The role of fibrinogen and related fragments in tumour angiogenesis and metastasis

Angiogenesis, the development of new blood vessels from existing vasculature, involves the migration, proliferation and differentiation of endothelial cells and is crucial for the growth and mestastasis of tumours. A specific association between cancer and the haemostatic system has long been recognised. Haemostatic mechanisms regulate blood flow by controlling platelet adhesion and fibrin deposition, and a number of haemostatic proteins have been shown to regulate angiogenesis, either directly, by interacting with endothelial cells themselves, or indirectly, by interacting with other regulators of angiogenesis. The polypeptide fibrinogen is the central protein in the haemostasis pathway and is found deposited in the majority of human and experimental animal tumours. In this review, the evidence for the ability of fibrinogen and various protein/peptide fragment derivatives to modulate angiogenic mechanisms in vitro and to affect tumour growth and metastasis in vivo is discussed.     

Human U251 glioma cell proliferation is suppressed by HET0016 [N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine], a selective inhibitor of CYP4A

We have previously reported that HET0016 [N-hydroxy-N'-(4-butyl-2 methylphenyl)formamidine], a selective inhibitor of CYP4A and thus 20-HETE (20-hydroxyeicosatetraenoic acid) synthesis, inhibits endothelial cell proliferation and decreases angiogenesis induced by human glioma cell U251. A stable 20-HETE agonist, WIT003 [20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (1 microM)], increased U251 cell proliferation from 3.9- to 4.8-folds from T(0) (time of the treatment). We examined the effects of HET0016 on the growth of U251. HET0016 inhibited U251 basal cell proliferation in a dose-dependent manner. 10 microM HET0016 suppressed 56% of U251 proliferation and significantly increased the proportions of the cells arrested in the G(0)/G(1) phase of the cell cycle. Exposure to HET0016 (as early as 4 h) reduced protein tyrosine and p42/p44 MAPK (mitogen-activated protein kinase) phosphorylation. Furthermore, HET0016 significantly inhibited the U251 proliferation and phosphorylation of both the epidermal growth factor (EGF) receptor and p42/p44 MAPK induced by EGF. CYP4A mRNA and proteins were both present in U251. This suggests that HET0016 inhibited U251 proliferation by inhibiting 20-HETE synthesis. However, U251 did not synthesize 20-HETE in the presence of arachidonic acid. This implies that HET0016 suppresses U251 proliferation by mechanisms that are not yet clear but may involve activities other than inhibition of 20-HETE synthesis. We concluded that HET0016 may be the prototype of novel compounds that suppress human glioma cell proliferation.     

Expression of CYP4A1 in U251 human glioma cell induces hyperproliferative phenotype in vitro and rapidly growing tumors in vivo

Exogenous 20-hydroxyeicosatetraenoic acid (20-HETE) increases the growth of human glioma cells in vitro. However, glioma cells in culture show negligible 20-HETE synthesis. We examined whether inducing the expression of a 20-HETE synthase in a human glioma U251 cell line would increase proliferation. U251 cells transfected with CYP4A1 cDNA (termed U251 O) increased the formation of 20-HETE from less than 1 to over 60 pmol/min/mg proteins and increased their proliferation rate by 2-fold (p < 0.01). Compared with control U251, U251 O cells were rounded, smaller, showed a disorganized cytoskeleton, exhibited reduced vinculin staining, and were easily detached from the growing surface. They showed a marked increase in dihydroethidium staining, suggesting increased oxidative stress. The expression of phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1/2, and vascular endothelial growth factor was markedly elevated in U251 O. The hyperproliferative and signaling effects seen in U251 O cells are abolished by selective CYP4A inhibition of 20-HETE formation with HET0016 [N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine], by small interfering RNA against the enzyme, and by the putative 20-HETE antagonist, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid. In vivo, implantation of U251O cells in the brain of nude rats resulted in a approximately 10-fold larger tumor volume (10 days postimplantation) compared with animals receiving mock-transfected U251 cells. These data show that elevations in 20-HETE synthesis in U251 cells lead to an increased growth both in vitro and in vivo. This suggests that 20-HETE may have proto-oncogenic properties in U251 human gliomas. Further studies are needed to determine whether 20-HETE plays a role promoting growth of some human gliomas.     

Tumor and non-tumor liver angiogenesis is traced and evaluated by hepatic arterial ultrasound in murine models

We studied the relationships between hepatic and mesenteric mean blood-flow velocities (mBFVs) measured by ultrasound imaging and (1) downstream tumor angiogenesis during liver metastasis induced by spleen injection of LS174 human colon cells overexpressing the antiangiogenic Netrin4 (LS174-NT4) or not (LS174-WT) and (2) downstream normal angiogenesis during hepatic regeneration after 50% hepatectomy. Liver volume and mBFVs were measured before and after surgery, at day 30 in the first model and at days 2, 7 and 16 in the second model. LS174-NT-4 vs. LS174-WT mice presented fewer metastases (25% vs. 90%, p < 0.001) and decreased hepatic mBFVs (16.5 ± 0.8 vs. 21.8 ± 1.4 cm s(-1), p < 0.01), without difference in mesenteric mBFVs. After partial hepatectomy, hepatic and mesenteric mBFVs increased at day 7, from 12.4 ± 1.7 and 11.8 ± 2.6 to 19.1 ± 1.8 and 17.5 ± 2.4 cm s(-1), respectively, (p < 0.01) then returned to baseline as liver volume. Duplex Doppler ultrasonography reliably assesses normal or tumor angiogenesis and may provide follow-up functional evaluation.     

Measurement of tumour protein synthesis in vivo in human colorectal and breast cancer and its variability in separate biopsies from the same tumour.

1. A method is described for measuring the rates of protein synthesis in vivo in human colorectal and breast tumours by the intravenous injection of L-[1-13C]leucine as a 'flooding dose'. 2. The incorporation of isotope into colorectal tumour protein was measured in six patients, whose tumours were biopsied after the injection. Fractional rates of protein synthesis were calculated from the enrichment of leucine in protein and the average free leucine enrichment in plasma. The range of rates obtained was 17.2-33.9%/day, with a mean rate (+/- SEM) of 22.5 +/- 2.6%/day. 3. Tumour protein synthesis rates were also measured in 15 patients with breast cancer. The range of rates obtained was 5.3-15.9%/day, with a mean rate (+/- SEM) of 10.3 +/- 0.8%/day. These rates are significantly lower than those obtained with colorectal tumours (P less than 0.001). 4. In 9 of the breast cancer patients, protein synthesis was measured in multiple random biopsies taken from the same tumour. The mean (+/- SEM) difference between the highest and lowest rates in biopsies from the same tumour was only 1.1 +/- 0.3%/day. Only 13% of the variation in protein synthesis between separate tumours could be explained by sampling error because of variation within the tumour itself, the remainder being genuine variation between individual tumours.     

AML1-ETO融合蛋白对BCL-2基因表达的影响

本研究旨在观察AML1-ETO在白血病细胞中对于抗凋亡基因日乳-2表达的影响,探讨其在白血病发生中的作用。应用流式细胞术检测急性单核细胞白血病细胞株U937-WT、U937-Mock和经AML1-ETO基因转染的U937-A／E1-4的细胞凋亡率;使用免疫印迹法检测cleavedcaspase-3蛋白的表达;荧光实时定量PCR检测转染细胞和对照组细胞以及AMLM2患者白血病细胞BCL-2mRNA的表达;染色质免疫沉淀技术研究转染细胞中AML1-ETO与BCL-2基因启动子之间直接的相互作用情况。结果表明：AML1-ETO转染细胞的凋亡率明显增加,且检测到cleavedcaspase-3蛋白的表达;转染了AML1—ETO 的U937细胞系和具有AML1—ETO融合基因的AML-M2患者中,BCL-2的mRNA表达水平显著下调;转染细胞沉淀富集的AML1-ETO直接结合的DNA中含有BCL-2基因的启动子序列。结论：BCL-2是AML-ETO的直接靶基因,AML1-ETO能下调BCL-2的表达。     

Detecting vascular changes in tumour xenografts using micro-ultrasound and micro-ct following treatment with VEGFR-2 blocking antibodies

Blockade of vascular endothelial growth factor (VEGF) binding to its receptors on endothelial cells has been shown preclinically to induce tumour growth inhibition. Using ultrasound biomicroscopy (UBM) or micro-ultrasound imaging and micro-computed tomography (micro-CT) analysis, we have examined the effects of DC101, a highly specific vascular endothelial growth factor receptor-2 (VEGFR-2)-targeting antibody, in inducing growth inhibition and functional vascular changes in established melanoma (MeWo) xenografts in mice. Postprocessing of UBM imaging loops for speckle variance was introduced to estimate the level of functional blood flow in tumours. Perfused tumour area visualized by speckle variance revealed decreased blood flow within 48 h after DC101 injection (control versus DC101: 1.90 +/- 0.25% versus 1.01 +/- 0.11%, p < 0.01) and following a 3-wk DC101 therapy (control versus DC101: 0.76 +/- 0.14% versus 0.45 +/- 0.05%, p = 0.04), suggesting that VEGFR-2 blockade mediates both early and long-term effects on tumour blood flow. The growth of xenografts was significantly inhibited after treating with DC101 for 3 wk compared with controls. In addition to UBM, we examined the tumour vasculature in three-dimension (3D) using contrast-enhanced Micro-CT imaging, which displayed a reduction in the number of tumour vessels following extended VEGFR-2 blockade (vascular density of control versus DC101: 48.4 +/- 5.4% versus 20.6 +/- 1.8%). Lastly, decreased microvessel density (MVD) was noted in DC101-treated xenografts (3 wk) by performing immunohistochemical staining of endothelial marker CD34. Our study investigates tumour response to DC101 using complementing micro-ultrasound and micro-CT imaging tools.     

Antigen recognition by human T cell receptor gamma-positive lymphocytes. Specific lysis of allogeneic cells after activation in mixed lymphocyte culture

These experiments were designed to define the ability of human TCR-gamma+ cells to recognize allogeneic cells. TCR-gamma+-enriched populations were obtained by treating peripheral blood E-rosetting cells with anti-CD4 and anti-CD8 mAbs. The resulting populations were CD2+4-8- expressed variable proportions of CD3+ cells (40-90%), and did not react with the WT31 mAb, which is specific for a framework determinant of the alpha/beta heterodimer that serves as receptor for antigen on most human T lymphocytes. After mixed lymphocyte culture with irradiated allogeneic cells for 7 d and 3 additional days in rIL-2 (100 U/ml), cells underwent proliferation in three of five individuals tested. In addition, MLC-derived cells lysed 51Cr-labeled PHA-induced blasts derived from the allogeneic cells used as stimulator, but not allogeneic unrelated or autologous blast cells. No cytotoxicity against autologous or allogeneic target cells could be induced by culturing CD3+4-8-WT31- lymphocytes in MLC with irradiated autologous cells. Surface iodination of allogeneic MLC-activated CD3+4-8-WT31- cells followed by lysis in 1% digitonin and immunoprecipitation with anti-CD3 mAb indicated that the CD3-associated molecules consisted of a major 45-kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNAs for alpha and beta chains were missing. These data support the notion that TCR-gamma rather than TCR-alpha/beta is expressed in allospecific CD3-4-8-WT31- cell populations. Clones were further derived from MLC-stimulated CD3+4-8-WT31- populations. All the seven clones studied in detail maintained the surface phenotype as well as the cytolytic pattern of the original MLC populations, thus only specific allogeneic PHA-induced blasts were lysed. NK-sensitive as well as NK-resistant tumor targets were variably susceptible to lysis; therefore, specific cytolytic activity against allogeneic cells was not necessarily linked to the expression of MHC-nonrestricted cytotoxicity against tumor cells.     

NOB1影响胶质瘤细胞增殖、凋亡的实验研究

目的 利用RNA干扰（RNAi）在人胶质瘤U251和U87-MG细胞中沉默NOB1基因的表达,探讨NOB1对恶性胶质瘤细胞增殖以及凋亡的影响.方法 构建NOB1基因的短发卡（shRNA）慢病毒表达载体,包装成病毒颗粒并感染人胶质瘤U251和U87-MG细胞,采用实时定量聚合酶链反应检测NOB1在恶性胶质瘤细胞系中的表达.采用甲基噻唑基四唑（MTT）比色法和克隆形成实验检测细胞增殖和克隆形成能力情况;同时利用PI染色流式细胞仪检测细胞凋亡情况,观察NOB1基因对U251和U87-MG细胞增殖、凋亡的影响.结果 NOB1基因在人胶质瘤U251、U87-MG细胞系中均明显高表达.NOB1-shRNA慢病毒能有效感染胶质瘤细胞,实验结果显示感染后U251和U87-MG细胞的增殖能力明显下降;U251克隆形成能力明显受到抑制;细胞周期在G0/G1停滞,而且细胞出现显著性凋亡;上述差异均具有统计学意义（P＜0.05）.结论 NOB1在人胶质瘤U251和U87-MG细胞中高表达;降低NOB1基因的表达,可以显著降低胶质瘤细胞的增殖,并促进胶质瘤细胞凋亡;NOB1基因在体外对胶质瘤细胞的发生发展可能具有癌基因的调节作用.     

siRNA抑制人胶质瘤细胞MDM2的表达及细胞增殖

目的研究siRNA对人胶质瘤U251细胞MDM2基因表达的抑制作用及对肿瘤细胞增殖和凋亡的影响。方法体外合成2对针对MDM2基因的siRNA,经脂质体转染导入U251细胞;用半定量RT-PCR检测siRNA MDM2对MDM2基因表达的抑制作用;并用MTT法检测siRNA MDM2对细胞增殖抑制作用,流式细胞仪检测细胞凋亡。结果转染siRNA MDM2的细胞的MDM2基因表达分别下调到对照组的31.61%和40.18%;转染阴性对照质粒的MDM2基因没有明显改变。MTT结果表明转染siRNA MDM2后细胞生长受到抑制,与对照组比差异具有显著性（P〈0.5）;同时细胞发生凋亡和细胞周期阻滞,转染siRNA MDM2-1,2的凋亡率分别为49.9%和40.0%,转染阴性对照质粒和对照组未检测出明显的增殖抑制和凋亡改变。结论 siRNA MDM2可以有效抑制U251细胞株中MDM2的表达,并抑制细胞增殖,诱导细胞凋亡。     

Interstitial albumin mass and transcapillary extravasation rate of albumin in DMBA-induced rat mammary tumours

Transcapillary extravasation rate for radio-iodinated human serum albumin (I-HSA) from plasma to interstitial fluid, interstitial albumin mass and interstitial albumin concentration have been determined in dimethyl-benz-alpha-anthracene (DMBA)-induced mammary tumours in rats. The plasma radioactivity of tracer was kept constant in awake, freely moving rats by a continuous infusion of I-HSA from 1 to 72 h after an initial priming dose. Capillary leakiness for I-HSA was calculated as the plasma equivalent volume of I-HSA located extravascularly after 1 h infusion divided by the blood volume in the tissue, i.e. as the fractional extravasation rate (FER). The experiments showed that FER was highest in tumours while intestine and skin had FER of about 70% and 25% that of tumour respectively. Heart and skeletal muscle had similar FER about 1/8 that of tumour. Interstitial albumin concentration in tumour averaged 42% of plasma albumin concentration in tumour. Intestine, heart and skeletal muscle had interstitial albumin concentrations between 34 and 37% of plasma albumin concentration while skin had an albumin concentration averaging 20% that of plasma. Lymph flow was calculated as the product of the fractional turnover rate constant for interstitial albumin and interstitial volume (extravascular 51Cr-EDTA volume) and averaged 36.7 X 10(-3) mg/g X h in tumour while intestine and heart had lymph flows of 19.1 X 10(-3) and 14.1 X 10(-3) ml/g X h respectively. In muscle and skin lymph flow was 2.4 X 10(-3) and 18.3 X 10(-3) ml/g X h respectively. It is suggested that the high interstitial fluid pressure previously observed in these tumours will contribute in maintaining interstitial volume in a steady state by opposing fluid filtration into the interstitium and maintaining a lymph flow high enough to remove excess fluid and protein from the interstitium.     

抑制STAT3表达增加H_2O_2诱导人胶质瘤U251细胞损伤

目的探讨RNA干涉技术抑制STAT3基因表达对过氧化氢(H2O2)复制的人胶质瘤U251细胞损伤模型的影响。方法转染pSilencer1.0-U6-SiRNA-STAT3重组质粒到U251细胞;Western blot方法观察转染重组质粒对STAT3蛋白表达的影响;MTT法检测细胞生存率,TUNEL法检测细胞凋亡,Western blot检测凋亡相关蛋白BCL-2、BAX表达。结果转染pSilencer1.0-U6-SiRNA-STAT3重组质粒能有效抑制U251细胞STAT3蛋白表达(抑制率50%);抑制STAT3表达能促进H2O2诱导U251细胞生存率下降,增加细胞凋亡率(P0.05)。STAT3抑制能促进H2O2作用下U251细胞BCL-2表达降低,BAX表达增加(P0.05)。结论pSilencer1.0-U6-SiRNA-STAT3可有效抑制U251细胞STAT3的表达,并促进H2O2诱导U251细胞凋亡。     

Resveratrol-induced apoptotic death in human U251 glioma cells

Resveratrol (trans-3,4',5-trihydroxystilbene) is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a variety of food sources. Resveratrol has antiinflammatory and antioxidant properties, and also has potent anticancer properties. Human glioma U251 cells were used to understand the molecular mechanisms by which resveratrol acts as an anticancer agent, since glioma is a particularly difficult cancer to treat and eradicate. Our data show that resveratrol induces dose- and time-dependent death of U251 cells, as measured by lactate dehydrogenase release and internucleosomal DNA fragmentation assays. Resveratrol induces activation of caspase-3 and increases the cleavage of the downstream caspase substrate, poly(ADP-ribose) polymerase. Resveratrol-induced DNA fragmentation can be completely blocked by either a general caspase inhibitor (Z-VAD-FMK) or a selective caspase-3 inhibitor (Z-DEVD-FMK), but not by a selective caspase-1 inhibitor. Resveratrol induces cytochrome c release from mitochondria to the cytoplasm and activation of caspase-9. Resveratrol also increases expression of proapoptotic Bax and its translocation to the mitochondria. Resveratrol inhibits U251 proliferation, as measured by MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], and induces G0/G1 growth arrest, as determined by flow cytometry. The cyclin-dependent kinase inhibitor, olomoucine, prevents cell cycle progression and resveratrol-induced apoptosis. These results suggest that multiple signaling pathways may underlie the apoptotic death of U251 glioma induced by resveratrol, which warrants further exploration as an anticancer agent in human glioma.     

Presence of tumour inhibits the normal post-operative response in arginine and NO production in non-cachectic mice

We have described recently that cancer patients have low plasma arginine concentrations, even without weight loss being present, suggesting that decreased arginine availability may be a specific feature of the presence of tumour. As arginine is important in post-operative repair, we hypothesized that abnormalities in arginine metabolism in cancer lead to an aberrant post-operative response in arginine and NO metabolism. To investigate this, we studied post-operative alterations in arginine and NO production and the acute-phase response in MCA (methylcholanthrene) sarcoma-bearing mice. Controls, mice with small MCA tumours (<15% of carcass weight) and large MCA tumours (>15% of carcass weight) were studied, either with or without undergoing laparotomy. The stable isotopes L-[guanidino-(15)N(2)-(2)H(2)]arginine and L-[ureido-(15)N]citrulline were used to study whole-body arginine and NO production rates. SAP (serum amyloid P component) concentrations were measured to assess the acute-phase response. Significance was tested using Mann-Whitney U test. In healthy FVB mice, laparotomy significantly increased whole-body arginine production (from 42+/-3 to 54+/-3 nmol x 10 g(-1) of carcass weight x min(-1)), NO production (from 1.1+/-0.1 to 1.4+/-0.2 nmol x 10 g(-1) of carcass weight x min(-1)) and levels of SAP (from 4+/-1 to 115+/-23 ng/ml), whereas in all MCA tumour-bearing mice baseline values of arginine metabolism and SAP concentration were already elevated and the response to laparotomy was absent. In conclusion, MCA tumour-bearing mice had a disturbed post-operative metabolic response, as evidenced by attenuated post-operative arginine and NO production, concomitant with an attenuated acute-phase response. This indicates that altered arginine metabolism may be an important characteristic of the metabolic changes in cancer.     

Tumor vascularity and tryptase-positive mast cells correlate with a poor prognosis in melanoma

BACKGROUND: The importance of angiogenesis in melanoma has been controversial and is not homogeneous. Mast cell density (MCD) is highly correlated with the extent of both normal and pathological angiogenesis, such as that in chronic inflammatory diseases and tumours. METHODS: We evaluated the prognostic significance of tumour microvascular density (MVD) and MCD in 25 advanced melanoma patients after resection and a 4-5-year follow up: 48% of the patients were alive and free of metastases (good prognostic subgroup); 16% had developed regional nodal metastases (intermediate prognostic subgroup); and 36% had died (poor prognostic subgroup). Tissues samples were investigated immunohistochemically to count microvessels and mast cells with an antifactor VIII and an antitryptase antibody, respectively. RESULTS: Immunohistological staining showed a higher number of microvessels and mast cells in melanoma lesions of poor prognosis as compared with intermediate prognosis and with good prognosis, respectively. CONCLUSIONS: These data agree with those showing a close relationship between MCD and angiogenesis during tumour progression and demonstrate, for the first time, a prognostic significance of MCD in human melanoma.     

CM通路AEA对人胶质瘤U251细胞增殖和凋亡的影响

目的观察在神经酰胺(CM)通路中,大麻受体激动剂花生四烯酸乙醇胺(AEA)抑制人胶质瘤U251细胞增殖及在诱导细胞凋亡中的作用。方法采用噻唑蓝(MTT)法,分别测定不同浓度的CM(5~20μmol/L)和烟曲霉毒素(FB1)(10μmol/L)预处理24h后对AEA(1~10μmol/L)抑制人胶质瘤U251细胞增殖作用的影响;流式细胞仪Annexin-V/PI双染色法定量分析FB1(10μmol/L)预处理24h后对AEA(10μmol/L)促细胞早期凋亡作用的影响。结果不同浓度的AEA对人胶质瘤U251细胞增殖的抑制作用不同,且与CM具有协同作用;10μmol/L FB1预处理24h后可显著对抗AEA对人胶质瘤U251细胞增殖的抑制作用;AEA(10μmol/L)可诱导人胶质瘤U251细胞发生早期凋亡,而FB1(10μmol/L)预处理24h后凋亡发生率明显下降。结论 AEA可通过CM从头合成通路,与CM呈浓度依赖性协同,从而抑制人胶质瘤U251细胞的增殖并诱导其早期凋亡。     

Effect of chronic D-Ala,2 D-Leu5-enkephalin or pertussis toxin treatment on the high-affinity state of delta opioid receptor in neuroblastoma x glioma NG108-15 hybrid cells.

Chronic treatment of neuroblastoma x glioma NG108-15 hybrid cells with the opioid agonist D-Ala,2 D-Leu5-enkephalin (DADLE) induces a homologous desensitization of the delta opioid receptors present in these cells. Since the Kd value of the delta opioid receptor's high-affinity state reflects the potency of the agonist, we examined the effect of receptor desensitization in NG108-15 cells on the percentage of receptor in the high-affinity state. When NG108-15 hybrid cells were treated with 10 or 100 nM DADLE for 4 hr at 24 degrees C, loss of DADLE's ability to inhibit adenylate cyclase was observed. However, when competition binding experiments were carried out with P2P3 membranes isolated from the delta opioid-desensitized hybrid cells, it was determined that 41.7 +/- 3.4% of the total binding sites remained in the high-affinity state, with no apparent alteration in the Kd value of either high- or low-affinity states. Similarly, when NG108-15 cells were treated with 100 ng/ml of pertussis toxin for 3 hr at 37 degrees C, 39.9 +/- 3.6% of the binding sites remained in the high-affinity state. This reduction in the percentage of receptor in high-affinity state was agonist specific, for chronic treatment of hybrid cells with levorphanol, a partial agonist, or the antagonist naloxone did not alter the percentage of opioid receptors in the high-affinity state. Furthermore, the delta opioid receptors remaining in the high-affinity state after chronic DADLE treatment were still sensitive to both Na+ and guanyldylimidodiphosphate, indicating that opioid ligand binding remained coupled to the G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential diagnosis of circumscribed growing breast tumors

The results of a comparison of clinical, roentgenological and cytological investigation of circumscribed growing tumours of the breast are reported - 43 carcinomas and 19 intracanalicular fibroadenomas - that are hard to differentiate. A number of diagnostically valuable symptoms are described: Significant for carcinoma are an age over 55, microcalcifications (60 +/- 13% of the cases) and different sizes of the tumour found by palpation and mammography (35 +/- 9% of the cases). The diagnostic accuracy of the methods was: clinical 44 +/- 8%, roentgenological 79 +/- 7%, cytological 89 +/- 3% and for their joint application in a special mammographic cabinet 98 +/- 2%.     

GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity

The role of gap junctional intercellular communication (GJIC) in bystander killing with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) was evaluated in U251 cells expressing a dominant-negative connexin 43 cDNA (DN14), and in HeLa cells, reportedly devoid of connexin protein. These cell lines both exhibited 0% GJIC when assayed by Lucifer Yellow fluorescent dye microinjection. Bystander cytotoxicity was still apparent in 50:50 cocultures of DN14 and HSV-TK-expressing U251 cells, but not in 50:50 cocultures of HeLa cells. However, the sensitivity of HeLa HSV-TK-expressing cells to GCV decreased nearly 100-fold (IC90=109 microM) when cocultured with bystander cells compared to results in 100% cultures of HSV-TK-expressing cells (IC90=1.2 microM). A more sensitive flow cytometry technique to measure GJIC over 24 h revealed that the DN14 and HeLa cells exhibited detectable levels of communication (29 and 23%, respectively). Transfer of phosphorylated GCV to HeLa bystander cells occurred within 4 h after drug addition, and GCV triphosphate (GCVTP) accumulated to 213+/-84 pmol/10(6) cells after 24 h. In addition, GCVTP levels were decreased in HSV-TK-expressing cells in coculture (867+/-33 pmol/10(6) cells) compared to 100% cultures of HSV-TK-expressing cells (1773+/-188 pmol/10(6) cells). The half-life of GCVTP in the HSV-TK-expressing cells was approximately four times that measured in the bystander cells (12.3 and 3.1 h, respectively). These data suggest that the lack of bystander cytotoxicity in HeLa cocultures is due to low transfer of phosphorylated GCV and a rapid half-life of GCVTP in the bystander cells. Thus, GCV phosphate transfer to non-HSV-TK-expressing bystander cells may mediate either bystander cell killing or sparing of HSV-TK-positive cells, depending upon the cell specific drug metabolism.     

Detection and quantification of small numbers of circulating tumour cells in peripheral blood using laser scanning cytometer (LSC).

The detection of circulating tumour cells disseminated from solid tumours requires extremely sensitive methods. Molecular genetic methods, which are most sensitive, are not applicable to solid tumours because no tumour-specific genetic markers are available. Detection of disseminated tumour cells by immunocytochemistry is time-consuming, whereas fluorimetry is fast and quantitative. The laser scanning cytometer (LSC) provides an automated microscopic procedure for screening up to 5x10(4) cells in suitable time. Using this system together with an enrichment procedure which allows up to ten thousand-fold enrichment, we have quantified minimal numbers of tumour cells. In a model system, breast cancer cell line cells diluted into peripheral blood mimicked seeding of tumour cells into the periphery. After staining with fluorochrome-conjugated anti-epithelial antibody, slides were screened for positive events directly or after enrichment with antibody-coated magnetic beads. One positive cell was unequivocally detectable in 10(4) cells and 50 out of 60 tumour cells were reliably recovered from a 20 ml blood volume, equal to 1-2 cells per 10(7), after magnetic bead enrichment. This method allows quantitation of tumour cells in peripheral blood and bone marrow in reasonable time and will, for the first time, enable extensive investigation of the seeding behaviour of tumours.