人白血病细胞癌性促凝物(CP)的分离纯化及其特性的初步研究

用三步纯化法从人M_3型白血病细胞中分离纯化出人类肿瘤癌性促凝物(CP)。促凝活性回收率为24％,CP纯化倍数为2481倍。纯化CP在SDS-PAGE上为单一区带,其理化和酶学特性类似于动物肿瘤CP,分子量约为70 000,PI为4.8,在FVⅡ缺乏血浆中以及在含有组织因子(TF)抑制剂情况下仍能激活FX。CP促凝活性能被半胱氨酸蛋白酶抑制剂HgCl_2抑制,纯化CP能与抗动物肿瘤CP抗体形成免疫沉淀反应。     

MDM2在原发性肝细胞癌中的表达

目的检测MDM2蛋白及其mRNA在原发性肝细胞癌中的表达,探讨MDM2在原发性肝细胞癌发生发展中作用及意义.方法采用免疫组织化学、原位分子杂交和细胞图像分析技术,检测肝细胞癌组织及其对应癌旁肝组织(各20例),正常肝组织(5例)中MDM2蛋白及其mRNA 表达情况.结果在肝细胞癌组织、癌旁肝组织和正常肝组织中,MDM2蛋白免疫组织化学阳性反应颗粒的平均光密度分别为:0.404±0.105, 0.302±0.067, 0.087±0.034.肝细胞癌组织与癌旁肝组织、正常肝组织相比,差异均有显著性意义(P<0.05).MDM2 mRNA在肝细胞癌组织中呈阳性表达,而癌旁肝组织及正常肝组织均呈阴性.结论 MDM2过度表达在原发性肝细胞癌的发生发展过程中可能发挥了重要作用.     

miRNA-181在肝癌中表达变化的研究

目的:观察微小RNA(miRNA)-181在正常人肝细胞L02、肝癌细胞株SMMC7221和Hep3B中的表达,以及在正常肝脏组织和肝癌组织中的表达,探讨其与肝癌发生发展的关系.方法:应用荧光实时定量PCR方法检测miRNA-181,包括miRNA-181a、miRNA-181b、miRNA-181c、miRNA-181d,在正常肝细胞和肝癌细胞的含量.同时收集并检测肝细胞癌患者术中取得的正常肝脏组织和癌症组织的含量.结果:MiRNA-181在肝癌细胞株中的表达明显高于正常人肝细胞(P＜0.05或P＜0.001).MiRNA-181在肝癌组织中表达明显高于正常肝脏组织(P＜0.05).结论:MiRNA-181在不同肝癌细胞株中表达不同,但都高于正常肝细胞,并且miRNA-181在肝癌组织中的表达高于正常肝脏组织.这说明miRNA-181与肝癌的发生发展有相关性,可能成为肝癌诊断和预后的一个指标.     

miRNA-492在肝癌中表达变化的研究

目的:通过观察miRNA-492在肝细胞株L02、肝胚细胞瘤HepT1细胞株和肝癌SMMC-7721细胞株中的表达差异,以及在正常肝脏组织、肝母细胞瘤组织和原发性肝癌组织中的含量差异,探讨其与不同类型肝脏肿瘤发生发展的关系.方法:应用Real time-PCR方法检测miRNA-492在正常L02肝细胞株和肝癌HepT1、SMMC-7721细胞株的含量.同时采集和检测原发性肝癌患者术中取得的正常肝脏组织和癌症组织,以及肝母细胞瘤患者瘤组织的含量.结果:1)与正常人L02肝细胞相比,miRNA-492在肝癌HepT1细胞株中的表达显著升高(P＜0.001),但在SMMC-7721细胞株中略升高,无统计学差异.2)与正常肝脏组织相比,miRNA-492在肝母细胞瘤的瘤组织中含量显著升高(P＜0.01),但原发性肝癌的癌组织略升高,无统计学差异.结论:miRNA-492在不同肝癌细胞株中表达不同,在肝胚细胞瘤中表达升高,且在肝母细胞瘤瘤组织中的表达高于正常肝脏组织.但原发性肝癌癌组织中变化不明显,这说明miRNA-492与某些类型肝癌,特别是肝母细胞瘤的发生发展有关.     

肝细胞癌及相关病变的计算机图像定量分析

为研究肝炎、肝硬变与肝细胞癌的关系,利用自动图像分析仪对６４例肝炎、肝硬变、癌旁肝硬变、肝细胞癌和正常肝组织进行了十三项参数的形态定量研究。结果：大多数参数随病变的发展而呈规律性变化,癌旁肝硬变的多数参数介于不伴肝癌的肝硬变与肝细胞癌之间。提示：（１）慢活肝、肝硬变与肝细胞癌密切相关;（２）癌旁肝硬变不同于不伴肝癌的肝硬变,与肝细胞癌的关系更密切。利用逐步判别分析选择出六项参数建立判别函数方程,回代正确率为９８．２％。     

泛素相关蛋白样因子2在肝癌中的表达及意义

目的:探讨肝癌中泛素相关蛋白样因子2(UBAP2L)的表达水平与预后的关系及其对肝癌细胞侵袭、转移能力的影响。方法:挖掘oncomine数据库,提取UBAP2L在肝癌与正常组织转录水平的变化及相关临床资料,采用Kaplan-Meier法分析UBAP2L表达水平与肝癌患者预后的关系。采用实时定量PCR、Western blot检测HCCLM3、MHCC97H、Hep3B、Huh7肝癌细胞株及正常肝细胞LO2中UBAP2L mRNA及蛋白水平,免疫组织化学染色法检测本院80例肝癌组织与癌旁组织中UBAP2L的表达水平加以验证。通过慢病毒载体使UBAP2L高表达的肝癌细胞株HCCLM3表达下调,采用克隆形成和划痕实验检测UBAP2L对肝癌细胞增殖能力和侵袭、转移的影响。结果:UBAP2L在oncomine数据库大多数队列呈高表达。UBAP2L在肝癌细胞株中的mRNA和蛋白的表达水平显著高于正常肝细胞(P0.05)。肝癌组织中UBAP2L阳性、强阳性表达率高于癌旁组织(P0.05),下调UBAP2L表达可明显抑制HCCLM3肝癌细胞的克隆形成能力和运动能力(P0.05)。UBAP2L高表达组的中位生存时间与UBAP2L低表达组比较差异无统计学意义(P0.05)。结论:UBAP2L在肝癌组织中呈高表达,下调UBAP2L表达可抑制肿瘤增殖和侵袭、转移表型,其可能成为肝癌早期诊断的生物标志物和治疗的潜在靶点。     

急性肝损伤小鼠中线粒体解耦联蛋白2的表达变化及意义

目的:观察C57小鼠急性肝损伤(acute liver injury,ALI)中解偶联蛋白2(Uncouple Protein2,UCP2)的表达,探讨ALI与UCP2表达变化的意义。方法:领取36只小鼠,随机分为对照组、ALI 1 d组、ALI 4 d组、ALI 7 d组。分别检测血清谷氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)的变化;肝组织病理变化用HE染色观察;利用Western Blot方法分别检测全肝组织蛋白和肝细胞线粒体提纯蛋白中的UCP2蛋白水平的变化;Real Time quality PCR检测UCP2在mRNA水平的表达变化。结果:ALI组1 d、4 d组与对照组相比,ALT(160.69±22.11 vs 34.43±5.19;96.37±15.39 vs 34.43±5.19)、AST(306.54±68.09 vs 97.74±14.49;173.94±26.74 vs 97.74±14.49)表达差异具有统计学意义(P0.05);肝组织病理学检查ALI组1 d和4 d组中肝细胞出现大面积坏死,7d组肝细胞坏死缓解;蛋白水平检测UCP2在ALI 1 d和4 d时全肝组织分别增加2.84倍和2.25倍,在肝线粒体中1 d、4 d和7 d时分别增加2.19倍、1.68倍和1.56倍,差异具有统计学意义(P0.05);mRNA水平检测UCP2在ALI 1 d和4 d与对照组相比明显升高,相对增加3.79倍和1.46倍(P0.05)。结论:急性肝损伤状态下UCP2在蛋白和mRNA水平高表达,UCP2可能对于治疗急性肝损伤具有重要意义。     

RICH2在肝细胞肝癌中蛋白表达的研究

目的:分析肝细胞肝癌与正常肝组织中RICH2蛋白表达之间的关系,探讨RICH2在肝细胞肝癌发生发展中的作用机理。方法:选取19例肝细胞肝癌患者作为研究对象,用免疫组织化学方法检测肝癌组织和癌旁正常组织中RICH2的表达情况并采用Friedrich免疫反应评分法作为判定标准。采用非参数Wilcoxon秩和检验对RICH2在肝细胞肝癌和正常组织中的评分结果进行统计学分析。结果:19例HCC病例癌旁正常组织中RICH2表达阳性率为100%(19/19),其中RICH2表达强阳性(+++)5例(26.3%),RICH2表达弱阳性(+)和中阳性(++)分别为3例(15.8%)和11例(57.9%);而HCC组织除了两例肝硬化组织未取到癌组织外,17例病例中RICH2表达阳性率仅为11.8%(2/17),表达为+和++各占50%。肝癌组织中RICH2的表达水平显著低于癌旁组织。结论:RICH2有可能通过某种或者其中几种因素作用失活,作用于信号通路,进而促进肝细胞肝癌的发生发展。研究其作用机理,可为临床诊断、治疗及预后评估提供理论依据。     

不同肝病变组织中CD34、CD31、Ki-67的表达及意义

目的比较正常肝组织、慢性肝炎、肝硬化、肝细胞肝癌组织及肝转移腺癌中CD34、CD31、Ki-67不同表达,寻找有助于鉴别不同性质病变的生物学标记物.方法正常肝及病变肝组织标本共104例;其中,正常肝组织10例;慢生C型肝炎组织73例;肝硬化组织7例;肝细胞肝癌7例;结肠癌肝转移5例;乳腺癌肝转移2例.73例慢性C型肝炎组织全部为肝穿活检标本,其余组织均为手术切除标本.所有病例标本分别行CD34、CD31、Ki-67免疫组织化学染色,半定量评分系统评价染色结果.统计学分析结果数据.结果在非肿瘤组织,抗CD34阳性染色主要存在于汇管区,亦可见于汇管区周围的肝实质内血窦.阳性染色内皮细胞呈点状、线状、半环状及环状,散在或簇状分布.肿瘤组织内抗CD34阳性染色特征与非肿瘤组织相似,阳性染色血管在肿瘤组织内散布分布.CD34指数在各病变组中的表达排列顺序依次为:肝细胞肝癌>乳腺癌肝转移>结肠癌肝转移>肝硬化>慢性C型肝炎>正常肝组织,从正常肝组织至慢性肝炎至肝细胞肝癌,CD34表达明显增强.组织中,抗CD31阳性染色分布、定位、形态特征与CD34相似.CD31在慢性肝炎、肝硬化、肝细胞肝癌、结肠癌肝转移及乳腺癌肝转移组织中阳性表达率分别为:6.8%(5/73)、100%(7/7)、100%(7/7)、100%(5/5)、100%(2/2);肝癌组织中CD31染色强度明显大于非癌组织中,组间比较具有显著差异(P<0.05).Ki-67阳性染色细胞呈棕黄色核着色,散在分布于肝实质内.阳性染色细胞无形态特殊性,亦无分布上的特殊性.Ki-67在各病变组间的阳性表达率分别为:64.4%(47/73)、28.6%(2/7)、100%(7/7)、100%(5/5)、100%(2/2),其中以在结肠癌肝转移组织中表达最明显;组间比较具有非常显著差异(P<0.05).在正常肝脏、慢性C型肝炎、肝硬化、肝细胞肝癌CD34、CD31、Ki-67三种生物学标记物在同一标本同时表达的阳性率分别为:0%(0/0)、4.1%(3/73)、28.6%(2/7)、100%(7/7),CD34、CD31、Ki-67其中任两种同时表达的阳性率分别为0%(0/10)、63.0%(46/73)、100%(7/7)、100%(7/7).结论 CD34是慢性肝病、肝癌临床病理评价的指标之一,CD34与CD31、Ki-67同时分析有助于建立可靠的诊断.     

载脂蛋白AI—CⅢ基因区域DNA多态性及单倍型与冠心病的关系

本文利用重组DNA技术,以人apoAI cDNA片段为探针,对50例正常血脂汉族个体和33例冠心病患者的载脂蛋白AⅠ—CⅢ基因区域DNA多态频率和特征进行了分析。发现中国汉族群体载脂蛋白AⅠ—CⅢ基因区域亦存在Sst-Ⅰ和Msp-Ⅰ多态位点,可产生S_2和M_22种多态片段,其等位基因频率分别为0.167和0.22,均高于高加索人但低于日本人,表明这2个多态部位的等位基因频率具有种族差异。我们还对44例正常血脂个体和27例冠心病患者进行了单倍型分析,没有发现S_1S_2M_1M_1,S_2S_2M_1M_2和S_2S_2M_1M_1基因型个体,因此,在中国汉族人群中,可能仅存在3种单倍型:S_1—M_1(0.81),S_2M_2(0.17)及S_1—M_2(0.02),而不存在S_2—M_1这种单倍型。同日本人和高加索一样,在中国汉族人群中,S_1S_2与M_1、M_2之间亦存在连锁不平衡。本研究结果表明,S_2与M_2等位基因在正常血脂的汉族人与冠心病病人之间的分布频率均没有显著差异,但冠心痛病人的S_1—M_2单倍型频率明显高于正常人,其有极显著的差异。提示S_1—M_2这种单倍型与冠心病有明显的关联,可做为一种假定的动脉粥样硬化致病基因的连锁标记,对有关个体进行连锁分析。     

The AP2M1 gene expression is a promising biomarker for predicting survival of patients with hepatocellular carcinoma

There is a growing need for the discovery of new prognostic factors for cases where the scoring and staging system of hepatocellular carcinoma (HCC) does not result in a clear definition. We analyzed whether AP-2 complex subunit mu (AP2M1) expression could be a new prognostic marker for HCC based on the roles of AP2M1 in influencing hepatocyte growth factor (HGF) promoter regulation and hepatitis C virus (HCV) assembly. Patient data were extracted from cohorts of the Gene Expression Omnibus (GSE10186), International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). Differential expression value between matched cancer and normal liver was identified using ICGC cohort. Subsequently, we compared AP2M1 expression as a prognostic gene with other well-known prognostic genes for HCC, using the time-dependent area under the curve (AUC) of the Uno's C-index, the AUC value of the receiver operating characteristics at 5 years, Kaplan-Meier survival curve, and multivariate analysis. Particularly, TCGA and GSE10186 patients were divided into subgroups based on alcohol intake, hepatitis B, and C viral infections, and analyzed in the same methods. The AP2M1 expression values in patients with cancer were much higher than matched normal liver. The AP2M1 level showed excellent prognosis predictions in comparison with existing markers in the three independent cohorts (n = 647). In particular, it was more predictive of prognosis than other markers in alcohol intake and HCV infections. In conclusion, we were confident that AP2M1 provides sufficient value as a new prognostic marker for HCC especially patients with HCV infection and/or alcohol intake.     

Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-protein-coupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src-/-Pyk2-/- cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src-/-Yes-/-Fyn-/- cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.     

The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics

The Cdc42-like GTPase Wnt responsive Cdc42 homolog 1 (Wrch1) has several atypical features; it has an N-terminal proline-rich extension that confers binding to SH3 domains, and it harbors an extremely high intrinsic nucleotide exchange activity, which overrides the normal GTPase activity. As a result, Wrch1 resides mainly in the active, GTP-loaded conformation under normal cellular conditions. We have previously shown that ectopic expression of Wrch1 in fibroblasts resulted in an altered cell morphology visible as a formation of filopodia, a loss of stress fibers, and a reduction in focal adhesions. Here, we show that Wrch1 binds to the nonreceptor tyrosine kinase Pyk2. The interaction required Wrch1 to be in a GTP conformation and also required an intact N-terminal proline-rich extension as well as an intact effector loop. Wrch1 requires Pyk2 in imposing the cytoskeletal effects, seen as the formation of filopodia, since treatment of cells with a Pyk2-specific small interfering RNA abrogated this response. Interestingly, we found that the presence and activity of Src were needed for the formation of a Wrch1-Pyk2 complex as well as for the Wrch1-induced formation of filopodia. We propose a model in which Pyk2 and Src function to coordinate the Wrch1-dependent effects on cytoskeletal dynamics.     

In vivo and in vitro phosphorylation of two isoforms of yeast pyruvate kinase by protein kinase A

Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HA-Tpk1.Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GST-Pyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1(w1)). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mm. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an n(H) value of 1.4, as compared with an n(H) of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.     

Suppression of Pyk2 kinase and cellular activities by FIP200

Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways. We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase-interacting protein of 200 kD) by using a yeast two-hybrid screen. In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK. However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm. FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays. FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases. Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2. Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200-Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells. Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain.     

Proline-rich tyrosine kinase 2 (Pyk2) promotes cell motility of hepatocellular carcinoma through induction of epithelial to mesenchymal transition

Aims

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells.

Methodology/Principal Findings

We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization.

Conclusion

These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT.     

Purification and properties of pig heart pyruvate kinase

Using essentially a two-step procedure involving phosphocellulose column chromatography followed by gel filtration on Sephadex G200, pig heart pyruvate kinase (PH PyK) was purified 267-fold to at least 97% purity. PH PyK co-sedimented with rabbit muscle PyK during sucrose density ultracentrifugation yielding an S_20,w of 10 and a corresponding molecular weight of about 237,000. Sodium docedyl sulfate polyacrylamide gel electrophoresis yielded a subunit molecular weight of approximately 59,000, suggesting that native PH PyK exists as a tetramer. The isoelectric point (pI) was determined to be 8.2, and thepH optimum (pHo) for the forward reaction is 7.2. Steady-state kinetics with phospho(enol)pyruvate (PEP) as the variable substrate show that there is a threefold decrease in the Km for PEP in the presence of 1.0 mM fructose-1,6-diphosphate (FDP), and that the activity of PH PyK is increased over fourfold by FDP at low (0.1 mM) PEP concentrations. Lineweaver-Burk plots are linear in the presence and absence of FDP, indicating that the Michaelis-Menten curves are hyperbolic. The amino acid composition for pig heart PyK shows close similarities between pig muscle and kidney PyKs, but not liver PyK. Among the data on pI,pHo, and FDP activation, only the activation by FDP is useful in tentatively designating pig heart PyK as an M₂ isozyme.Presented in partial fulfillment for the Master of Science degree.     

Organometallic 99mTc(III) '4 + 1' bombesin(7-14) conjugates: synthesis, radiolabeling, and in vitro/in vivo studies

Bombesin (BBN) peptide exhibits high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). The GRPr is overexpressed on many human cancer cell types, thus making BBN a potent delivery vehicle for radionuclide targeting. In this study, the biologically active minimal sequence BBN(7-14) was labeled using the novel Tc '4 + 1' mixed-ligand system, [Tc(NS3)(CN-R)], in which Tc(III) is coordinated by a monodentate isocyanide linker bearing the peptide and the tetradentate, tripodal chelator, 2,2',2'-nitrilotriethanethiol (NS3). BBN(7-14) was N-terminally modified with Gly-Gly-Gly, betaAla, and Ser-Ser-Ser spacer groups (X) and functionalized with 4-(isocyanomethyl)benzoic acid (L1) or 4-isocyanobutanoic acid (L2), resulting in a series of [M(NS3)(L-X-BBN(7-14))] conjugates (M = 99mTc, Re). The isocyanide ligand frameworks were introduced using novel bifunctional coupling agents. The spacer groups (X), the monodentate isocyanide units, and a tetradentate NS3 chelator bearing a pendant carboxylic acid (NS3COOH) were proposed as pharmacological modifiers. 99mTc-labeling was performed in a two-step procedure by first preparing 99mTc-EDTA/mannitol followed by reactions with the isocyanides and NS3 or NS3COOH ligand frameworks. The 99mTc complexes were obtained with a radiochemical yield of 30-80% depending on the amount of the isocyanide (20-100 nmol) used. These new conjugates were purified by reversed-phased high-performance liquid chromatography (RP-HPLC) to give a radiochemical purity of >or=95%. The 99mTc conjugates exhibited high in vitro stability (>90%, 24 h). Analogous nonradioactive Re conjugates were synthesized and characterized by electrospray ionization mass spectrometry (ESI-MS). RP-HPLC analyses of the Re conjugates indicated that they exhibited identical retention times to the corresponding 99mTc conjugates under identical HPLC conditions, demonstrating structural similarity between the two metalated species. The [Re(NS3)(L-X-BBN(7-14))] conjugates exhibited GRPr affinity in the nanomolar range as demonstrated by in vitro competitive binding assays using PC-3 human prostate cancer cells. In vitro internalization/externalization assays indicated that approximately 65% of [99mTc(NS3)(L2-betaAla-BBN(7-14))] conjugate was either surface-bound or internalized in PC-3 cells. Cell-associated activity for all other 99mTc conjugates was below 20%. Biodistribution studies of [99mTc(NS3)(L-betaAla-BBN(7-14))], L = L1 or L2, in normal, CF-1 mice showed minimal accumulation in normal pancreas (a tissue expressing the GRPr in high density in rodent models) and rapid hepatobiliary elimination. Introduction of a carboxyl group onto the NS3 ligand framework had only minimal effects to increase renal excretion. Activity distribution and accumulation was highly dominated by the relatively lipophilic '4 + 1' complex unit.