A post hoc analysis of dalteparin versus oral anticoagulant (VKA) therapy for the prevention of recurrent venous thromboembolism (rVTE) in patients with cancer and renal impairment.

Venous thromboembolism (VTE) is a common and serious complication in patients with cancer; treatment guidelines recommend extended therapy of ≥6 months with low-molecular-weight heparin (LMWH) for treatment and prevention of recurrent VTE (rVTE) in this population. This post hoc analysis used data from the CLOT study-a phase III, randomized, open-label, controlled study (N = 676)-to compare the efficacy and safety of dalteparin, a LMWH, versus vitamin K antagonist (VKA) for prevention of rVTE in patients with cancer and renal impairment (creatinine clearance <60 ml/min). Overall, 162/676 (24 %) patients had renal impairment at baseline. Patients received subcutaneous dalteparin 200 IU/kg once daily during month 1, followed by 150 IU/kg once daily for months 2-6; or VKA once daily for 6 months, with initial overlapping subcutaneous dalteparin 200 IU/kg once daily for ≥5 days until international normalized ratio was 2.0-3.0 for 2 consecutive days. Endpoints included the rates of rVTE (primary) and bleeding events. Overall, fewer dalteparin-treated patients (2/74 [2.7 %]) experienced ≥1 adjudicated symptomatic rVTE compared with VKA-treated patients (15/88 [17.0 %]; hazard ratio = 0.15 [95 % confidence interval 0.03-0.65]; p = 0.01). Bleeding event rates for both treatments were similar (p = 0.47). In summary, compared with VKA, dalteparin significantly reduced risk of rVTE in patients with cancer and renal impairment (p = 0.01) while exhibiting a comparable safety profile. This analysis supports dosing patients with renal impairment in accordance with patients with normal renal function; however, anti-Xa monitoring could be considered to further support safety in selected patients, particularly those with very severe renal impairment.

Probiotic Lactobacillus acidophilus and L. casei mix sensitize colorectal tumoral cells to 5-fluorouracil-induced apoptosis.

To assess the potential of Lactobacillus acidophilus and Lactobacillus casei strains to increase the apoptosis of a colorectal cancer cell line in the presence of 5-fluorouracil (5-FU), LS513 colorectal cancer cells were treated for 48 h with increasing concentrations of these lactic acid bacteria (LAB) in the presence of 100 mu g/ml of 5-FU. In the presence of 10(8) CFU/ml of live LAB, the apoptotic efficacy of the 5-FU increased by 40%, and the phenomenon was dose dependent. Moreover, irradiation-inactivated LAB caused the same level of induction, whereas microwave-inactivated LAB reduced the apoptotic capacity of the 5-FU. When cells were treated with a combination of live LAB and 5-FU, a faster activation of caspase-3 protein was observed, and the p21 protein seems to be downregulated. These results suggest that live L. acidophilus and L. casei are able to increase the apoptosis-induction capacity of 5-FU. The mechanisms of action are still not elucidated, and more research is needed to understand them. This is the first set of experiments demonstrating that some strains of LAB can enhance the apoptosis-induction capacity of the 5-FU. Based on these results, it is possible to speculate that LAB or probiotics could be used as an adjuvant treatment during anticancer chemotherapy.

A prostate secretory protein94-derived synthetic peptide PCK3145 inhibits VEGF signalling in endothelial cells: implication in tumor angiogenesis.

We have previously observed that the synthetic peptide corresponding to amino acids 31-45 (PCK3145) of PSP94 can reduce prostate tumor growth in vivo. Moreover, a recently concluded phase IIa clinical trial with patients with hormone refractory prostate cancer indicated that PCK3145 down-regulates the levels of plasma matrix metalloproteinase (MMP)-9, a MMP involved in metastasis and tumor angiogenesis. The purpose of our study was to investigate the molecular mechanisms of action of PCK3145 and whether this peptide could antagonize tumor neovascularization. We show that, in a syngeneic in vivo model of rat prostate cancer, the expression of endothelial cell (EC) specific CD31, a marker of tumor vessel density, was decreased by 43% in PCK3145-treated animals. In vitro, PCK3145 specifically antagonized in a dose-dependent manner the VEGF-induced ERK phosphorylation as well as the phosphorylation of the VEGFR-2 in cultured EC (HUVEC). These anti-VEGF effects were partly reproduced by pharmacological inhibitors such as PD98059 and PTK787, suggesting that PCK3145 inhibits the tyrosine kinase activity associated to VEGFR-2, which in turn prevents intracellular signalling through the MAPK cascade. Moreover, PCK3145 was also found to inhibit the PDGF-induced phosphorylation of PDGFR in smooth muscle cells. Finally, PCK3145 inhibited in vitro EC tubulogenesis and VEGF-induced MMP-2 secretion suggesting its potential implication as an antiangiogenic agent. Our study demonstrates that PCK3145 interferes with the tyrosine kinase activity associated with VEGF signalling axis in EC. The antiangiogenic properties of this peptide could be highly beneficial and exploited in novel antiangiogenic therapies, for patients with various cancers.

Identification, purification and characterization of a novel human blood protein with binding affinity for prostate secretory protein of 94 amino acids.

PSP94 (prostate secretory protein of 94 amino acids), an abundant protein within semen, has reported local functions within the reproductive tract and reported systemic functions. Mechanisms of action remain poorly understood, but binding to undefined molecules within the prostate, pituitary, testis and blood may initiate some of these actions. PSP94 serum measurements, especially of bound and free forms, have potential clinical utility in prostate cancer management. Identification of the binding molecules will help in the understanding of PSP94's action, and enable further development of PSP94 serum assays. PSPBP (PSP94-binding protein) was purified from human serum by ammonium sulphate fractionation, ion-exchange and affinity chromatography. The glycosylated protein ran as two bands on SDS/PAGE (70 and 95 kDa). N-terminal sequencing yielded a 30-amino-acid sequence, identical with the translated N-terminal region of a previously published cDNA (GenBank accession number AX136261). Reverse transcriptase PCR and plaque hybridization demonstrated PSPBP mRNA in peripheral blood leucocytes and in a prostate cDNA library. Northern blotting showed 2 kb mRNA species in prostate, testis, ovary and intestine. Immunohistochemistry demonstrated PSPBP in tissues, including pituitary and Leydig cells, supporting a role for PSP94 in hormonal control at the pituitary gonadal axis. ELISA demonstrated that PSPBP levels were significantly lower (P=0.0014) in the serum of a prostate cancer population (n=65) compared with a control population (n=70). PSPBP identification will help the understanding of PSP94's functions and facilitate ELISA development to address the clinical value of PSP94 serum assays.

NAP-2 is part of multi-protein complexes in HeLa cells.

We previously reported that a complex of nuclear proteins from HeLa cells, among them histone H1 and casein kinase 2 co-eluted from immobilized nucleosome assembly protein 2 (NAP-2)-Sepharose. Here, using HeLa cell nuclear extracts, we found NAP-2 migrates in a blue-native polyacrylamide gel with an apparent molecular weight of 300 kDa. HeLa cell NAP-2, labeled in vivo with radioactive orthophosphate, co-precipitated with at least two phosphoproteins, with an apparent mass of 100 and 175 kDa, respectively, as determined by SDS-PAGE. NAP-2 from total HeLa cell extract co-purified with other proteins through two sequential chromatographic steps: first, a positively charged resin, Q-Sepharose, was used, which purified NAP-2 more easily with other proteins that eluted as a single peak at 0.5 M NaCl. This fraction possessed both relaxing and supercoiling activities, and it was able to assemble regularly spaced nucleosomes onto naked DNA in an ATP-dependent manner. Second, a negatively charged resin (heparin) was used, which retained small amounts of NAP-2 (a very acidic polypeptide) and topoisomerase I. This fraction, although able to supercoil relaxed DNA, did so to a lesser extent than the Q-Sepharose fraction. The data suggest that NAP-2 is in complex(es) with other proteins, which are distinct from histones.

Differential DNA binding of Ku antigen determines its involvement in DNA replication.

Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.

Ku antigen, an origin-specific binding protein that associates with replication proteins, is required for mammalian DNA replication.

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.

14-3-3sigma is a cruciform DNA binding protein and associates in vivo with origins of DNA replication.

A human cruciform binding protein (CBP) was previously shown to bind to cruciform DNA in a structure-specific manner and be a member of the 14-3-3 protein family. CBP had been found to contain the 14-3-3 isoforms beta, gamma, epsilon, and zeta. Here, we show by Western blot analysis that the CBP-cruciform DNA complex eluted from band-shift polyacrylamide gels also contains the 14-3-3sigma isoform, which is present in HeLa cell nuclear extracts. An antibody specific for the 14-3-3sigma isoform was able to interfere with the formation of the CBP-cruciform DNA complex. The effect of the same anti-14-3-3sigma antibody in the in vitro replication of p186, a plasmid containing the minimal replication origin of the monkey origin ors8, was also analyzed. Pre-incubation of total HeLa cell extracts with this antibody decreased p186 in vitro replication to approximately 30% of control levels, while non-specific antibodies had no effect. 14-3-3sigma was found to associate in vivo with the monkey origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, as assayed by a chromatin immunoprecipitation (ChIP) assay that involved formaldehyde cross-linking, followed by immunoprecipitation with anti-14-3-3sigma antibody and quantitative PCR. The association of 14-3-3sigma with the replication origins was maximal at the G(1)/S phase. The results indicate that 14-3-3sigma is an origin binding protein involved in the regulation of DNA replication via cruciform DNA binding.

DNA-PKcs-OBA/Ku associate in the absence of DNA, as revealed by two-dimensional capillary gel electromobility shift assay.

Ors-binding activity (OBA) has been previously purified by its ability to specifically interact with A3/4, a 36-bp mammalian origin consensus sequence [1]. Peptide sequence analyses identified OBA as Ku86, the largest subunit of Ku antigen, a heterodimeric protein (Ku70/Ku86) involved in several autoimmune disorders [2-5]. The affinity-purified fraction containing OBA/Ku is also enriched for DNA-dependent protein kinase DNA-PKcs, the catalytic subunit of the DNA-PK holoenzyme, of which Ku antigen is the DNA-binding subunit [6-8]. Glycerol-gradient sedimentation analyses have demonstrated the presence of OBA/Ku in a high-molecular-weight complex. In order to investigate whether OBA/Ku and DNA-PKcs are associated in this fraction, we have used a modification of the two-dimensional gel electrophoresis technique originally described [9]. Electromobility shift assays were developed in native capillary gels, which were subsequently used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. The gels were then processed for Western blotting using the Ku70, Ku86 and DNA-PKcs antibodies. This approach has revealed the association of OBA/Ku and DNA-PKcs to give rise to the DNA-PK holoenzyme irrespective of the presence, or the absence of DNA. Altogether, we have proven the utility of this technique for the study of protein-protein and protein-DNA interactions.