Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy.

Genome-wide mechanisms that coordinate expression of subsets of functionally related genes are largely unknown. Recent studies show that receptor tyrosine kinases and components of signal transduction cascades including the extracellular signal-regulated protein kinase (ERK), once thought to act predominantly in the vicinity of plasma membrane and in the cytoplasm, can be recruited to chromatin encompassing transcribed genes. Genome-wide distribution of these transducers and their relationship to transcribing RNA polymerase II (Pol2) could provide new insights about co-regulation of functionally related gene subsets. Chromatin immunoprecipitations (ChIP) followed by deep sequencing, ChIP-Seq, revealed that genome-wide binding of epidermal growth factor receptor, EGFR and ERK pathway components at EGF-responsive genes was highly correlated with characteristic mitogen-induced Pol2-profile. Endosomes play a role in intracellular trafficking of proteins including their nuclear import. Immunofluorescence revealed that EGF-activated EGFR, MEK1/2 and ERK1/2 co-localize on endosomes. Perturbation of endosome internalization process, through the depletion of AP2M1 protein, resulted in decreased number of the EGFR containing endosomes and inhibition of Pol2, EGFR/ERK recruitment to EGR1 gene. Thus, mitogen-induced co-recruitment of EGFR/ERK components to subsets of genes, a kinase module possibly pre-assembled on endosome to synchronize their nuclear import, could coordinate genome-wide transcriptional events to ensure effective cell proliferation.

DNA methylation status is more reliable than gene expression at detecting cancer in prostate biopsy.

BACKGROUND: We analysed critically the potential usefulness of RNA- and DNA-based biomarkers in supporting conventional histological diagnostic tests for prostate carcinoma (PCa) detection. METHODS: Microarray profiling of gene expression and DNA methylation was performed on 16 benign prostatic hyperplasia (BPH) and 32 cancerous and non-cancerous prostate samples extracted by radical prostatectomy. The predictive value of the selected biomarkers was validated by qPCR-based methods using tissue samples extracted from the 58 prostates and, separately, using 227 prostate core biopsies. RESULTS: HOXC6, AMACR and PCA3 expression showed the best discrimination between PCa and BPH. All three genes were previously reported as the most promising mRNA-based markers for distinguishing cancerous lesions from benign prostate lesions; however, none were sufficiently sensitive and specific to meet the criteria for a PCa diagnostic biomarker. By contrast, DNA methylation levels of the APC, TACC2, RARB, DGKZ and HES5 promoter regions achieved high discriminating sensitivity and specificity, with area under the curve (AUCs) reaching 0.95-1.0. Only a small overlap was detected between the DNA methylation levels of PCa-positive and PCa-negative needle biopsies, with AUCs ranging between 0.854 and 0.899. CONCLUSIONS: DNA methylation-based biomarkers reflect the prostate malignancy and might be useful in supporting clinical decisions for suspected PCa following an initial negative prostate biopsy.

Chemometric methods for studying the relationships between trace elements in laryngeal cancer and healthy tissues.

A quick and reliable method for the evaluation and classification of two types of tissues is presented. Several chemometric methods were applied to evaluate multivariate data of the tissue samples with respect to the content of trace elements. The content of Pb, Al, Zn, Cd, Cu, Ni and Co was determined in samples of healthy and cancerous tissue obtained from 26 patients. Determination was done at milligram/kilogram level with inductively coupled plasma optical emission spectrometry (ICP-OES) and atomic absorption spectroscopy (AAS) techniques. Contents of trace metals in studied tissues are not normally distributed; however, normal distribution was confirmed for log values. There is a statistically significant difference in the content of Zn, Cd, Cu and Al (p<0.01) and Ni and Co (p<0.05) when healthy tissue is compared to cancerous one. Correlation between contents of trace elements for studied tissues was positive; the highest was found between Zn and Cu. A chemometric methodology seems to be a promising tool for classifications of the tissue samples.

Genomic Grade Index predicts postoperative clinical outcome of GIST.

BACKGROUND: Prognosis of localised gastrointestinal stromal tumour (GIST) is heterogeneous, notably for patients with AFIP intermediate or high risk of relapse, who are candidates to adjuvant imatinib. We hypothesised that gene expression profiles might improve the prognostication and help to refine the indications for imatinib. METHODS: We collected gene expression and histoclinical data of 146 pre-treatment localised GIST samples treated with surgery alone. We searched for a gene expression signature (GES) predictive for relapse-free survival (RFS) and compared its performances to that of three published prognostic proliferation-based GES (Genomic Grade Index (GGI), 16-Kinase, and CINSARC) and AFIP classification. We also analysed a data set from 28 patients with advanced GIST treated with neo-adjuvant imatinib. RESULTS: We identified a 275-gene GES (gene expression signature) predictive of RFS in a learning set and validated its robustness in an independent set. However, the GGI outperformed its prognostic performances, and those of the two other signatures and the AFIP intermediate-risk classification in two independent tests sets in uni- and multivariate analyses. Importantly, GGI could split the AFIP intermediate/high-risk samples into two groups with different RFS. Genomic Grade Index 'high-risk' tumours were more proliferative and genetically unstable than 'low-risk' tumours, and more sensitive to imatinib. CONCLUSION: GGI refines the prediction of RFS in localised GIST and might help tailor adjuvant imatinib.

Do low-molecular-weight heparins influence the healing process in colon anastomosis?

Anastomosis leakage is one of the most serious complications of colorectal surgery. A role for extracellular matrix remodelling in the healing process of the colon wall has been recently postulated. Changes in matrix metalloproteinase (MMP) activity in the intestinal wall occurring prior to elective resection and primary anastomosis appear to be responsible for dehiscence leading to anastomosis. Thrombophylaxis using low-molecular-weight heparins is routinely administered to all patients during the perioperative period. However, adverse antiproliferative and proapoptotic effects such as limitation of bioavailability of growth factors and angiogenesis inhibition have been characterized in various cell types as a result of heparin administration. It is also likely that relationships exist between extracellular matrix homeostasis and the coagulation/fibrinolysis system. We hypothesize that subcutaneous administration of LMWHs (low-molecular-weight heparins) may influence matrix metalloproteinase activity in the colon wall and increase the risk of postoperative leakage.

Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.

BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

Editing of hnRNP K protein mRNA in colorectal adenocarcinoma and surrounding mucosa.

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein is an RNA-binding protein involved in many processes that compose gene expression. K protein is upregulated in the malignant processes and has been shown to modulate the expression of genes involved in mitogenic responses and tumorigenesis. To explore the possibility that there are alternative isoforms of K protein expressed in colon cancer, we amplified and sequenced K protein mRNA that was isolated from colorectal cancers as well as from normal tissues surrounding the tumours. Sequencing revealed a single G-to-A base substitution at position 274 that was found in tumours and surrounding mucosa, but not in individuals that had no colorectal tumour. This substitution most likely reflects an RNA editing event because it was not found in the corresponding genomic DNAs. Sequencing of RNA from normal colonic mucosa of patients with prior resection of colorectal cancer revealed only the wild-type K protein transcript, indicating that G274A isoform is tumour related. To our knowledge, this is the first example of an RNA editing event in cancer and its surrounding tissue, a finding that may offer a new diagnostic and treatment marker.

A randomised comparison of treosulfan and carboplatin in patients with ovarian cancer: a study by the Scottish Gynaecological Cancer Trials Group (SGCTG).

The management of older and unfit women with advanced ovarian cancer requires post-operative chemotherapy but many of these patients are not suitable for high-dose cisplatin-based regimes. Carboplatin has been an easier alternative and can be given in the ambulatory setting. Historical data suggests that oral alkylating agents to be just effective with similar efficacy. In this study we have compared platinum-based carboplatin to the alkylating agent treosulfan in a population unfit to receive high-dose cisplatin. The trial randomised patients to either intravenous carboplatin or treosulfan as single agent. The trial was stopped prematurely after the interim analysis showed improved survival and response rates in the carboplatin arm. We conclude that carboplatin is a safe and effective drug in a population that is unfit for high-dose cisplatin. Treosulfan showed limited activity but may be considered along with other oral drugs in limited circumstances. With the exception of myelosuppression, toxicity was mild in both arms. Carboplatin remains the gold standard in this older and less fit group of patients.

Expression of pendrin in benign and malignant human thyroid tissues.

The Pendred syndrome gene (PDS) encodes a transmembrane protein, pendrin, which is expressed in follicular thyroid cells and participates in the apical iodide transport. Pendrin expression has been studied in various thyroid neoplasms by means of immunohistochemistry (IHC), Western blot and RT-quantitative real-time PCR. The expression was related to the functional activity of the thyroid tissue. Follicular cells of normal, nodular goitre and Graves' disease tissues express pendrin at the apical pole of the thyrocytes. In follicular adenomas, pendrin was detected in cell membranes and cytoplasm simultaneously in 10 out of 15 cases. Pendrin protein was detected in 73.3 and 76.7% of the follicular (FTC) and papillary (PTC) thyroid carcinomas, respectively, where pendrin was solely localised inside the cytoplasm. An extensive intracellular immunostaining of pendrin was observed in six out of 11 (54.5%) of positive FTCs and 19 out of 23 (82%) of PTCs. Focal reactivity was detected in one follicular- and three papillary carcinomas, whereas pendrin protein was absent in three of 15 FTC and four of 30 PTC; mRNA of pendrin was detected in 92.4% of thyroid tumours. The relative mRNA expression of pendrin was lower in cancers than in normal thyroid tissues (P<0.001). The pendrin protein level was found to parallel its mRNA expression, which was not, however, related to the tumour size and tumour stage. In conclusion, pendrin is expressed in the majority of differentiated thyroid tumours with high individual variability but its targeting to the apical cell membrane is affected.

Sclerotherapy with bleomycin for recurrent massive inguinal lymphoceles following partial vulvectomy and bilateral lymphadenectomy-Case report and literature review.

BACKGROUND: Formation of lymphoceles following radical vulvectomy presents a formidable problem that is associated with high degree of morbidity. A variety of approaches have been described in the literature to treat this condition. CASE: An 82-year-old woman developed massive inguinal lymphoceles following partial vulvectomy and inguinal lymphadenectomy for cancer vulva. The lymphoceles involved wide surface areas extending to both flanks, and accumulation of lymph was very rapid at a rate of 1 l daily. The condition failed to respond to continuous drainage and compression for 6 weeks, but responded quickly to sclerotherapy using bleomycin without any significant side effects. CONCLUSION: Intracavitary bleomycin could be used safely and effectively in huge rapidly accumulating lymphoceles.

Nuclear shift of hnRNP K protein in neoplasms and other states of enhanced cell proliferation.

The heterogeneous nuclear ribonucleoprotein K (hnRNP K), is a ubiquitously expressed protein that interacts with signal transducers, proteins that modulate gene expression and selective RNA and DNA motifs. K protein is modified in response to extracellular signals and directly regulates rates of transcription and translation. We used serum-treated hepatocyte culture, liver after partial hepatectomy and hepatic neoplasms as systems to compare expression, subcellular distribution and tyrosine phosphorylation of K protein in quiescent and dividing cells. The results show that expression of K protein mRNA was increased in states of enhanced proliferation. Levels of nuclear K protein were also higher in proliferating compared to resting cells. In contrast, levels of cytoplasmic K protein were the same or lower in dividing compared to quiescent cells. States of enhanced proliferation were also associated with increased levels of K protein tyrosine phosphorylation. Nuclear shift of K protein in dividing cells may reflect involvement of K protein in signalling multiple events that regulate expression of genes in proliferating cells.

Do altering in ornithine decarboxylase activity and gene expression contribute to antiproliferative properties of COX inhibitors?

Two isoforms of cyclooxygenase (COX) participate in growth control; COX-1 is constitutively expressed in most cells, and COX-2 is an inducible enzyme in response to cellular stimuli. An induction of COX-2 found in neoplastic tissues results in increased cell growth, inhibition of apoptosis, activation of angiogenesis, and decreased immune responsiveness. Although both COX-1 and COX-2 inhibitors are suppressors of cell proliferation and appear to be chemopreventive agents for tumorigenesis, the molecular mechanisms mediating antiproliferative effect of COX inhibitors are still not well defined. This study contrasts and compares the effects of aspirin and celecoxib, inhibitors of COX-1 and COX-2, in rat hepatoma HTC-IR cells. The following were assessed: cell proliferation and apoptosis, ornithine decarboxylase (ODC) activity, and pattern expression of three immediate-early genes, c-myc, Egr-1, and c-fos. We have shown that the treatment of hepatocytes in vitro with the selective COX-2 inhibitor, celecoxib, was associated with induction of apoptosis and complete inhibition of cellular proliferation. Aspirin exhibited a small antiproliferative effect that was not associated with apoptosis. Treatment with celecoxib produced dose- and time-dependent decrease in ODC activity. In addition, at higher drug concentration the decrease in ODC activity was greater in proliferating than in resting cells. Much lesser inhibitory effect on ODC activity was observed in aspirin-treated cells. The two COX inhibitors did not change c-myc expression, significantly decreased the expression of Egr-1, and differentially altered expression of c-fos; aspirin did not change, but celecoxib dramatically decreased the levels of c-fos-mRNA. Our study revealed that celecoxib and aspirin share the ability to inhibit ODC activity and alter the pattern of immediate-early gene expression. It seems that some of the observed effects are likely to be related to COX-independent pathways. The precise mechanisms of action of COX inhibitors should be defined before using these drugs for cancer chemopreventive therapy.

[Postero-medial release in surgical treatment of congenital clubfeet: a comment to Turco's method based on personal experience]. / Leczenie operacyjne wrodzonej stopy konsko-szpotawej technika uwolnienia tylno-przysrodkowego--odniesienie do metody turco na podstawie wlasnych obserwacji.

Complete correction of congenital clubfeet by conservative treatment is often impossible. Surgical treatment plays a major role in treatment of this deformity. At the Department of Pediatric Orthopedics in Lublin between 1970 and 1999 1041 children (1253 feet) were treated surgically with Turco's method, in the authors' own modification. The paper presents the technically optimal procedure, the range of tendon elongation. The way the wound is closed is particularly stressed, as well as the need to achieve muscle balance, along with a description of proper post-op care. The material was analysed as a whole, although particular attention was given to three periods: 1970-1975, 1980-1985, 1990-1995. The results were assessed according to the Turco classification, the Magone classification in accordance with the injunctions of the Scientific Committee Meeting of the Pediatric Section of the Polish Orthopedic Society held in Poznan. Good and very good results were achieved in 65-67% of the cases, while satisfactory and bad results were found in 23-30% of the cases.

The retinoic acid receptor antagonist, BMS453, inhibits normal breast cell growth by inducing active TGFbeta and causing cell cycle arrest.

We have previously shown that a retinoic acid receptor (RAR) antagonist BMS453, which does not activate RAR-dependent gene transcription in breast cells, inhibits normal breast cell growth. In this study we have investigated the mechanisms by which this retinoid receptor antagonist inhibits cell growth. Both all trans retinoic acid (atRA) and BMS453 inhibited the proliferation of normal breast cell growth without significantly inducing apoptosis. Both retinoids caused a G1 block in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase. We then investigated the effects of the retinoids on molecules that regulate the G1 to S transition. These studies demonstrated that both atRA and BMS453 induce Rb hypophosphorylation and decrease CDK2 kinase activity. We then studied the effect of the retinoids on the expression of CDK inhibitors. atRA and BMS453 increased total p21 protein levels and CDK2-bound p21 protein, but did not change CDK4-bound p21. These results suggest that atRA and BMS453 increase p21, decrease CDK2 kinase activity, which in turn leads to hypophosphorylation of Rb and G1 arrest. Because transforming growth factor beta (TGFbeta) has been proposed as a mediator of retinoid-induced growth inhibition, we next investigated whether TGFbeta mediates the anti-proliferative effect of atRA and BMS453 in normal breast cells. These studies showed that atRA and BMS453 increased total TGFbeta activity by 3-5-fold. However, BMS453 increased active TGFbeta activity by 33-fold while atRA increased active TGFbeta activity by only threefold. These results suggest that BMS453 treatment induces conversion of latent TGFbeta to active TGFbeta. To investigate whether this increase in active TGFbeta mediates the anti-proliferative effects of these retinoids, a TGFbeta-blocking antibody was used in an attempt to prevent retinoid-induced growth inhibition. Results from these experiments showed that the anti-TGFbeta antibody prevented the inhibition of cell proliferation induced by BMS453, but did not prevent the inhibition of cell proliferation induced by atRA. These results demonstrate that BMS453 inhibits breast cell growth predominantly through the induction of active TGFbeta, while atRA inhibits growth through other mechanisms. These results suggest that retinoid analogs that increase active TGFbeta may be promising agents for the prevention of breast cancer.

Insulin alters heterogeneous nuclear ribonucleoprotein K protein binding to DNA and RNA.

The interaction of the multimodular heterogeneous nuclear ribonucleoprotein (hnRNP) K protein with many of its protein and nucleic acid partners is regulated by extracellular signals. Acting as a docking platform, K protein could link signal-transduction pathways to DNA- and RNA-directed processes such as transcription, mRNA processing, transport, and translation. Treatment of hepatocyte culture with insulin increased K protein tyrosine phosphorylation. Insulin altered K protein interaction with RNA and DNA in vitro. Administration of insulin into mice had similar effects on K protein in liver. Coimmunoprecipitations of RNA with K protein revealed preferential in vivo K protein binding of a subset of transcripts, including the insulin-inducible c-fos mRNA. These results suggest a class of insulin pathways that signal nucleic acid-directed processes that involve K protein.

Mutations, methylation and expression of CDKN2a/p16 gene in colorectal cancer and normal colonic mucosa.

The status of CDKN2a gene, coding for p16 and p19ARF proteins, was examined in 55 colorectal cancers. Polymerase chain reaction (PCR), single stranded conformational polymorphism and sequencing revealed 1 case of CDKN2a mutation. Methylation-specific PCR detected p16 locus methylation in 37 (73%) of 51 normal samples and 29 (53%) of 55 cancers (P=0.035). p16 transcript absence (assessed by reverse transcription-polymerase chain reaction) was noted in 10 (45%) of 22 normal samples and four (14%) of 29 cancers (P=0.012) and correlated with gene methylation (P=0.036). The decreasing frequency of p16 silencing in cancer comparing to normal mucosa does not support the postulated role of p16 in colorectal carcinogenesis.

Peptide analog of fibronectin that inhibits cell migration and ERK 1/2 activity.

Two analogs of the peptide mimicking the 1977-1991 C- terminal part of fibronectin have been synthesized and tested. AWLI simulated human fibronectin fragment 1977-1991, whereas AWLII hybridized to both RGD and 1977-1991 fragments. AWLI and AWLII peptides inhibited the migration of the ovarian carcinoma cell line OVP10 regardless of the presence RGD. AWLI peptide inhibited spontaneous and fibronectin-activated cell migration and ERK1/2 activity. Neither AWLI nor fibronectin induced changes in FAK proteins, as could be judged from Western blots. In conclusion, it seems that the C-terminal fragment of fibronectin inhibits ERK1/2-dependent (random) migration of ovarian carcinoma cells.

Idarubicin and cyclophosphamide--an active oral chemotherapy regimen for advanced breast cancer.

UNLABELLED: Between October 1993 and September 1994, 33 women with metastatic breast cancer aged between 29 and 74 years with a median age of 58 were entered into a study of oral chemotherapy from three UK centres. Patients by definition had metastatic disease and were fit and well with performance status 0 or 1 in 23 cases, 2 in seven cases and 3 in two cases (one missing). Five patients had received prior adjuvant CMF chemotherapy, nine first line non-anthracycline containing chemotherapy for relapse, eight patients second line non-anthracycline containing chemotherapy and all patients had had hormone therapy either as adjuvant or for relapsed disease. Adjuvant radiotherapy had been given to 17 and palliative radiotherapy to 12 patients. In nine patients there was one site of disease at start of therapy, in 10 two sites, in 11 three sites and in three patients four or more sites. The regimen comprised oral idarubicin 15 mg/m2 on day 1, 10 mg/m2 on days 2 and 3 and oral cyclophosphamide 250 mg/m2 (maximum 400 mg) on days 1, 2 and 3. Treatment was continued until disease progression or toxicity. RESULTS: Overall 25% of 32 evaluable patients responded objectively including one complete response; 50% of patients had stable disease and 25% of patients progression. Among patients who had had no prior chemotherapy the objective response rate was 37.5%; 45% of patients had symptomatic improvement. The most common severe toxicity was granulocytopenia WHO grade 3 or more in 69.7% of patients. Thrombocytopenia grade 3 or 4 was seen in four patients. Six patients had documented infections and all but four patients had alopecia. All patients complained of mild or moderate fatigue. Nausea and vomiting occurred in 75% of patients but only four individuals had grade 3 toxicity. Two patients stopped therapy after myocardial infarction and one after impaired cardiac function was noted. The median time to progression was 2.7 months (1-11.5 months) and median survival time 8.8 months (1-13+ months). CONCLUSION: The combination chemotherapy is active in heavily treated patients with manageable toxicity but there are problems in heavily pre-treated patients. There was good compliance in taking medication and at the doses chosen the drugs appear to be suitable for younger fitter patients.