Evaluation of the prognostic value of fibrinolytic elements in invasive breast carcinoma patients.

Breast cancer (BrC) is one of the most serious oncological problems in the world. The aim of the study was to evaluate concentrations of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) and their ratios: t-PA/PAI-1 and PAI-1/t-PA in breast cancer patients and in healthy individuals and to estimate the ability of fibrinolytic parameters in predicting neoplasm disease and disease relapse. One hundred and five women were enrolled in the study, including 60 cases with primary BrC, (M0) and 45 healthy females. Follow-up was completed in all BrC patients with a 16.7% recurrence rate. An immunoassay of t-PA, PAI-1 in all cases was made as well as the immunohistochemistry of estrogen and progesterone receptors, human epidermal growth factor receptor 2, E-cadherin, and Ki-67 was performed in BrC subjects. A significantly higher PAI-1 concentration in breast cancer patients below the age of 55 than in controls was obtained. According to the ROC curve analysis, the PAI-1 concentration demonstrates the most accurate prognostic value with the cut-off point at 33.91 ng/ml, with 90% sensitivity and 36% specificity, which discriminates between controls and cancer patients. However, t-PA presents the highest area under the receiver-operating characteristic curves (AUCROC)=0.634 in predicting disease relapse with the cut-off value of 5.3 ng/ml. According to the Kaplan-Meier curves, a high concentration of t-PA (>5 ng/ml) and a lower PAI-1/t-PA ratio (<7.5) are associated with shorter survival. Evaluation of plasma t-PA and PAI-1 concentrations may deliver relevant prognostic information for breast cancer patients.

The redox status of human breast cancer cell lines (MCF-7 and MDA-MB231) treated with novel dinuclear berenil-platinum(II) complexes.

This study compared the effects of cisplatinum and novel berenil-platinum(ll) complexes on the redox status of breast cancer cells that were estrogen receptor-positive (MCF-7) or estrogen receptor-negative (MDA-MB231). Both cell lines were treated with cisplatinum or the following berenil-platinum(ll) complexes: Pt2(isopropylamine)4(berenil)2, Pt2(piperidine)4(berenil)2, Pt2(2-picoline)4(berenil)2, Pt2(3-picoline)4(berenil)2, and Pt2(4-picoline)4(berenil)2. Changes in levels of reactive oxygen species, levels and activities of antioxidants, and lipid peroxidation products levels were measured. All investigated compounds enhanced ROS generation, reduced the activity of antioxidant enzymes (e.g., glutathione peroxidase and glutathione reductase), and decreased levels of small-molecule antioxidants (GSH, vitamins E and A). Such conditions are conducive to generating oxidative stress and phospholipids peroxidation. Cellular phospholipids in MCF-7 cells were most sensitive to the Pt2(isopropylamine)4(berenil)2 complex, whereas MDA-MB231 cells were not particularly sensitive to any berenil-platinum(ll) complex. These findings will facilitate future anticancer drug design strategy for breast cancer pharmacotherapy.

Micro-mechanical fingerprints of the rat bladder change in actinic cystitis and tumor presence.

Tissue mechanics determines tissue homeostasis, disease development and progression. Bladder strongly relies on its mechanical properties to perform its physiological function, but these are poorly unveiled under normal and pathological conditions. Here we characterize the mechanical fingerprints at the micro-scale level of the three tissue layers which compose the healthy bladder wall, and identify modifications associated with the onset and progression of pathological conditions (i.e., actinic cystitis and bladder cancer). We use two indentation-based instruments (an Atomic Force Microscope and a nanoindenter) and compare the micromechanical maps with a comprehensive histological analysis. We find that the healthy bladder wall is a mechanically inhomogeneous tissue, with a gradient of increasing stiffness from the urothelium to the lamina propria, which gradually decreases when reaching the muscle outer layer. Stiffening in fibrotic tissues correlate with increased deposition of dense extracellular matrix in the lamina propria. An increase in tissue compliance is observed before the onset and invasion of the tumor. By providing high resolution micromechanical investigation of each tissue layer of the bladder, we depict the intrinsic mechanical heterogeneity of the layers of a healthy bladder as compared with the mechanical properties alterations associated with either actinic cystitis or bladder tumor.

Assessment of leptin-to-adiponectin ratio in prediction of insulin resistance and nutrition status in a geriatric female population.

Both obesity and malnutrition leading to cachexia and sarcopenia are relevant risk factors in the development of many diseases. They also increase mortality, also prolong hospitalisations and convalescence, and undoubtedly increase the cost of treatment, mostly in the elderly populations. The aim of the study was to assess the relationship between the levels of leptin and adiponectin with regard to insulin resistance and malnutrition status by studying a senior female population and to evaluate predictors of insulin resistance and malnutrition. A total of 88 elderly females were enrolled prospectively with a median age of 75 years. Anthropometric and biochemical parameters (fasting glucose, insulin, folic acid, vitamin B12 concentrations, lipid profile, complete blood count) were recorded along with a full geriatric assessment, have been made in all participants. A comprehensive nutritional phenotype has been established. Leptin and adiponectin concentrations were measured by applying immunoassay techniques. Lipid profile and other parameters were performed by biochemical methods. We observed significant decreases of albumin, alanine aminotransferase, insulin, and triglycerides concentrations with age. The risk of insulin resistance based on HOMA-IR index was decreased with age. Significantly higher concentrations of leptin, leptin-to-adiponectin ratio (LAR), hsCRP, fasting glucose, insulin in the insulin resistant subgroup in respect of normal sensitivity insulin cases were noted. The concentrations of albumin, aspartate aminotransferase, alanine aminotransferase and total cholesterol were significantly lower in those patients at risk of malnutrition than in the well-nourished subjects. LAR reached the most accurate AUCROC = 0.705 for insulin resistance prediction, with a cut-off value at 3.85. The greatest diagnostic power was presented by the albumin concentration with AUCROC = 0.761 and then LAR 0.718 in discriminating between well-nourished patients and those at risk of malnutrition. We suggest that the leptin-to-adiponectin ratio is suitable as a marker of insulin resistance and nutritional status in the elderly.

Heparanase link between vasculogenesis and angiogenesis as well as a predictive factor of a shorter survival rate.

Heparanase concentration is low in normal epithelia cells but its overexpression is reported in many carcinomas, including sarcomas and haematological malignancies. The purpose of this study was to investigate the association with selected angiogenic parameters as well as in the number of circulating endothelial progenitors (EPCs) in respect to low, moderate and high concentrations of heparanase. Also, we estimated the diagnostic usefulness of the heparanase concentration for disease recurrence prediction in breast cancer cases. Eighty-six patients with IA-IIB stage invasive breast carcinoma who passed a comprehensive clinicopathologic evaluation were included in the study. The median tumour diameter was 1.5 cm. Twenty cases showed lymph node metastasis (N1). Follow-up was completed in all patients a median follow-up was 33.5 months with a 11.6% recurrence rate. An immunoassay of selected angiogenic parameters, heparanase, as well as an immunohistochemistry of oestrogen and progesterone receptors, human epidermal growth factor receptor 2 (HER2), Ki67 and E-cadherin was performed in all cases. Circulating EPCs were determined by flow cytometry. Higher levels of heparanase in oestrogen and progesterone receptor negative cancers than in positive ones were noted. A higher concentration of heparanase was observed in T2 cases than T1 subjects. Significant positive associations between circulating EPCs, soluble forms of VEGF receptors and increasing plasma levels of heparanase were obtained. Follow-up revealed a significantly higher incidence of disease relapse in breast cancer patients with high baseline concentrations of heparanase. Heparanase was the most accurate biomarker with an AUCROC = 0.72. The cut-off value of 213.74 pg/mL was identified in order to discriminate between disease recurrence patients and those without disease relapse. We suggest, that a high concentration of heparanase next to tumour size and oestrogen and progesterone receptor expression may serve as an indicator of a more an aggressive character of tumour cells and a shorter survival rate.

Human DNA topoisomerase inhibitors from Potentilla argentea and their cytotoxic effect against MCF-7.

Two polyphenolics, kaempferol 3-O-beta-D-(6"-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) and methyl brevifolincarboxylate (2) isolated from aerial parts of Potentilla argentea L. (Rosaceae) were evaluated for their cytotoxicities against human breast carcionoma cell line (MCF-7) and their DNA-binding ability. The DNA-binding ability of these compounds was studied by means of the human DNA topoisomerase I and II inhibition assay and ethidium displacement assay using calf thymus DNA, poly(dA-dT)2 and poly(dG-dC)2. Compound 2 was much more active and showed a higher level of cytotoxic potency than compound 1, with IC50 values of 1.11 +/- 2 microM and 21.60 +/- 2 microM, respectively. In DNA topoisomerase I and II inhibition in vitro assays both investigated compounds 1 and 2 were more effective against topoisomerase II than I. The results of DNA binding studies reveal that methyl brevifolincarboxylate had a greater DNA binding affinity that tiliroside, which correlates with its greater potency as a topoisomerase I/II inhibitor.

Elevated plasma levels of tissue factor as a valuable diagnostic biomarker with relevant efficacy for prediction of breast cancer morbidity.

The aim of this study was to evaluate the concentrations of tissue factor (TF) and its inhibitor (TFPI), vascular endothelial growth factor A (VEGF-A), soluble forms of VEGF receptors type 1 and 2 (sVEGFR1 and aVEGFR2) in patients diagnosed with luminal A breast cancer (BrC) and in healthy individuals and to find associations of analyzed factors with demographic, clinical and pathological characteristics in a homogeneous breast cancer group. Study group consisted of 60 women aged 40 - 69 years, diagnosed with luminal-A subtype of BrC, without distant metastases (M0). Control group comprised 40 healthy women aged 45 - 63 years. Blood samples were collected from each patient in order to determine plasma levels of TF, TFPI, VEGF-A and sVEGFR1 and sVEGFR2. The examined parameters were measured by enzyme-linked immunosorbent assay (ELISA). The capacity of angiogenic and hemostatic parameters in predicting neoplasm disease was analyzed using receiver operating characteristic (ROC) curve analysis. According to ROC curve analysis, the optimum cut-off point for TF was 304.58 pg/ml, with 100% sensitivity and 100% specificity, which was calculated to discriminate between controls and malignancy patients. In luminal A BrC patients there were significantly higher concentrations of VEGF-A and TF than in controls. On the contrast the levels of sVEGF receptors type 1 and 2 as well as TFPI in luminal-A BrC cases were significantly lower in respect to healthy volunteers. Levels of examined factors in the study group varied depending on age, menopausal status, lymph node involvement and histological type. We concluded that altered levels of examined factors in patients diagnosed with luminal-A breast cancer indicate increased activation of angiogenesis and hemostasis. The results obtained may be indicative of a mutual connection between angiogenesis and hemostasis processes in tumor development and progression. Clinical and pathological parameters may possibly affect levels of angiogenic and coagulation factors.

Increased number of endothelial progenitors in peripheral blood as a possible early marker of tumour growth in post-menopausal breast cancer patients.

The aim of the study was to evaluate the number of circulating endothelial progenitor cells (circulating EPCs) in the blood of patients diagnosed with breast cancer and to make an attempt at finding associations with the number of circulating EPCs and selected clinic-pathological factors; TNM and histological grading, molecular subtype of breast cancer, hormonal status, the expression of Ki-67 and the size of tumour. The study involved 96 Caucasian ethnicity post-menopausal women. Sixty-six women aged 48 - 63 (mean age 55) with breast cancer diagnosis without distant metastases (M0). The median value of the tumour diameter was 1.51 cm. The control group consisted of 30 healthy, non-smoking, post-menopausal women, mean age 49, range 44 - 54 years of age. The exclusion criteria for all the participants were hypertension, hyperlipidaemia, and hyperglycaemia, acute and chronic infection. With regard to the fresh blood samples the number of circulating endothelial progenitors was determined using flow cytometry. The fluorescence of 100,000 cells was measured during the analysis. Circulating EPCs were identified with the immune-phenotype CD45-, CD34+, CD133+, CD31+. A significantly higher number of circulating EPCs in the study group, as compared to the controls (P = 0.0001) and a significantly higher number of circulating EPCs in women over 60 with breast cancer than in the younger women (P = 0.0029) were reported. A positive correlation was noted between circulating EPCs and age as well as between circulating EPCs and HER-2 (P = 0.0231, P = 0.0414, respectively), and a negative correlation between circulating EPCs and histological grading of breast cancer (P = 0.0272). The study has shown a higher number of circulating EPCs in breast cancer patients, which indicates stimulation of neovascularization. Additionally, since bone morrow-derived circulating EPCs are more intensively mobilised in older and overweight breast cancer patients, we can speculate that more aggressive neo-angiogenesis can occur in those patients.

Involvement of the host initiator function dnaA in the replication of coliphage lambda.

We demonstrate that the initiation of coliphage lambda DNA replication is dependent on the host initiator function dnaA, provided that the lambdoid prophage Rac is absent. Presence of Rac compensated the absence of dnaA function, causing initiation of replication. In dnaAts rac+ cells at 43 degrees, most of parental phage DNA molecules, after one round of theta replication, switched to a replication with features of the sigma mode and produced progeny at high yield. Initiation of replication of the lambda Pts1 mutant at 43 degrees was blocked by dnaA function; however, under dnaA-rac+ conditions all parental phage DNA molecules, after one round of theta replication, switched to the sigma mode and produced progeny at high yield. Taking into account our recent finding that transcriptional activation of ori lambda seems to be dnaA-regulated (to be published elsewhere), we suggest that the DnaA-lambda Pts1 incompatibility occurs at the insertion of the ori lambda-bound lambda O-lambda P-DnaB preprimosome between the complementary lambda DNA strands. The role of Rac and the mechanism of the switch from theta to sigma mode of lambda phage DNA replication are discussed.

The diverse signaling network of EGFR, HER2, HER3 and HER4 tyrosine kinase receptors and the consequences for therapeutic approaches.

The HER family of receptor tyrosine kinase couples binding of extracellular growth factor ligands to intracellular signal transduction pathways, contributing in this fashion to the ability of the cell to respond correctly to its environment. The HER family and its ligands are critically involved in the carcinogenesis of the mammary gland. Abnormal function of the members of HER family resulting in receptor hyper-activation (due to gene amplification, protein overexpression or abnormal transcriptional regulation) has been linked with breast cancer prognosis. It is also extensively studied as the predictive factor and target for therapy. There are clinical indications supporting the concept that none of the receptors: EGFR, HER2, HER3 and HER4 can be considered as the stand-alone receptor in breast cancer development and clinical course of the disease. There is a growing body of evidence that cooperation between them contributes to more aggressive tumor phenotype and influences the response to therapy. This underlines the importance of quantification of all HER family members and indicates the urgent need for implementation of methods that can efficiently and reliably examine four HER receptors as a whole panel in breast cancer patients.

An epidermal growth factor receptor intron 1 polymorphism in healthy women in Poland.

The frequency of CA allele combinations was assessed in healthy women from Poland and compared to previously published polymorphism data of individuals from Germany and a Caucasian reference group. There were close similarities between these three geographically and ethnically similar populations. By contrast, the distribution of these alleles in European and Asian (Japan) populations proved to be different. There might therefore be major ethnic differences in allelic frequencies of EGFR intron 1 polymorphism. Our results provide new data on EGFR microsatellite instability and may contribute to the understanding of EGFR gene expression regulation. The clinical relevance of these findings warrants further evaluation.

Dynamics of hepatitis B virus quasispecies heterogeneity in association with nucleos(t)ide analogue treatment determined by MALDI-TOF MS.

Minor drug-resistant variants may preexist in every subject infected with hepatitis B virus (HBV). However, understanding the dynamic of genotypic evolution within the HBV population requires accurately following allele frequencies through time. We used MALDI-TOF MS (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry) for localization and quantitative allele frequency detection to investigate preexisting HBV quasispecies and the genotypic evolution of drug-resistant variants during nucleos(t)ide analogue therapy. We found a significant difference between the genotypic evolution of drug-resistant variants depending on response to treatment.

SacNI, an isoschizomer of BanII isolated from Streptomyces achromogenes recognizes the 5'-GRGCY/C sequence.

SacNI, an isoschizomer of the restriction endonuclease, BanII [Sugisaki et al., Nucleic Acids Res. 10 (1982) 5747-5752], has been isolated from Streptomyces achromogenes N-J-H. SacNI recognizes the palindromic sequence, 5'-GRGCY/C, and cleaves within the recognition sequence, generating a 3' protruding RGCY end (where R = A or G, and Y = C or G).

Amplification of erbB-4 oncogene occurs less frequently than that of erbB-2 in primary human breast cancer.

ErbB-4 protein is a recently discovered member of the ErbB family. The role of ErbB-4 protein in mammary-gland tissue has not been definitively established. To date, the expression of erbB-4 in breast tissue has been determined in only a few cases and, to the best of our knowledge, its amplification has not been examined. We therefore used the double differential polymerase chain reaction (ddPCR) for determination of the amplification profile of erbB-4 and erbB-2, another gene from the ErbB family, in human primary breast cancer specimens. We examined the amplification of the genes in 20 normal breasts and 176 invasive breast cancer samples. Amplification of erbB-2 was detected in 19% and erbB-4 in 13% of the samples studied. Co-amplification of the two oncogenes was found in only five out of 176 samples. Human breast cancer-derived cell lines in most cases overexpress both erbB-2 and erbB-4 (Beerli et al., 1995. Mol. Cell Biol. 15, 6496-6505; Han et al., 1995. Proc. Natl. Acad. Sci. USA 92, 9747-9751), but data on separate erbB-2 overexpression, without overexpression of erbB-4, were also reported (Wosikowski et al., 1997. Clin. Cancer Res. 3, 2405-2414). At the gene level, we found that co-amplification of the genes in the case of human breast cancer is rare. Moreover, an inverse association of the erbB-4 amplification with estrogen receptor activity and direct correlation with the tumor size were found. Due to these correlations, erbB-4 oncogene amplification can be assumed to be of prognostic or predictive value in the diagnosis of breast cancer.

p53 Gene status in relation to ex vivo chemosensitivity of non-small cell lung cancer.

PURPOSE: About 40% of non-small cell lung cancer (NSCLC) patients are candidates for systemic chemotherapy, despite the fact that at diagnosis most NSCLC are usually chemoresistant both in vivo and ex vivo. It is important to develop sufficient methods of prediction of the response to chemotherapy and to find molecular markers that may prognose this response. Therefore, a study on the relationship of p53gene status to the ex vivo chemosensitivity of primary human NSCLC was performed. METHODS: Three drug combinations (carboplatin/etoposide, cyclophosphamide/etoposide/epirubicin, and paclitaxel/carboplatin) were tested in a modified ATP cell viability assay. A group of 28 cases of primary human NSCLC was assessed. RESULTS: Ex vivo chemosensitivity testing showed that tumors with p53 mutations were significantly more resistant to the cyclophosphamide/etoposide/epirubicin regimen than with normal p53 gene ( P = 0.012). However, no correlation was observed for two other treatment regimens. CONCLUSION: Mutations in the p53gene can lead to enhanced chemoresistance, confirming the hypothesis that the p53 gene may serve as a marker of tumor response to treatment in NSCLC. However, the data also illustrate that some additional factors might contribute to drug resistance of the examined tumors.

Characterization of restriction endonuclease AjoI from Acinetobacter johnsonii.

A new type II restriction endonuclease, named AjoI, was detected in Acinetobacter johnsonii. The enzyme AjoI, an isoschizomer of PstI, recognized the hexanucleotide sequence [5'-CTGCA/G-3'], with a cleavage site generating fragments of DNA with protruding cohesive 3' termini.

Drastically decreased transcription from CII-activated promoters is responsible for impaired lysogenization of the Escherichia coli rpoA341 mutant by bacteriophage lambda.

It was demonstrated previously that a mutation, rpoA341, in the gene encoding the alpha subunit of Escherichia coli RNA polymerase prevents lysogenization by bacteriophage lambda. The rpoA341 allele is known to be responsible for impaired transcription of some positively regulated E. coli chromosomal operons. Here we demonstrate that the inhibition of lysogenization of the rpoA341 mutant is a result of drastically decreased transcription from positively regulated phage promoters. We were unable to detect any transcripts originating from the CII-activated pE, pI and paQ promoters (important for lysogenic development) in the phage-infected rpoA341 mutant, in contrast to an otherwise isogenic rpoA+ strain. The results are discussed in the light of other reports showing that activation of the pE promoter by CII protein in vitro is decreased only about fivefold when the native alpha subunit is replaced by truncated alpha polypeptides.

Isolation and characterization of the restriction endonuclease PpeI from Phormidium persicinum.

PpeI is a type II restriction endonuclease isolated from cyanobacterial strain Phormidium persicinum. The endonuclease PpeI, an isoschizomer of ApaI, recognizes the hexanucleotide sequence (5'-GGGCC/C-3') and cleaves, after the second C, producing four nucleotide 3'-cohesive ends.

Inhibitory effects of pentamidine analogues on protein biosynthesis in vitro.

Pentamidine despite its rather high toxicity, is currently in clinical use. For development of new drugs of this type it is important to know the mechanism of their action. Two new amidines (I and II) and 4',6-diamidino-2-phenylindole (DAPI) were found in preliminary experiments to inhibit protein synthesis in vitro in the cell-free rat liver system. The three compounds differed in the precise mode of action. The inhibitory effect of I on the activity of the eukaryotic elongation factor eEF-2 and ribosomes seems to suggest that the binding site of eEF-2 on the ribosome was blocked by this compound. eEF-2 has been identified as the primary target of II and eEF-1 as the primary target of DAPI in the system studied.

Molecular modelling of the interaction of carbocyclic analogues of netropsin and distamycin with d(CGCGAATTCGCG)2.

A molecular mechanics and molecular dynamics approach was used to examine the structure of complexes formed between the d(CGCGAATTCGCG)2 duplex and netropsin, distamycin, and four carbocyclic analogues of netropsin and distamycin (1-4). The resulting structures of the ligand-DNA model complexes and their energetics were examined. It is predicted that the compounds 1-4 should have a decreased affinity for the minor groove of AT-rich regions in comparison to netropsin and distamycin. From the energetic analysis it appears that van der Waals and electrostatic interactions are more important than specific hydrogen bonds in stabilizing the ligand-duplex complexes. We predict that compounds 1 and 2 are effectively isohelical with the DNA minor groove. The superior DNA-binding afforded by 1 and 2 in comparison to 3 and 4 results from their more effective penetration into the minor groove and smaller perturbation of molecular structure upon complex formation.