Basal Tumor Cell Isolation and Patient-Derived Xenograft Engraftment Identify High-Risk Clinical Bladder Cancers.

Strategies to identify tumors at highest risk for treatment failure are currently under investigation for patients with bladder cancer. We demonstrate that flow cytometric detection of poorly differentiated basal tumor cells (BTCs), as defined by the co-expression of CD90, CD44 and CD49f, directly from patients with early stage tumors (T1-T2 and N0) and patient-derived xenograft (PDX) engraftment in locally advanced tumors (T3-T4 or N+) predict poor prognosis in patients with bladder cancer. Comparative transcriptomic analysis of bladder tumor cells isolated from PDXs indicates unique patterns of gene expression during bladder tumor cell differentiation. We found cell division cycle 25C (CDC25C) overexpression in poorly differentiated BTCs and determined that CDC25C expression predicts adverse survival independent of standard clinical and pathologic features in bladder cancer patients. Taken together, our findings support the utility of BTCs and bladder cancer PDX models in the discovery of novel molecular targets and predictive biomarkers for personalizing oncology care for patients.

Oncogenic CXCL10 signalling drives metastasis development and poor clinical outcome.

BACKGROUND: The CXCL10/CXCR3 signalling mediates paracrine interactions between tumour and stromal cells that govern leukocyte trafficking and angiogenesis. Emerging data implicate noncanonical CXCL10/CXCR3 signalling in tumourigenesis and metastasis. However, little is known regarding the role for autocrine CXCL10/CXCR3 signalling in regulating the metastatic potential of individual tumour clones. METHODS: We performed transcriptomic and cytokine profiling to characterise the functions of CXCL10 and CXCR3 in tumour cells with different metastatic abilities. We modulated the expression of the CXCL10/CXCR3 pathway using shRNA-mediated silencing in both in vitro and in vivo models of B16F1 melanoma. In addition, we examined the expression of CXCL10 and CXCR3 and their associations with clinical outcomes in clinical data sets derived from over 670 patients with melanoma and colon and renal cell carcinomas. RESULTS: We identified a critical role for autocrine CXCL10/CXCR3 signalling in promoting tumour cell growth, motility and metastasis. Analysis of publicly available clinical data sets demonstrated that coexpression of CXCL10 and CXCR3 predicted an increased metastatic potential and was associated with early metastatic disease progression and poor overall survival. CONCLUSION: These findings support the potential for CXCL10/CXCR3 coexpression as a predictor of metastatic recurrence and point towards a role for targeting of this oncogenic axis in the treatment of metastatic disease.

[The influence of general air cryotherapy on the serum lipid spectrum].

Analysis of the lipid status and oxidation of serum of blood lipids by free radicals was performed in the study involving practically healthy volunteers who underwent a course of general air cryotherapy. It was ahown that oxidized and modified lipoproteins play an important role in the etiopathogenetic mechanisms underlying the development of vasoconstrictive reactions and heat production processes in response to the short-term exposure to extremely low temperatures.

[Oxygen-transporting function of the blood following general air cryogenic therapy].

The present clinical and laboratory study of the effects of general air cryogenic therapy on the characteristics of blood supply included practically healthy subjects. The study has demonstrate that dosed cold stress promotes adaptive capacities of the organism by modulating the oxygen-transporting function of the blood that in its turn stimulates the adaptive mechanisms and leads to decoupling tissue respiration. It is concluded that general air cryogenic therapy can be recommended as an instrument for the improvement of endurance of and resistibility to the action of hypoxemia in special groups of people under the low temperature conditions.

Cooperativity of the MUC1 oncoprotein and STAT1 pathway in poor prognosis human breast cancer.

Signal transducer and activator of transcription 1 (STAT1) is activated in the inflammatory response to interferons. The MUC1 oncoprotein is overexpressed in human breast cancers. Analysis of genes differentially expressed in MUC1-transformed cells has identified a network linking MUC1 and STAT1 that is associated with cellular growth and inflammation. The results further show that the MUC1-C subunit associates with STAT1 in cells and the MUC1-C cytoplasmic domain binds directly to the STAT1 DNA-binding domain. The interaction between MUC1-C and STAT1 is inducible by IFNgamma in non-malignant epithelial cells and constitutive in breast cancer cells. Moreover, the MUC1-STAT1 interaction contributes to the activation of STAT1 target genes, including MUC1 itself. Analysis of two independent databases showed that MUC1 and STAT1 are coexpressed in about 15% of primary human breast tumors. Coexpression of MUC1 and the STAT1 pathway was found to be significantly associated with decreased recurrence-free and overall survival. These findings indicate that (i) MUC1 and STAT1 function in an auto-inductive loop, and (ii) activation of both MUC1 and the STAT1 pathway in breast tumors confers a poor prognosis for patients.

Translational strategies exploiting TNF-alpha that sensitize tumors to radiation therapy.

TNFerade is a radioinducible adenoviral vector expressing tumor necrosis factor-alpha (TNF-alpha) (Ad.Egr-TNF) currently in a phase III trial for inoperable pancreatic cancer. We studied B16-F1 melanoma tumors in TNF receptor wild-type (C57BL/6) and deficient (TNFR1,2-/- and TNFR1-/-) mice. Ad.Egr-TNF+IR inhibited tumor growth compared with IR in C57BL/6 but not in receptor-deficient mice. Tumors resistant to TNF-alpha were also sensitive to Ad.Egr-TNF+IR in C57BL/6 mice. Ad.Egr-TNF+IR produced an increase in tumor-associated endothelial cell apoptosis not observed in receptor-deficient animals. Also, B16-F1 tumors in mice with germline deletions of TNFR1,2, TNFR1 or TNF-alpha, or in mice receiving anti-TNF-alpha exhibited radiosensitivity. These results show that tumor-associated endothelium is the principal target for Ad.Egr-TNF radiosensitization and implicate TNF-alpha signaling in tumor radiosensitivity.

Dose-dependent and independent temporal patterns of gene responses to ionizing radiation in normal and tumor cells and tumor xenografts.

U87 cells derived from human malignant gliomas and growtharrested human embryonic lung (HEL) fibroblasts were examined with respect to their response to ionizing radiation by profiling their RNAs. In the first series of experiments, cells grown in vitro were harvested and the RNAs were extracted 5 h after exposure to 1, 3, or 10 Gy. In the second series of experiments the U87 tumors were implanted in nude mice and subjected to the same doses of irradiation. The xenografts were harvested at 1, 5, or 24 h after irradiation and subjected to the same analyses. We observed and report on (i) cell-type common and cell-type specific responses, (ii) genes induced at low levels of irradiation but not at higher doses, (iii) temporal patterns of gene response in U87 xenografts that varied depending on radiation dose and temporal patterns of response that were similar at all doses tested, (iv) significantly higher up-regulation of cells in xenografts than in in vitro cultures, and (v) genes highly up-regulated by radiation. The responding genes could be grouped into nine functional clusters. The representation of the nine clusters was to some extent dependent on dose and time after irradiation. The results suggest that clinical outcome of ionizing radiation treatment may benefit significantly by taking into account both cell-type and radiation-dose specificity of cellular responses.

Angiostatin effects on endothelial cells mediated by ceramide and RhoA.

Angiostatin is a cleavage product of plasminogen that has anti-angiogenic properties. We investigated whether the effects of angiostatin on endothelial cells are mediated by ceramide, a lipid implicated in endothelial cell signaling. Our results demonstrate that angiostatin produces a transient increase in ceramide that correlates with actin stress fiber reorganization, detachment and death. DNA array expression analysis performed on ceramide-treated human endothelial cells demonstrated induction of certain genes involved in cytoskeleton organization. Specifically, we report that treatment with angiostatin or ceramide results in the activation of RhoA, an important effector of cytoskeletal structure. We also show that treatment of endothelial cells with the antioxidant N-acetylcysteine abrogates morphological changes and cytotoxic effects of treatment with angiostatin or ceramide. These findings support a model in which angiostatin induces a transient rise in ceramide, RhoA activation and free radical production.

Quantitative analysis of DNA array autoradiographs.

DNA arrays and chips are powerful new tools for gene expression profiling. Current arrays contain hundreds or thousands of probes and large scale sequencing and screening projects will likely lead to the creation of global genomic arrays. DNA arrays and chips will be key in understanding how genes respond to specific changes of environment and will also greatly assist in drug discovery and molecular diagnostics. To facilitate widespread realization of the quantitative potential of this approach, we have designed procedures and software which facilitate analysis of autoradiography films with accuracy comparable to phosphorimaging devices. Algorithms designed for analysis of DNA array autoradiographs incorporate 3-D peak fitting of features on films and estimation of local backgrounds. This software has a flexible grid geometry and can be applied to different types of DNA arrays, including custom arrays.

LINE L1 retrotransposable element is targeted during the initial stages of apoptotic DNA fragmentation.

Using a directional cloning strategy, DNA sequence information was obtained corresponding to the site of early radiation-induced apoptotic DNA fragmentation within the human lymphoblastoid cell line TK6. Data were obtained from 88 distinct clones comprising approximately 65 kbp of sequenced material. Analysis of all cloned material showed that sequences in the 10 bp immediately adjacent to the cleavage sites were enriched in short oligoT tracts. The proportion of repetitive DNA within the entire cloned material was found to be within the normal range. However the distribution of Alu and LINE repetitive DNA were biased to positions at or adjacent to the apoptotic cleavage site. In particular, a non-random distribution of five cleavage sites was found clustered within the second ORF of the LINE L1 that partially overlapped with two binding sites for the nuclear matrix-associated protein SATB1. Three other clones, containing alpha satellite elements, were also linked to a DNA matrix binding function. These data indicate that the site of chromatin loop formation at the nuclear matrix may be a specific target for early DNA fragmentation events during apoptosis.

Accumulation of specific RNAs encoding transcriptional factors and stress response proteins against a background of severe depletion of cellular RNAs in cells infected with herpes simplex virus 1.

Herpes simplex virus 1 encodes several functions to preclude the shutoff of host response to infection, including degradation of mRNA immediately after infection. To determine whether any cellular mRNAs accumulate in infected cells against a background of severe loss of host RNA, we hybridized cDNAs derived from three different cell lines infected with wild type and a mutant virus to a DNA array containing probes for 588 human genes representing different functional groups. The results were that (i) infected cells accumulated at levels above those of mock-infected cells, a small number of transcripts representing transcriptional factors that could regulate gene expression both positively and negatively, and one stress response protein (GADD45), (ii) the amount and nature of the accumulated transcripts showed limited variability depending on the cell and virus, and (iii) at least some of the proteins encoded by the accumulated transcripts could benefit either the virus or the host.

Abortive apoptosis as an initiator of chromosomal translocations.

Apoptosis is a well-recognized regulator of a cell populations size and structure. Irreversible stages of apoptosis lead to activation of different enzymatic cascades, changes in cell morphology and DNA fragmentation. However, little is known about nuclear events which accompany the initial stages of apoptosis. These events are connected with introduction of limited amounts of double strand breaks into genomic DNA, some of which may be subsequently rejoined. We hypothesize here that the initial stages of apoptotic DNA fragmentation may be reversible and connected with the initiation of recombinational events and certain chromosomal translocations. The factors influencing apoptosis reversibility and cell survival after delivery of apoptotic stimuli may provide new insights into mechanisms of lymphocyte development and tumorigenesis.

Synchronous block in DNA synthesis initiation with change in chromatin topology mediated by VP16.

The impact of chromatin topology on the DNA synthetic process was studied in the human squamous-cell carcinoma cell line SQ-20B. A 1-h exposure < or = 10 microM VP16 produced an increase in DNA supercoil tension, measured by recording laser light scatter from salt-extracted nuclei. This change was precisely paralleled by a decrease in DNA synthesis. The effects on both DNA supercoiling and DNA synthesis were suppressed at VP16 concentrations between 10 and 20 microM. The changes in DNA supercoiling and synthesis at VP16 concentrations -10 microM were eliminated by coincubation with mimosine, a DNA synthesis initiator poison. We conclude that brief exposure to low concentrations of VP16 disturbs the balance of torsional energy within discrete replicon domains by affecting normal topoisomerase II activity at sites of replication initiation. The resultant increase in negative supercoil tension mediates a topologic checkpoint, limiting the initiation of DNA synthesis. Such a checkpoint may be a common pathway for control, both during the normal replicative cycle and subsequent to DNA damage.

Association between DNA cleavage during apoptosis and regions of chromatin replication.

We have addressed the association between the site of DNA cleavage during apoptosis and DNA replication. DNA double strand breaks were introduced into chromatin containing pulse labeled nascent DNA by the induction of apoptosis or autocleavage of isolated nuclei. The location of these breaks in relation to nascent DNA were revealed by Bal31 exonuclease digestion at the cut sites. Our data show that Bal31 accessible cut sites are directly linked to regions enriched in nascent DNA. We suggest that these regions coincide with the termini of replication domains, possibly linked by strong DNA-matrix interactions with biophysically defined topological structures of 0.5-1.3 Mbp in size. The 50 kbp fragments that are commonly observed as products of apoptosis are also enriched in nascent DNA within internal regions but not at their termini. It is proposed that these fragments contain a subset of replicon DNA that is excised during apoptosis through recognition of their weak attachment to the nuclear matrix within the replication domain.

Mechanisms of induction of apoptotic DNA fragmentation.

PURPOSE: Despite its common use as an indicator of apoptosis, little is known about the mechanisms controlling apoptotic DNA fragmentation in irradiated cells. This review discusses the pathways of chromatin fragmentation, and the role of both nucleases and chromatin structure in this process. DEFINITIONS: DNA fragmentation linked to apoptosis is a combination of cleavage events excising both large DNA fragments within the range 0.4-1.0 Mbp and 50 kbp followed by random cuts within internucleosomal regions (i.e. DNA laddering). The first two cleavage steps can be detected in virtually all apoptotic cells, but DNA laddering is not ubiquitously observed. Endonucleases that mediate this cleavage of chromatin may be classified by substrate specificity, mode of DNA cleavage and their cofactor requirements. CONCLUSIONS: Three major pathways of DNA fragmentation are proposed and discussed: (1) upregulation of endonucleases, (2) their intranuclear/intracellular redistribution and (3) primary changes of chromatin structure.

Multiple forms of endonuclease activity linked with radiation induced apoptosis in C4-1 cervical carcinoma cells.

Irradiation of C4-1 cervical carcinoma cells induced apoptosis, as determined by their morphology and the presence of oligonucleosomal DNA fragmentation, with the formation of 5'- P and 3'-OH termini. Extracts of nuclear proteins from both control and irradiated cells possessed similar metallodependent endonucleolytic activity which cleaved target plasmid DNA with the same specificity as that found in apoptotic cells. Fractionation of the nuclear extracts revealed that the predominant endonuclease activity of unirradiated cells was a protein of approximately 40 kDa. After irradiation, the predominant activity was found to be associated with a 70 kDa fraction, with a reduction in the 40 kDa form. The activity of each endonuclease was found to be Ca2+ and Mg2+ dependent. It is proposed that the changes in molecular weight observed for these enzymes may be linked to the final step in apoptosis execution, irreversible chromatin fragmentation, and thus offer a potentially novel target for manipulating the effector pathway of apoptosis in these cells.

Mycoplasma infection can sensitize host cells to apoptosis through contribution of apoptotic-like endonuclease(s).

Mycoplasma infection may lead to various pathologies in a broad range of hosts. It has been shown that Mycoplasma may trigger cell death in cell cultures; however, the mechanism remains unknown. In the present paper we show that Mycoplasma infection of different lymphocyte and epithelial tumour cell lines leads to the inhibition of proliferation, and increased cell death, accompanied by DNA fragmentation and the morphological features of apoptosis. We also showed that this infection leads to an increased sensitivity of cells to various inducers of apoptosis targeting different signalling pathways. Finally, we show that increased apoptosis is associated with overexpression of an endonuclease produced by Mycoplasma. This endonuclease is recovered in the nuclear fraction of host cells, introduces mostly DSB and is active at neutral pH in the presence of divalent cations. Activation of this endonuclease is connected with limited proteolysis, which may be reproduced in vitro by snake venom serine proteinase.

Topologically constrained domains of supercoiled DNA in eukaryotic cells.

The size of supercoiled, topologically constrained DNA domains within the squamous carcinoma cell line SQ-20B were determined by direct comparison with a panel of irradiated supercoiled plasmid DNAs. Loss of supercoiling in plasmids was determined by gel electrophoresis and in cells by nucleoid flow cytometry. Comparison of dose-response data for plasmid relaxation with that obtained from SQ-20B cells enabled a direct estimation of supercoil target size in these cells. Plasmids pUCD9P (3.9 kbp), pXT-1 (10.1 kbp), pdBPV-MMT-neo (14.6 kbp), pRK290 (20.0 kbp), and R6K (38 kbp) were used and analyzed under the same exposure conditions as nucleoid DNA. Two sizes of topologically closed domains were found in nucleoids of 0.51+/-0.17Mbp and 1.34+/-0.3 Mbp. In an attempt to relate these large-scale organizations of DNA with function, cells were exposed to the DNA topoisomerase II inhibitor, VP16 and the G1/S cell cycle blocking agent mimosine. A 1 h exposure to VP16 was effective in reducing DNA synthesis which was associated with a parallel increase in nucleoid supercoiling. Addition of the G1 > S inhibitor mimosine enhanced both responses. It is concluded that chromosomes and interphase nuclei are organized into at least two sizes of topologically constrained domains of DNA which may have functional relevance to the control and execution of DNA synthesis.

Fibronectin, Cryoglobulinemia and Immune Complexes in Sjogren's Syndrome Patients.

The problem of proper diagnosis, patient's status and disease progression is very actual for autoimmune diseases. Still the diagnosis of Sjogren's syndrome is more state of art than of science. Our preliminary investigations showed that serum fibronectin of elevated molecular weight may be probably considered as one of the diagnostic criteria in Sjogren's syndrome patients. The increase of molecular weight of FN chains from 220 kDa to 250 kDa derives from relative and absolute elevation in a content of N-acetylglucosamine in FN carbohydrate chains. The present results concern the presence of FN in cryoprecipitates and circulating immune complexes in Sjogren's syndrome patients and as it has been demonstrated this parameter may be used as a prognostic sign.