all examined children significant changes in the promoter region of the IGF-I gene (P1 IGF-I). Nucleotide sequence changes, such as CC-->GT and A-->G, and their localization are described. The results obtained excluded mutations in the coding sequence of the IGF-I gene. We conclude that testing the IGF-I P1 region, using PCR/SSCP analysis, could be useful in the diagnosis of growth disorders.

Study on the zona pellucida 4 (ZP4) gene sequence and its expression in the ovaries of patients with polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine disorder of unknown pathology, involving reproductive and metabolic abnormalities. Oocyte-specific genes are a group of genes expressed exclusively in ovarian tissue; therefore, they can play an important role in ovarian pathologies such as PCOS. The zona pellucida 4 (ZP4) gene encodes glycoprotein which is a part of the extracellular matrix of oocyte.Materials and methodsWe analyzed 87 patients with PCOS, which were divided into four groups depending on their phenotype. In each patient, we performed profound clinical and biochemical analysis, including the measurement of serum androgens. The ovarian tissue samples were used to perform a real-time polymerase chain reaction and immunohistochemical staining using anti-ZP4 monoclonal antibodies. The ZP4 gene was sequenced from peripheral lymphocytes.ResultsThe expression of ZP4 was present in early antral follicles and was stronger in mature follicles. The subgroup of patients with eumenorrhea and without hyperandrogenism presented the highest expression of ZP4 in ovarian tissue. In one case, we found a mutation of the ZP4 gene. No correlations were found between the ZP4 expression level and biochemical or clinical indices.ConclusionsData from this and animal studies suggest a possible relationship between androgens and ZP4 expression. ZP4 expression is highest among patients with PCOS and a regular cycle, and this is a consequence of the presence of mature follicles in this group. In some patients with PCOS and infertility, ZP4 mutation can be found.

Analysis of the human growth hormone receptor and IGF-I coding sequences in children with growth disorders.

Analysis of GHR and IGF-I coding sequences in 47 children with normal serum levels of GH, low IGF-I and growth disorders generally did not show mutation in the genes studied. Only one boy had a mutation located in the fifth exon of the GHR gene (C-->T in codon 88). This suggests that the growth disorders in this group of children might be due to a defect in a DNA region regulating expression of the GHR and IGF1 genes or genes involved in their regulation.

Presence of irregularity in region between −1115 and −784 nt in P1 promoter of Insulin-Like Growth Factor-1 gene may indicate beneficial effect on coronary arteries in a group of patients with stable angina: preliminary data

Insulin-like growth factor-1 (IGF-1) plays an important role in arterial homeostasis. Its properties seem to depend on circulating IGF-1 level changes. The various IGF-1 levels are caused by varied expression of IGF-1 gene, due to the polymorphic structure of IGF-1 gene or its regulatory sequences. We examined the P1 promoter, being responsible for most IGF-1 transcripts, in patients with stable angina, to evaluate its sequence changes and to assess its influence on protein synthesis as well as on the degree of arteriosclerosis. For that purpose we evaluated the DNA isolated from blood cells. The DNA was amplified by using polymerase chain reaction (PCR), then analyzed using the SSCP (single-strand conformation polymorphism) technique. Products of every stage were verified by electrophoresis on agarose gel. In addition, every patient had coronary angiography performed and IGF-1, IGFBP3, and lipid levels measured. The SSCP in the region between −1115 and −784 nt was less commonly observed among subjects with positive MI (myocardial infarction) familial history (P = 0.0008) and with MI history (P = 0.012) than in patients without these conditions. Subjects with this irregularity tended towards higher circulating IGF-1 levels. In addition high Gensini scores — over 95th percentile, 105 points in our study — were more frequent in SSCP patients (P = 0.03). We presume that presence of SSCP in the P1 region between −1115 and −784 nt may positively affect coronary arteries by increasing circulating IGF-1 levels, but its clinical importance requires molecular verification and further studies.

Analysis of cytosine-adenine repeats in P1 promoter region of IGF-1 gene in peripheral blood cells and cervical tissue samples of females with cervical intraepithelial lesions and squamous cervical cancer.

High oncogenic risk human papillomaviruses (HPVs) are closely associated with cancer of the cervix. However, HPV infection alone may not be sufficient to cause cervical cancer, and other factors or cofactors may have a cumulative effect on the risk of progression from cervical HPV infection to cancer. The present study investigates the cytosine-adenine (CA) repeat polymorphism in the P1 promoter region of the insulin-like growth factor-1 (IGF-1) gene among cervical precancerous and cancer patients and healthy control females. The association between these polymorphisms, tissue and blood serum levels of IGF-1, and cervical cancer risk and progression is evaluated. The material for analysis consisted of blood cells and postoperative tissues from patients diagnosed with low-grade squamous intraepithelial lesions (L-SILs), high-grade squamous intraepithelial lesions (H-SILs) and invasive cervical cancer (ICC). A polymerase chain reaction amplification and the sequencing of DNA were used for the identification of (CA)n repeats in the IGF-1 P1 region and detection of HPV DNA. The blood serum concentration of IGF was determined by enzyme-linked immunosorbent assay. The identification of the IGF-1 protein in the cervical tissues was performed by immunohistochemical analysis. The range of the length of the CA repeats in the study DNA was 11 to 21. However, the most common allele length and genotype in the control and study patients from serum and tissues was 19 CA repeats and a homozygous genotype of CA19/19. Statistically significant differences in the concentration of IGF-1 in the blood serum were observed between H-SILs and controls, only (p=0.047). However, the concentration of IGF-1 in the group of females with CA19/19, CA19<19 and CA19>19 was significantly higher in the group of patients with H-SIL (P=0.041) and ICC (P=0.048) in comparison with the control group. An association was detected between CA repeat length <19 and/or >19, IGF concentration in blood serum and tissues and the development of cervical cancer.