Deficient activity of von Willebrand's factor-cleaving protease in patients with disseminated malignancies

An aberrant platelet immunorelated glycoprotein Ib (GPIb) receptor expressed by human tumor cells appears to participate in primary adhesive interactions required for the metastatic process. Hence, we questioned whether plasma von Willebrand's factor (vWf), its adhesive ligand, manifested comparable anomalies in patients with disseminated tumors. Plasma specimens from patients with disseminated metastases showed 68% (P < 0.013), 91% (P < 0.0009), and 207% (P < 0.0009) enhancements in FVIII:C activity, vWf-related antigen levels, and ristocetin co-factor activity, respectively, whereas their SDS-agarose electrophoretic analysis demonstrated a 165% (P < 0.001) increase in the highly polymeric forms of vWf compared to control preparations from patients with corresponding, localized solid tumors. Substantially reduced levels of vWf-cleaving protease activity were observed in study patient specimens, with no plasma inhibitors detectable. The clinical presence and absence of tumor metastases correlated significantly with vWf-cleaving enzyme activities of < or = 15% and > or = 88%, respectively (n = 20; P < 0.0001). Finally, with an in vitro model system, tumor-induced platelet aggregation was enhanced by 127% (P < 0.001) in study patient platelet-rich plasma (PRP) compared to control PRP and could be completely inhibited (P < 0.0009) when both tumor cells and their PRP substrates were incubated with monoclonal antibodies directed against the vWf binding epitope of GPIb alpha and against the GPIb binding epitope of plasma vWf, respectively. Unusually large vWf multimers observed in patients with disseminated tumors probably result from deficient vWf-cleaving protease activity and may represent a novel mechanism regulating primary platelet-tumor adhesive interactions involved in the metastatic process.     

Impact of surgery and chemotherapy on von Willebrand factor and vascular endothelial growth factor levels in colorectal cancer

Introduction von Willebrand factor (vWf) is thought to mediate binding of tumour cells to platelets and to favour their systemic spreading capacity. Platelets involved in tumour angiogenesis are capable of releasing vascular endothelial growth factor (VEGF). Hence, levels of vWf and VEGF may correlate with cancer stage. The objectives are determine the impact of surgery and chemotherapy on vWf and VEGF in colorectal cancer (CRC) patients. Material and methods Twenty healthy volunteers (group 1), 14 patients with locally advanced CRC (group 2) and 12 patients with metastatic CRC (group 3) were enrolled. Blood samples were taken at recruitment in group 1, and before and after surgery and chemotherapy in groups 2 and 3, respectively. Blood levels of vWf, VEGF, platelet count, C-reactive protein (CRP), ceruloplasmin and carcinoembrionary antigen (CEA) were measured. Results At baseline, group 3 showed higher concentrations of vWf than the other groups (p<0.05). In group 2, vWf became elevated 40% post-surgery (p=0.016), independently of changes in CRP or ceruloplasmin. In group 3, chemotherapy caused a 42% reduction in VEGF (p=0.015). Conclusions There was a strong correlation between higher vWf levels and more advanced CRC stage at diagnosis. These levels were elevated post-surgery in patients with locally advanced CRC. Chemotherapy significantly decreased VEGF in metastatic CRC patients before CEA showed any significant change.     

Identification and Characterisation of a Platelet GPIb/V/IX-like Complex on Human Breast Cancers: Implications for the Metastatic Process

The glycoprotein (GP) Ib/V/IX receptor complex is an important adhesion molecule, originally thought to be unique to the megakaryocytic lineage. Recent evidence now indicates that GPIb/V/IX may be more widely expressed. In this study we report the presence of all subunits of the complex on four breast cancer cell lines, and 51/80 primary breast tumours. The surface expression of GPIb/V/IX was confirmed by flow cytometry, and by immunoprecipitation of biotin surface-labelled tumour cells. Western blotting of cell lysates under reducing conditions revealed that tumour cell-GPIba had a relative molecular weight of 95 kDa as compared to 135 kDa on platelets. Despite the discrepant protein size, molecular analyses on the tumour cell-GPIba subunit using RT-PCR and DNA sequencing revealed 100% sequence homology to platelet GPIba. Tumour cell-GPIb/V/IX was capable of binding human von Willebrand factor (vWf), and this binding caused aggregation of tumour cells in suspension. Tumour cells bound to immobilised vWf in the presence of EDTA and demonstrated prominent filapodial extensions indicative of cytoskeletal reorganisation. Furthermore, in a modified Boyden chamber assay, prior exposure to vWf or a GPIba monoclonal antibody, AK2, enhanced cell migration. The presence of a functional GPIb/V/IX-like complex in tumour cells suggests that this complex may participate in the process of haematogenous breast cancer metastasis     

Identification and characterisation of a platelet GPIb/V/IX-like complex on human breast cancers: implications for the metastatic process.

The glycoprotein (GP) Ib /V/IX receptor complex is an important adhesion molecule, originally thought to be unique to the megakaryocytic lineage. Recent evidence now indicates that GPIb /V/IX may be more widely expressed. In this study we report the presence of all subunits of the complex on four breast cancer cell lines, and 51 / 80 primary breast tumours. The surface expression of GPIb /V/IX was confirmed by flow cytometry, and by immunoprecipitation of biotin surface-labelled tumour cells. Western blotting of cell lysates under reducing conditions revealed that tumour cell-GPIb alpha had a relative molecular weight of 95 kDa as compared to 135 kDa on platelets. Despite the discrepant protein size, molecular analyses on the tumour cell-GPIb alpha subunit using RT-PCR and DNA sequencing revealed 100% sequence homology to platelet GPIb alpha. Tumour cell-GPIb /V/IX was capable of binding human von Willebrand factor (vWf), and this binding caused aggregation of tumour cells in suspension. Tumour cells bound to immobilised vWf in the presence of EDTA and demonstrated prominent filapodial extensions indicative of cytoskeletal reorganisation. Furthermore, in a modified Boyden chamber assay, prior exposure to vWf or a GPIb alpha monoclonal antibody, AK2, enhanced cell migration. The presence of a functional GPIb /V/IX-like complex in tumour cells suggests that this complex may participate in the process of haematogenous breast cancer metastasis.     

Functional role of platelets in experimental metastasis studied with cloned murine fibrosarcoma cell variants

The involvement of platelets in experimental metastasis was studied with cloned cell lines derived from PAK 17, a recently induced methylcholanthrene-induced C57BL/6 mouse fibrosarcoma. Tumor cell-induced platelet aggregation and lung colonization assays were used to distinguish three major stable phenotypes among the clones: a low metastatic-low platelet aggregating type, e.g., clone PAK 17.12; a low metastatic-high platelet aggregating type, e.g., clone PAK 17.14; and a high metastatic-high platelet aggregating phenotype, e.g., clone PAK 17.15. Clones with high metastatic but low platelet aggregating potential were not observed in the study. Intravenously injected PAK 17.14 and PAK 17.15 cells, but not PAK 17.12 cells, induced greater than 50% reductions in circulating platelet levels in C57BL/6 mice. Since highly metastatic clone PAK 17.15 cells consistently induced high levels of tumor cell-induced platelet aggregation regardless of the platelet donor, it was selected to study the relationship between its tumor cell-induced platelet aggregation and lung colonizing abilities. (a) A 93% decrease in lung colony number resulted in mice injected with 100 micrograms of prostacyclin immediately before injection of clone PAK 17.15 cells. Prostacyclin was also able to inhibit, in a dose dependent fashion (0-5 ng), platelet aggregation induced by clone PAK 17.15 cells in vitro. (b) A 92% reduction in lung colony number occurred in mice showing marked thrombocytopenia following injection of 100 micrograms of rabbit anti-mouse platelet antibody 24 h before tumor cell injection. (c) A greater than 80% reduction in clone PAK 17.15 lung colony number was observed in mice rendered thrombocytopenic by i.v. injection of 0.038 units of neuraminidase 24 h before i.v. injection of 10(5) tumor cells. These results suggest that platelets are required for successful lung colonization by clone PAK 17.15 cells. However, the presence in this fibrosarcoma of high platelet aggregating-poorly metastatic cells, such as clone PAK 17.14, demonstrates that while the ability to aggregate platelets is necessary for successful metastasis by some tumor cells, it is insufficient if tumor cells lack other critical properties required for completion of the metastatic cascade.     

Differential effects of tumor–platelet interaction in vitro and in vivo in glioblastoma

An elevated platelet count is considered an independent predictor of short survival in glioblastoma and various other tumor entities. Prothrombotic activity of the tumor microcirculation resulting in platelet activation and release of cytokines from activated platelets has been suggested to play a role. This study was designed to analyze the effects of platelet-released cytokines on glioblastoma and endothelial cell proliferation and migration in vitro, and the influence of platelet count on glioblastoma growth and angiogenesis in vivo. In cultured human glioblastoma, umbilical cord and cerebral microvascular endothelial cells platelet-released cytokines significantly stimulated proliferation and migration as well as sprouting and formation of capillary-like structures. In vivo, glioblastoma cells were implanted in mice followed by platelet depletion starting 1 or 8 days later. Tumor volume, proliferative index, and vessel density analyzed 14 days after engraftment did not differ between animals with a normal and a low platelet count. Likewise, no effect of platelet depletion over 20 days upon the volume of intracerebrally growing tumors was observed in mice. Additionally, proliferative activity and vessel density determined in tumor samples from patients operated upon glioblastoma did not show any correlation with the patients’ preoperative platelet count. Thus, we conclude that distinct proliferation- and chemotaxis-stimulating effects of platelet-derived cytokines can be achieved in vitro, while the platelet count does not exert a major influence on tumor growth and tumor angiogenesis in GBM in vivo.     

Muscarinic acetylcholine and histamine-receptor mediated calcium mobilization and cell-growth in human ovarian-cancer cells

The effects of carbachol and histamine on changes in cytosolic-free calcium ([Ca2+]i) and cell proliferation have been characterized in human ovarian cancer cells (OVCAR-3) and non-tumourigenic Chinese hamster ovary cells (CHO). The muscarinic agonist carbachol increased [Ca2+]i significantly with a rapid biphasic response due to both influx of extracellular calcium and release of calcium from intracellular stores. None of these effects were however seen in CHO cells. The increase in cellular calcium by carbachol was also confirmed by calcium uptake experiments using Ca-45. Carbachol increased Ca-45 uptake by 25% in OVCAR-3 cells but had no effect in CHO cells. Histamine also stimulated calcium mobilization in OVCAR-3 cells but had no effect in CHO cells. The response to histamine was also biphasic although the calcium increase was smaller than with carbachol. Data obtained with selective histamine antagonists showed that the response to histamine was mediated by H-1 histaminergic receptors. Both carbachol and histamine also stimulated cell growth of OVCAR-3 cells but were without effect on CHO cells. The cell proliferating effect of carbachol and of histamine on OVCAR-3 cells as well as the increase in [Ca2+], was totally blocked by atropine and selective H-1 histaminergic receptor antagonist pyrilamine, respectively. Fetal calf serum (FCS) which increased [Ca2+]i in both cell lines also caused a substantial increase in cell growth in the two cell lines. Verapamil partially and TMB-8 totally blocked carbachol stimulated release of calcium from intracellular stores, whereas prenylamine had only a minor inhibitory effect on calcium influx. The effect of verapamil and TMB-8 were most likely resulted from their inhibition of cholinergic receptors rather than a direct inhibition of intracellular calcium release. The carbachol induced effects on calcium transients were also partially inhibited by pertussis toxin and the phorbol ester PMA. Our data suggest that the mitogenic action of carbachol occurs through an increase in [Ca2+]i which promote DNA synthesis and cell growth. These data also indicate the involvement of both a pertussis toxin sensitive and insensitive G-protein as well as protein kinase C in the signal transduction pathway induced by carbachol.     

Photofrin and light induces microtubule depolymerization in cultured human endothelial cells.

Endothelial cells were cultured from human umbilical veins and incubated with Photofrin (1 microgram/ml). Cells were then exposed to light, and cytoplasmic microtubule (MT) status was monitored by immunofluorescence microscopy using alpha-tubulin antibody. As early as 15 min following irradiation, a light dose-dependent depolymerization of MT was observed. At sublethal light doses, this effect was transient, with MT repolymerizing within 2-3 h. Cellular ATP levels were monitored to determine whether diminished ATP levels were correlated with MT depolymerization. No correlation was found, since ATP levels remained at a constant value near 50% of unirradiated controls during a time interval in which transient MT depolymerization was observed. Cell viability was monitored by trypan blue exclusion. Transient MT depolymerization occurred at photodynamic doses that produced essentially no decrease in cell viability, while at higher doses, irreversible MT depolymerization was observed prior to loss of viability. Since MT are unstable at intracellular calcium levels greater than 1 microM, we postulate that MT depolymerization results from increases in intracellular calcium caused by photodynamic insult. MT are important in maintaining cell shape. Disruption of MT in endothelial cells due to photodynamic therapy could result in or contribute to exposure of the thrombogenic subendothelium or could alter vascular permeability in the treatment area.     

Development of a slow release system of anti-cancer drugs retained in calcium-hydroxyapatite ceramic

A new type of anticancer material with sustained release by enclosure of cisplatin (CDDP) into porous calcium hydroxyapatite ceramic (CDDP-CHA) was developed. A slow release of anticancer drug from CDDP-CHA was confirmed in in vitro experiments. When this material was implanted into normal thigh muscle of mouse, a sustained release of CDDP was observed during over 8 weeks after implantation. The diffusion of drug into blood and other organs from the implanted site was very small. CDDP-CHA placed into the implanted tumor of mouse allowed a slow release and high drug level from the material in the tumor. The concentration of the drug in other organs such as liver and kidney was very low compared with that of the tumor. These results suggest that this material has an important role for cancer chemotherapy, particularly in bone tumors for the reasons of calcium hydroxyapatite ceramic carrier having a mechanical strength.     

Effects of thromboxane A2 inhibition on osteogenic sarcoma cell-induced platelet aggregation

There is evidence that tumors may stimulate platelet aggregation, causing release of thromboxane A2. Thromboxane A2 may potentiate tumor metastasis by stimulating tumor cell growth and proliferation and by enhancing platelet-tumor cell aggregate formation. Despite potential significance of thromboxane A2 in tumor metastasis, agents which inhibit thromboxane A2 synthesis have not been uniformly effective in reducing tumor metastasis. We, therefore, evaluated the effects of a thromboxane A2 receptor antagonist SQ-29,548 compared to those of a thromboxane A2 synthetase inhibitor OKY-046 on osteogenic sarcoma-induced platelet aggregation and thromboxane A2 release. Osteogenic sarcoma cells added to platelet-rich plasma caused complete and irreversible platelet aggregation as well as thromboxane A2 release. Preincubation of platelet-rich plasma with SQ-29,548 (2 to 20 nM) decreased platelet aggregation induced by tumor cells, but it had no effect on thromboxane A2 release. In contrast, preincubation of platelet-rich plasma with OKY-046 (0.1 to 10 microM) had no effect on platelet aggregation despite a decrease in thromboxane A2 synthesis. These results suggest that thromboxane A2 receptor blockers, rather than synthetase inhibitors, may prevent tumor cell-induced platelet aggregation.     

Calcium-dependent photodynamic action of di- and tetrasulphonated aluminium phthalocyanine on normal and tumour-derived rat pancreatic exocrine cells.

Important differences exist in the responses to photodynamic agents of normal and tumour-derived pancreatic acinar cells. In the present study amylase release has been used to assess the mechanisms by which the photodynamic drugs tetra- and disulphonated aluminium phthalocyanine (A1PcS4, A1PcS2) act on pancreatic cells via energy and calcium-dependent activation and transduction pathways. The photodynamic release of amylase was found to be energy dependent and inhibited by the chelation of free cytoplasmic calcium but not by the removal of extracellular calcium. In contrast to their effects on normal acinar cells, the photodynamic action of A1PcS4 and A1PcS2 was to inhibit amylase secretion from pancreatoma AR4-2J cells. Removal of extracellular calcium reversed this inhibitory effect on AR4-2J cells and produced a significant increase in amylase release, but chelation of free cytoplasmic calcium did not affect the inhibitory photodynamic action of the phthalocyanines on amylase release from the tumour cells. Overall, these results demonstrate further important distinctions between the photodynamic action of sulphonated aluminium phthalocyanines on normal versus tumour exocrine cells of the pancreas and indicate that calcium plays an important role in photodynamic drug action, since these agents affected intracellular calcium mobilisation at some distal point in the membrane signal transduction pathway for regulated secretion. Furthermore, the photodynamic inhibition of constitutive secretion in tumour cells may involve a calcium-dependent membrane target site or modulation of membrane calcium channels by activation of protein kinase C.     

Matrix metalloproteinase 2 in tumor cell-induced platelet aggregation: regulation by nitric oxide

A correlation exists between the ability of tumor cells to aggregate platelets and their tendency to metastasize. Tumor cell-induced platelet aggregation (TCIPA) facilitates the embolization of the vasculature with tumor cells and the formation of metastatic foci. It is well documented that matrix metalloproteinases (MMPs) play an integral part in tumor spread and the metastatic cascade. Therefore, we have examined the role of MMPs during TCIPA and its regulation by nitric oxide (NO) in vitro. Human HT-1080 fibrosarcoma and A549 lung epithelial cancer cells induced TCIPA in a concentration-dependent manner that was monitored by aggregometry. This aggregation resulted in the release of MMIP-2 from platelets and cancer cells, as measured by zymography. HT-1080 cells released significantly more MMP-2 than A549 cells and were more efficacious in inducing TCIPA. Inhibition of MMP-2 with phenanthroline (1-1000 microM), a synthetic inhibitor of MMPs, and by neutralizing anti-MMIP-2 antibody (10 microg/ml) reduced TCIPA induced by HT-1080 cells. TCIPA was abolished by simultaneous inhibition of platelet function with acetylsalicylic acid (100 microM; thromboxane pathway inhibitor), apyrase (250 microg/ml; ADP pathway inhibitor), and phenanthroline. NO donors such as S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione (both at 0.01-100 microM) inhibited TCIPA and MMP-2 release from platelets and tumor cells. The inhibitory actions of S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione were reversed by 1H-[1,2,4]oxadiazole[4,3]quinoxalin-1-one (0.01-30 microM), a selective inhibitor of the soluble guanylyl cyclase. We conclude that (a) human fibrosarcoma cells aggregate platelets via mechanism(s) that are mediated, in part, by MMP-2; (b) NO inhibits TCIPA, in part, by attenuating the release of MMP-2; and (c) these effects of NO are cGMP-dependent.     

Platelets promote osteosarcoma cell growth through activation of the platelet‐derived growth factor receptor‐Akt signaling axis

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation‐inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor‐platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma‐platelet interactions induce the release of platelet‐derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co‐culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho‐antibody arrays revealed that co‐culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma‐platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated‐PDGFR and ‐Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet‐induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma‐platelet interactions may eradicate osteosarcomas.     

Multiparametric flow cytometry of the modulation of tumor cell membrane permeability by developmental antitumor ether lipid SRI 62-834 in EMT6 mouse mammary tumor and HL-60 human promyelocytic leukemia cells

(+-)-2-[Hydroxy[tetrahydro-2-(octadecyloxy)methylfuran-2- yl]methoxyl]phosphinyloxy-N,N,N-trimethylethaniminium hydroxide, inner salt (SRI 62-834) is a tetrahydrofuran analogue of platelet activating factor (PAF) that is currently entering clinical trial. Like other ether lipids it is of interest as a membrane-active antitumor agent. Here, we have used two-color multiparameter flow cytometry to study simultaneously its effects on cell membrane permeability, intracellular pH, and cell size/structure of EMT6 mouse mammary tumor cells and HL-60 human promyelocytic leukemia cells in vitro. Concentrations as low as 1 microM up to 100 microM SRI 62-834 caused a rapid, dose-dependent increase in membrane permeability, initially towards outward efflux of the preloaded fluorescein probe bis(carboxyethyl)carboxyfluorescein (green fluorescence) and then towards influx of extracellular propidium (red fluorescence). At the same time, median cell size from light scatter was reduced with an increased coefficient of variation, and the proportion of cell debris was elevated. In vitro antitumor activity was seen over the same concentration range, as measured by tetrazolium dye reduction and cell growth curves. Neither low concentrations of PAF (50 nM) nor the potent PAF antagonist 3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f][1,2,4]tria- zolo[4,3a][1,4]diazepin-2-yl]-1-(4-morpholinyl)-1-propanone (0.5-100 microM) had any influence on the membrane effects of SRI 62-834, and at higher concentrations (1-200 microM) PAF mimicked the behavior of SRI 62-834. In addition, the PAF antagonist did not modulate the cytotoxicity of SRI 62-834 or PAF. HL-60 cells were more sensitive to SRI 62-834 than were EMT6 cells in terms of both cytotoxicity and membrane permeability. However, PAF was more potent than SRI 62-834 in causing membrane permeabilization with both cell lines, whereas PAF was less active than SRI 62-834 in cytotoxicity assays. The results support a membrane-damaging role in the cytotoxicity of SRI 62-834 but suggest that additional factors are also involved. Membrane permeabilization may be related to its reported effects on protein kinase C-dependent intracellular calcium signaling but apparently does not involve a conventional PAF receptor in HL-60 or EMT6 cells.     

Human rhabdosarcoma cell-induced aggregation of blood platelets

The ability of tumor cells shed into the circulation to cause adhesion and aggregation of blood platelets may be involved in successful metastasis of primary tumors. Rhabdosarcoma is a rare, early metastasizing tumor previously uncharacterized for ability to alter platelet function. It was found that human rhabdosarcoma cells (American Type Culture Collection) dose dependently induce biphasic aggregation of human blood platelets in heparinized platelet-rich plasma; aggregation responses could also be elicited in citrated plasma. Aggregation caused by rhabdosarcoma can be inhibited by apyrase treatment of either rhabdosarcoma or platelets, and by pretreatment of platelets with prostacyclin, cilostamide, inhibitors of thromboxane A2 production, or TMB-8; only apyrase and prostacyclin inhibited both phases of aggregation. Tumor cell supernatant contained only enough ADP to cause a negligible, reversible aggregation response. Hirudin, verapamil, and triazolam do not inhibit rhabdosarcoma-induced aggregation. Aggregation of platelets by rhabdosarcoma cells thus appears to involve ADP, from tumor cells and/or platelets, and platelet calcium mobilization and thromboxane A2 synthesis and release.     

Subcellular concentrations of calcium, zinc, and magnesium in benign nodular hyperplasia of the human prostate: X-ray microanalysis of freeze-dried cryosections

Biopsies from human prostates were obtained from normal and hyperplastic glands. The intracellular concentrations of calcium, zinc, and magnesium were analyzed using X-ray microanalysis of freeze-dried cryosections. Two prostate biopsies were obtained from kidney donors, ages 19 and 50 years, without any sign of benign nodular hyperplasia. The normal tissues were frozen within 15 min after circulatory arrest. The central part of biopsies from eight elderly men suffering from benign nodular hyperplasia were frozen within 30 s after excision. Adjacent tissue was processed for light microscopy and histopathological diagnosis. All samples were fresh-frozen using liquid nitrogen cooled pliers, without the use of any freeze-protection, fixation, or staining. In both the normal and the hyperplastic prostates high concentrations (up to above 100 mmol/kg dry weight) of zinc were present in electron dense bodies in the cytoplasm of the epithelial cells. Together with zinc, about equal concentrations of magnesium were found. Calcium was detected in 4 to 8 times the concentration of zinc. Significant, positive correlation between calcium and zinc as well as between calcium and magnesium in the cytoplasm was a typical finding in both normal and hyperplastic glands. In six of eight patients, older than 60 years of age, high levels of calcium (17.0-38.8 mmol/kg dry weight) were observed in the nuclei of the epithelial cells, while very low values were found in the remaining two. In the two younger cases (19 and 50 years of age), the nuclear calcium level in prostatic epithelium was relatively low (about 10 mmol/kg dry weight). These observations suggest that an increase of intranuclear calcium with advancing age may be of pathogenetic significance to growth disturbances in the prostate.     

Release of intracellular calcium primes chronic myeloid leukaemia cells for tyrosine kinase inhibitor-induced apoptosis

Imatinib is a substrate for hOCT1 (SLC22A1) and inhibitors of this influx transporter, such as amantadine and prazosin, have previously been shown to decrease cellular imatinib uptake. However, here we report that in longer term experiments, both drugs paradoxically increase the cytotoxicity of all three currently licensed tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib. This effect is due to release of intracellular calcium from the endoplasmic reticulum (ER), with changes in mitochondrial calcium and alterations in mitochondrial membrane permeability, resulting in caspase-mediated apoptosis. The effect is confined to BCR-ABL-positive cells, and is greater in primary cells than in cell lines. Furthermore, in primary cells at original diagnosis, the effect is only seen in samples from patients destined to become complete cytogenetic responders to imatinib. These results indicate that calcium release from the ER, here induced by amantadine or prazosin, may prime BCR-ABL-positive cells to TKI-induced apoptosis. Amantadine/prazosin primed TKI cytotoxicity in vitro may be a useful test for the level of ER-releasable calcium, and may be of prognostic value.     

Establishment of megakaryoblastic cell lines by coinfection of Abelson murine leukemia virus and recombinant SV40-retrovirus

Murine embryonic cells including yolk sac prepared from 8-day embryos were co-infected with Abelson murine leukemia virus (A-MuLV) and/or a recombinant retrovirus containing large T and small t antigens, and early region of simian virus 40 (M-SV40). By coinfection with A-MuLV and M-SV40, megakaryoblastic cells were obtained in addition to mast cells and fibroblastic cells. However, following infection with A-MuLV or M-SV40 alone, no megakaryoblastic cells were detected, although mast cells and/or fibroblastic cells developed. The same results were obtained in several experiments. By single-cell transfer, 6 acetyl-cholinesterase (AchE)-positive clonal cell lines were established. Characteristics of megakaryocytes, such as AchE, glycoproteins IIb and IIIa, and platelet peroxidase were detected in two representative cells (C1 and C8). More significant changes expressing differentiation were observed following treatment with phorbol myristate acetate or pokeweed mitogen-stimulated murine spleen cell conditioned medium, although release of platelets was not observed. This is the first report showing development of megakaryocytic cells as the result of coinfection with retroviruses.     

Vascular endothelial growth factor expression and neovascularisation in non-small cell lung cancer

Many recent studies have demonstrated that tumour angiogenesis is a potent prognostic factor for various malignant tumours, but this has not been clearly shown in non-small cell lung carcinoma (NSCLC). The purpose of this study was to re-evaluate the prognostic value of MVD associated with VEGF in patients with NSCLC by comparing the immunohistochemical results obtained for CD34 with those obtained for vWf. Microvessel density (MVD) and the expression of vascular endothelial growth factor (VEGF) were investigated in 108 cases of NSCLC by immunohistochemistry. The correlation between von Willebrand factor (vWf) and CD34 staining for MVD was not strong, and vWf staining did not correlate with VEGF expression, but CD34 staining did. Staining for CD34 significantly correlated with survival in adenocarcinoma, distant metastasis and postoperative recurrence, but staining for vWf did not. CD34 was more sensitive and specific than vWf for staining endothelial cells associated with VEGF expression. It is suggested that research on neovascularisation should be investigated on every histological subtype or should focus on the early stages of NSCLC which are not under the influence of a variety of complications facilitating tumour neovascularisation.     

Ultra-violet irradiation induces apoptosis via mitochondrial pathway in pancreatic cancer cells

Pancreatic cancer is a highly lethal disease and gemcitabine is considered to be the standard of care for the treatment of advanced pancreatic cancer. However, the outcome of the patients treated with gemcitabine is still unstatisfactory and further development of new treatments is required. We recently found that short wavelength ultra-violet (UV-C) suppresses cell proliferation with downregulation of epidermal growth factor receptor (EGFR) in human pancreatic cancer cells, but not in normal pancreatic epithelial (PE) cells. In this study, we investigated the effect of UV-C on apoptosis in several cell lines derived from the pancreas. UV-C induced poly(ADP-ribose) polymerase (PARP) cleavage, which is a marker of cells undergoing apoptosis, in Panc1, MiaPaca2, KP3 and BxPC3 pancreatic cancer cells, but not in PE cells. We also observed similar effects in Hoechst 33258 staining, which shows DNA fragmentation. While p53, a tumor suppressor protein, plays a critical role in UV-C-induced cell damage, we did not observe the correlation between the sensitivity to UV-C and p53 status. Thapsigargin, an agent that promotes endoplasmic reticulum (ER) stress by depletion of lumenal calcium stores, as well as cis-diamineplatinum (II) dichloride, a classical anti-cancer drug that causes DNA damage, induced PARP cleavage even in PE cells. Moreover, UV-C-induced apoptosis in Panc1 and KP3 cells was associated with the release of cytochrome c, indicating that it was mediated via mitochondrial pathway. Taken together, UV-C has a potent anti-cancer effect on pancreatic cancer cells without adverse effect on normal cells and it could be useful for the treatment of human pancreatic cancers.