豚鼠Ⅱ型前庭毛细胞乙酰胆碱敏感性大电导钙依赖性钾通道与L型钙通道共存

目的 研究豚鼠Ⅱ型前庭毛细胞乙酰胆碱(acetylcholine,ACh)敏感性大电导钙依赖性钾通道(big conductance,calcium-dependent potassium channel,BK)激活过程中的钙离子内流机制.方法 健康杂色豚鼠52只,断头后取出前庭终器,经胶原酶IA消化后获取Ⅱ型前庭毛细胞.采用全细胞膜片钳技术检测新鲜单离的Ⅱ型前庭毛细胞ACh-敏感性BK电流对细胞外钙通道阻断剂和激动剂的敏感性.结果 ①细胞外ACh激活一缓慢持久的外向性电流,其反转电位为(-70.5±10.6)mV(-x±s,下同,n=10);-50 mV钳制电压下,100 μmol/L ACh激活电流的幅值为(267±106)pA(n=11).②ACh-敏感性钾电流对细胞外IBTX(iberiotoxin,200 nmol/L)敏感,而细胞外蜂毒明肽(apamin,1 μmol/L)对ACh-敏感性钾电流幅值无抑制作用.③ACh-敏感性BK对细胞外钙通道阻断剂NiCl2敏感,可被细胞外钙通道阻断剂CdCl2强力阻断.NiCl2和CdCl2对ACh-敏感性BK的半数抑制浓度分别为(135.5±18.5)μmol/L(n=7)和(23.4±2.6)μmol/L(n=7).④L型钙通道激动剂(-)-Bay-K 8644激活Ⅱ型前庭毛细胞产生一种与ACh-敏感性BK类似的外向性电流,并能被IBTX强力阻断.结论 Ⅱ型前庭毛细胞中ACh-敏感性BK与细胞膜L型钙通道共存,可能具有重要的生理学作用和意义.     

硝普钠对豚鼠单离耳蜗外毛细胞全细胞电流的影响

目的研究并探讨硝普钠(sodium nitroprusside,SNP)对豚鼠耳蜗外毛细胞(outer hair cells,OHC)全细胞电流的影响及其作用机制.方法利用急性分离的OHC和特异性的离子通道阻断剂作为工具药,通过膜片钳电压钳记录技术观察了SNP对豚鼠耳蜗OHC全细胞电流的影响,结果①在以 40mV的指令电压刺激时,10-3mol/L的SNP抑制15.34±6.59%的细胞电流(n=5);②SNP对全细胞电流的抑制作用有电压依赖性,在刺激高于0mV时作用明显,10-2mol/L的SNP在 10mV时抑制率为4.97±1.74%,而在 40mV时的抑制率为33.82±1.61%;③SNP对全细胞电流的抑制呈量效关系,在浓度大于10-3mol/L时,抑制作用逐渐明显,10-1mol/L的SNP时接近达到最大抑制效应;④分离离子电流成分后,SNP仅对Ca2 电流有抑制作用.结论SNP对豚鼠耳蜗外毛细胞的全细胞电流的抑制作用,主要通过抑制细胞外Ca2 的内流,进而影响Ca2 依赖性K 通道而实现.     

EphA2调控头颈部鳞状细胞癌血管新生及转移的体内实验

目的 研究EphA2对头颈部鳞状细胞癌(简称鳞癌)血管新生及颈淋巴转移的影响.方法 利用慢病毒介导的短发夹RNA( short hairpin RNA,shRNA)沉默EphA2在高转移性头颈鳞癌细胞株M2中的表达,嘌呤霉素筛选稳定克隆,反转录PCR及Western blot技术检测EphA2沉默效果;构建头颈鳞癌高转移动物模型,通过HE染色验证其成瘤及颈淋巴转移情况;免疫组织化学技术检测移植瘤微血管密度,Western blot技术检测移植瘤标本中EphA2及血管内皮生长因子(VEGF)的表达情况.结果 筛选出EphA2基因稳定沉默的M2细胞(定义为M2 EphA2 RNA+即实验组),并成功构建头颈鳞癌高转移动物模型,成瘤率达100％.25 d后,实验组移植瘤体积为(430.7±190.0) mm3(x±s,以下同)较对照组的(1179.0 ±289.4) mm3明显减少(t =5.597,P＜0.01),质量显著减轻(t=-4.560,P＜0.05),且双侧颈淋巴转移率明显降低(Mann-Whitney U=10.0,P＜0.05).Western blot 显示实验组移植瘤组织中EphA2及VEGF蛋白表达显著下调,免疫组织化学技术发现实验组瘤体微血管密度为(4.74±0.67)个/视野,显著少于对照组的(14.17±0.59)个/视野(t=26.751,P＜0.01).结论 EphA2沉默能抑制头颈部鳞癌细胞的生长及转移,并能明显减少头颈部鳞癌组织内的肿瘤新生血管,且该肿瘤新生血管的抑制可能与促血管生成因子VEGF的减少有关.     

豚鼠耳蜗外毛细胞乙酰胆碱敏感性钾电流特性

目的 研究豚鼠耳蜗外毛细胞乙酰胆碱(acetylcholine,ACh)敏感性钾电流离子特性及其受体的药理学特性.方法 健康豚鼠38只,断头后取出基底膜,经胶原酶Ⅳ消化后获取外毛细胞.采用全细胞记录膜片钳技术检测新鲜单离外毛细胞ACh-敏感性钾电流对细胞外钙依赖性钾电流阻断剂和N型胆碱能受体抑制剂的敏感性.结果 ①细胞外ACh激活一快速去敏感化的外向性钾电流,其平均(-x±s,以下同)反转电位为(-67.3±8.2)mV(n=10);-50 mV钳制电压下,100 μmol/LACh激活电流的幅值为(506.6±186.3)pA(n=9).②ACh-敏感性钾电流对细胞外四乙铵(tetraethylammonium,TEA,10 mmol/L)、蜂毒明肽(apamin,1 μmol/L)敏感,而细胞外的IBTX(iberiotoxin,200 nmol/L)对ACh-敏感性钾电流幅值无抑制作用.③ACh-敏感性钾电流的半数激活浓度(EC50)为(33.5±5.7)μmol/L(n=7).④ACh-敏感性钾电流对细胞外γ-氨基丁酸(gammaaminobutyric acid,GABA)-A受体阻断剂荷包牡丹碱(bicuculline)和α9-N型胆碱能受体(α9受体)特异性抑制剂士的宁(strychnine)敏感.士的宁和荷包牡丹碱对ACh-敏感性钾电流的抑制作用具有浓度依赖性,其半数抑制浓度(IC50)分别为(22.3±2.6)nmol/L(n=7)和(1.2±0.4)μmol/L(n=6).结论 细胞外ACh激活豚鼠耳蜗外毛细胞产生小电导钙依赖性钾电流(SK),此电流可能由α9受体介导.     

白细胞介素-2基因与单纯疱疹病毒胸苷激酶基因联合治疗小鼠头颈鳞状细胞癌的研究

目的观察白细胞介素-2(interleukin-2,IL-2)基因与单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase,HSV-TK)基因联合治疗小鼠头颈鳞状细胞癌的疗效.方法建立小鼠头颈鳞状细胞癌动物模型后在荷瘤部位分别注射表达小鼠IL-2基因的重组腺病毒(recombinantadenovirus, Ad)和表达HSV-TK基因的重组腺病毒Ad HSV-TK及联合注射组,对照组分别注射不带目的基因的腺病毒或磷酸盐缓冲液(phosphatic buffered solution,PBS).对注射有Ad HSV-TK基因组和联合治疗组每日腹腔注射抗病毒药物更西罗韦(ganciclovir,GCV) 25 mg/kg体重,每日2次连续7 d.观察肿瘤大小变化并检测脾脏自然杀伤细胞和颈淋巴结细胞毒性T淋巴细胞的活性,探讨其抗肿瘤机制.结果 Ad IL-2和Ad HSV-TK联合治疗组,肿瘤生长明显受抑制,疗效显著优于单独治疗组和对照组(P<0.05),在注射有Ad IL-2基因的治疗组中,IL-2蛋白水平明显升高,自然杀伤细胞活性和细胞毒性T淋巴细胞杀伤活性增强,肿瘤组织中可见大片坏死,并含有大量的CD+4、CD+8淋巴细胞浸润.结论 Ad IL-2基因治疗可提高肿瘤局部和全身的抗肿瘤免疫应答,能加强自杀基因AdHSV-TK的抗肿瘤效果,两者联合应用能显著抑制小鼠头颈鳞状细胞癌的生长.     

锌指转录因子Snail在头颈鳞状细胞癌组织中的表达及与血管生成的关系

目的观察头颈鳞状细胞癌(简称鳞癌)组织中锌指转录因子Snail的表达及与血管生成的关系,探讨其对血管生成的作用。方法采用免疫组织化学方法检测62例头颈鳞癌及20例正常软腭黏膜组织中Snail的表达情况,并计数微血管密度(microvascular density,MVD)。结果 Snail在头颈鳞癌中的表达率为66.13%,显著高于正常软腭黏膜组织,MVD在头颈鳞癌中的表达率显著高于正常软腭黏膜组织,且二者均随病理分化程度降低而升高,并在有淋巴结转移组中显著高于无淋巴结转移组,头颈鳞癌组织中MVD与Snail的表达呈正相关。结论 Snail高表达与头颈鳞癌的发生发展密切相关,并可能在肿瘤的血管生成中起重要作用。     

阻断氯通道对人喉癌裸鼠移植瘤ERK1/2和AKT1的影响

目的研究氯通道阻断剂5-硝基-2-(3-苯丙氨基)苯甲酸[5-nitro-2-(3-phenylpropylamino)benzoicacid,NPPB],对人喉癌细胞系Hep-2细胞裸鼠移植瘤细胞外调节激酶(extracellulat regulated kinase,ERK)ERK1/2和AKT1蛋白磷酸化的影响,探讨阻断氯通道对喉癌移植瘤抑制作用的可能机制。方法以人喉癌Hep-2细胞建立裸鼠皮下移植瘤模型,应用不同浓度的NPPB分组进行治疗实验,研究移植瘤生长变化,Westernblot法检测移植瘤ERK1/2和AKT1蛋白磷酸化水平的变化。结果与对照组比较,各治疗组移植瘤ERK1/2和AKT1总蛋白水平无显著性差异,50、100、150μmol NPPB组ERK1/2和AKT1蛋白磷酸化水平明显降低,差异均有显著性,NPPB浓度依赖性地抑制ERK1/2和AKT1蛋白磷酸化。结论体内阻断氯通道可以抑制人喉癌裸鼠移植瘤的生长,其机制可能与抑制移植瘤细胞ERK1/2和AKT1蛋白磷酸化有关。     

人乳头状瘤病毒阳性的头颈鳞状细胞癌特异性基因生物信息学研究

目的分析人乳头状瘤病毒(HPV)阳性的头颈鳞状细胞癌(鳞癌)特异表达基因及关键信号通路,为HPV相关头颈鳞癌筛选有价值的基因标记物,并为进一步的肿瘤机制研究提供参考。方法从GEO高通量基因芯片数据库中筛选出头颈鳞癌具有HPV感染信息的芯片,从中筛出差异基因进行基因本体分析及京都基因和基因组(KEGG)信号通路富集分析,并筛出头颈鳞癌的特征基因簇和通路,以及关键基因并进行蛋白质相互作用网络可视化分析。通过Cbioportal信息门户以及癌症基因组图谱(TCGA)数据库验证这些特异基因在HPV(+)与HPV(-)头颈鳞癌中的表达差异并分析特异基因与头颈鳞癌患者生存预后的相关性。结果从数据集GSE52088与GSE39366中筛选出42个共同差异基因,其中上调基因25个,下调基因17个,经Cytoscape两轮筛选确定白介素-6(IL-6)、细胞表面标记物CD44、基质金属蛋白酶1(MMP1)、CXC趋化因子配体基序1(CXCL1) 4个特异基因。信号通路富集分析显示共同差异基因参与细胞周期、NOD样受体信号通路、肿瘤坏死因子(TNF)信号通路途径等信号通路(P < 0.01)。经TCGA数据库以及Cbioportal检验证实特异基因在HPV(+)与HPV(-)头颈鳞癌中的表达差异,且IL-6、CD44表达水平与头颈鳞癌生存预后呈负相关(P < 0.01)。结论HPV(+)头颈鳞癌具有特异性基因表达,并可能参与关键信号通路调控肿瘤的发生发展。IL-6、CD44、MMP1、CXCL1 4个特异基因可能参与HPV(+)头颈鳞癌发展及侵袭过程,其中MMP1、CXCL1有望作为诊断及预后的标志物,IL-6、CD44与头颈鳞癌预后存在相关性,有望成为治疗HPV(+)头颈鳞癌的潜在靶点。     

反义表皮生长因子受体纳米颗粒对小鼠头颈鳞状细胞癌放疗增敏的实验

目的应用反义表皮生长因子受体（epidermal growth factor receptor,EGFR）纳米颗粒,研究阻断EGFR表达,对小鼠头颈部鳞状细胞癌（简称头颈鳞癌）敏感性的作用。方法载反义EGFR寡核苷酸纳米颗粒转染SCC VII细胞株,通过Western—blot研究其蛋白抑制效应。放射治疗千预后,细胞克隆试验和MTT试验检测细胞的放射敏感性,流式细胞仪检测细胞周期分布和凋亡情况：构建小鼠头颈癌荷瘤模型,瘤体注射反义EGFR纳米颗粒,予以4Gy放射治疗,观察肿瘤生长抑制情况。结果反义EGFR纳米颗粒明显抑制EGFR蛋白的表达情况：反义EGFR纳米颗粒与放疗联合降低了肿瘤细胞克隆形成能力及生长能力（P〈0．05）：肿瘤细胞发生G1期阻滞,细胞凋亡率增加（P〈0．05）。体内实验发现反义EGFR纳米颗粒组肿瘤生长明显延缓。结论反义EGFR纳米颗粒通过下调EGFR的表达,使SCC VII细胞发生G1期阻滞,具有放疗增敏效应。     

The independent and combined effects of RAR-, RXR-, and VDR-selective ligands on the growth of squamous cell carcinoma in vitro

OBJECTIVE: The purpose of our study was to analyze the independent and combined effects of RAR-, RXR-, and VDR-selective ligands on the growth of squamous cell carcinoma to develop new, more effective, and less toxic chemopreventive therapy. METHOD: The effects of 13-cis retinoic acid (13-cis RA), LG1069 (a highly selective RXR ligand), and vitamin D3 (D3) were analyzed in vitro in the SCC-25 human squamous cell carcinoma cell line. SCC-25 cells were grown in culture medium containing vehicle or hormone at the indicated concentrations. Growth of surviving cells was then assessed using a hemocytometer. The presence of functional vitamin D3 receptor (VDR) was confirmed using gene-transfer experiments with a promoter-lac Z reporter plasmid. RESULTS: D3 and 13-cis RA have equipotent antiproliferative effects on SCC-25 cells. Furthermore, equimolar LG1069 completely blocks the growth-suppressive effects of D3 but has no effect on the action of 13-cis RA. CONCLUSION: D3 and its analogs, administered alone or in combination with 13-cis RA, may provide more effective and less toxic chemopreventive therapy for the prevention of second primary carcinomas of the head and neck.     

Inhibition of Head and Neck Tumor Cell Growth With Arachidonic Acid Metabolism Inhibition

Head and neck tumors are known to synthesize arachidonic acid metabolites. The authors have postulated that these substances may confer growth advantage to cancer cells, and by interfering with arachidonic acid metabolism squamous cancer growth may be altered. The tongue derived squamous carcinoma cell line, SCC-25, was treated with three leukotriene synthesis inhibitors and indomethacin. A dose-dependent decrease in DNA synthesis occurred with leukotriene inhibition, but not prostaglandin inhibition. All leukotriene synthesis inhibitors produced a dramatic and immediate effect (>70% inhibition by 4 hours) without cytotoxicity (>90% trypan blue exclusion). Cell populations at 96 hours were decreased when compared to control populations. In conclusion, leukotrienes or other lipoxygenase products may play a role as growth factors for squamous cell carcinoma, and arachidonic acid inhibition may be a novel target for chemotherapeutic intervention.     

Expression of vascular endothelial growth factor receptors on tumor cells in head and neck squamous cell carcinoma

BACKGROUND: Angiogenesis is essential for the growth of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Angiogenesis is regulated by angiogenic factors such as vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) 1, 2, and 3 known to be located on vascular endothelial cells (VECs). We hypothesize that VEGFRs are also expressed on HNSCC tumor cells in vitro and in vivo and likely control tumor function in vivo. DESIGN: Immunohistochemical analysis for VEGFR-1 (n = 13), VEGFR-2 (n = 21), and VEGFR-3 (n = 16) was performed on human HNSCC tumor samples. Specimens were analyzed for receptor expression and staining intensity. A cultured oral SCC cell line (SCC-25) and a pharyngeal SCC cell line (FADU) were also studied for receptor expression. RESULTS: The HNSCC tumor cells expressed VEGFR-1, VEGFR-2, and VEGFR-3 in all specimens evaluated. Staining for all 3 receptors was also found on tumor-associated macrophages and fibroblasts, except that VEGFR-2 was not present on fibroblasts. Staining intensity for VEGFR-1 and VEGFR-2 was significantly higher in tumor cells and macrophages than in VECs stained for the same receptor. Both cultured HNSCC cell lines demonstrated expression of all 3 receptors. CONCLUSIONS: This represents the first report of all 3 VEGFRs being expressed by HNSCC cells. These findings indicate that VEGF may be an autocrine regulator of tumor cell activity in addition to its known angiogenic effects on VECs. The presence of VEGFRs on tumor-associated macrophages and fibroblasts contributes to the complexity of the VEGF/VEGFR system in human cancer.     

Development of monoclonal antibodies with specificity to oral squamous cell carcinoma

A panel of five monoclonal antibodies have been produced that show binding when directed against human oral head and neck squamous cell carcinomas. These monoclonal antibodies are derived from antibody-secreting cells obtained using cell hybridization techniques. The activity of the antibody was tested in vitro against oral head and neck squamous cell carcinoma (n = 10). Control groups included normal oral mucosa (n = 7), hyperkeratosis (n = 5), mild dysplasia (n = 4), and severe dysplasia (n = 3). Results using immunoperoxidase staining confirmed the binding of the five monoclonal antibodies to human head and neck squamous cell carcinoma cells, with predominant reactivity (50% to 90%) in the majority of the specimens, compared with negative (0%) to weak (less than 5%) or focal (5% to less than 50%) reactivity in the control specimens.     

Inhibition of epidermal growth factor receptor signaling decreases p63 expression in head and neck squamous carcinoma cells

OBJECTIVES/HYPOTHESIS: Both the epidermal growth factor receptor (EGFR) and the p53 homologue p63 are overexpressed in a significant number of cases of head and neck squamous cell carcinoma (HNSCC). Epidermal growth factor receptor and p63 both possess oncogenic properties, including the potential to increase cell proliferation and antagonize apoptosis. ZD1839 ("Iressa") is an adenosine triphosphate-competitive inhibitor specific to the EGFR tyrosine kinase currently under evaluation as a chemotherapeutic agent in HNSCC. The objective was to investigate whether p63 expression is decreased after treatment of HNSCC cells with ZD1839. Downregulation of p63 by ZD1839 would identify a potential molecular relationship between EGFR signaling and p63 and could provide insight into the mechanism of action of ZD1839. STUDY DESIGN: In vitro examination of p63 expression after ZD1839 treatment. METHODS: A human HNSCC cell line, SCC-012, was treated with varying doses of ZD1839. p63 protein and messenger RNA levels were analyzed by Western and Northern blot analyses. The effect of ZD1839 on SCC-012 cell cycle was analyzed by flow cytometric analysis. RESULTS: In SCC-012 cells there was a dose-dependent decrease in p63 protein and messenger RNA levels over the course of ZD1839 treatment. Levels of phosphorylated MAPK decreased and p27KIP-1 levels increased after ZD1839 treatment. ZD1839 treatment induced a twofold increase in G1-phase cells and a 3.5-fold decrease in S-phase cells consistent with growth arrest. CONCLUSION: ZD1839 downregulates p63 expression at the messenger RNA level, suggesting that p63 is a downstream target of EGFR signaling.     

Enhancement of cytarabine sensitivity in squamous cell carcinoma cell line transfected with deoxycytidine kinase

BACKGROUND: Cytarabine is the most effective agent known for the treatment of acute myeloid leukemia. Its antitumor effect is expressed by combining with DNA during replication and then destroying the DNA chain. However, cytarabine has only limited activity against most solid tumors, including squamous cell carcinoma of the head and neck. The reason for this is thought to be that in cell lines of solid tumors the expression of cytidine deaminase, an enzyme that degrades cytarabine, is high, whereas the expression of deoxycytidine kinase (dCK), which phosphorylates cytarabine (a prodrug), is weak. OBJECTIVE: To determine whether head and neck squamous cell carcinomas can be made more sensitive to the cytotoxic effects of cytarabine by shifting the balance from the degradative to the activation pathway. METHODS: Human SCC-25 squamous carcinoma cells were transfected by either retroviral vector or adenoviral vector containing DCK gene and were identified for dCK expression by Northern blot analysis. In vitro cytotoxic assay after cytarabine exposure was performed using these cells. RESULTS: Both retroviral and adenoviral vector-mediated transduction of the dCK complementary DNA resulted in marked sensitization of tongue squamous carcinoma cell lines to the cytotoxic effects of cytarabine in vitro. CONCLUSION: The dCK-cytarabine system may be a useful approach for gene therapy of squamous cell carcinomas of the head and neck.     

小干扰RNA对喉鳞状细胞癌裸鼠移植瘤增殖细胞核抗原及p53蛋白表达的影响

目的：应用小干扰RNA（siRNA）技术抑制裸鼠喉癌皮下移植瘤人端粒酶逆转录酶（hTERT）基因表达,探讨移植瘤中增殖细胞核抗原（PCNA）及p53蛋白表达的变化。方法：根据hTERT cDNA序列构建表达短发夹RNA（shRNA）的、靶向hTERT mRNA的真核表达质粒pshRNA,其载体质粒含荧光素报告基因。建立人喉鳞状细胞癌Hep-2细胞株裸鼠皮下接种模型,将pshRNA转染入荷瘤裸鼠瘤体内,观察肿瘤生长情况。以激光共聚焦显微镜观察质粒在瘤体内的表达;以免疫组织化学SP法检测PCNA及p53蛋白在肿瘤内的表达。结果：所有裸鼠均接种成功,5d后可见皮下肿瘤形成,14d左右肿瘤直径达5～7mm。pshRNA及空质粒载体转染入瘤体后,共聚焦显微镜下见癌组织中有绿色荧光表达。病理学检查发现：pshRNA组肿瘤生长受到抑制,细胞分裂少见,可见大量癌细胞坏死。与注射生理盐水的对照组比较,转染质粒完毕后7d抑瘤率为76．50％,P〈0．01。pshRNA治疗后瘤体内PCNA蛋白表达显著下调（P〈0．05）,p53蛋白表达显著上调（P〈0．05）。结论：靶向hTERT mRNA的shRNA可显著抑制人喉癌裸鼠移植瘤的生长,其机制可能是诱导PCNA下调,促进p53蛋白表达所致。     

Monocyte tumor necrosis factor production in head and neck squamous cell carcinoma.

Changes in monocyte and macrophage function have been demonstrated in patients with head and neck squamous cell carcinoma. The purpose of this study was to evaluate tumor necrosis factor (TNF) production by activated monocytes from patients with head and neck squamous cell carcinoma and to evaluate its relation to cancer stage, weight loss, and performance status. Monocytes from patients (n = 10) and controls (n = 10) were isolated from peripheral blood mononuclear cells by plastic adherence and incubated with lipopolysaccharide (10 micrograms/mL). The TNF concentration of supernatants was assayed by TNF alpha-specific immunoassay. The TNF production by monocytes from head and neck squamous cell carcinoma patients was significantly higher (P less than .001) than those of controls. No significant relationship was found to cancer stage, weight loss, and performance status. These findings indicate that, in head and neck squamous cell carcinoma patients, an increased TNF production by activated monocytes takes place which does not correlate with cancer stage, cancer-related weight loss, and performance status.     

Enhancement of cyclophosphamide sensitivity in squamous cell carcinoma transfected with cytochrome P-450 2B1

PURPOSE: Cyclophosphamide (CPA) has been shown to be quite effective and safe in the treatment of leukemia, but it had shown almost no efficacy in the treatment of solid tumors such as head and neck cancers. The purpose of this study was to determine whether head and neck squamous cell carcinomas can be made more sensitive to the cytotoxic effect of CPA by shifting the balance from the degradative to the activation pathway. MATERIALS AND METHODS: Human SCC-25 squamous carcinoma cells were transfected by adenoviral vector containing P-450 2B1 gene and were identified for P-450 2B1 expression by Northern blot analysis. In vitro cytotoxic assay after CPA exposure was performed using these cells. Human KB oral base squamous cell carcinoma cells were injected subcutaneously to the backs of nude mice. Then Ad.CMV-P-450 2B1 was injected intratumorally. At 48 hours after the injection of Ad.CMV-P-450 2B1, CPA was injected into the peritoneal cavity of the animals and the volume of the tumor was determined with the passage of time. All the animal procedures were performed under the guidance of the committee in our animal care facility. RESULTS: The in vitro experiments found that transduction of the P-450 2B1 gene caused a nearly 30-fold increase in the sensitivity of the host cell line to CPA on the basis of the 50% inhibitory concentration. The in vivo experiments, which were conducted in nude mice, found that the combination of P-450 2B1 gene transduction and administration of CPA showed an inhibitory effect on tumor growth. CONCLUSIONS: The P-450 2B1- cyclophosphamide system may be useful approach for gene therapy of squamous cell carcinomas of head and neck.     

Urokinase-type plasminogen activator expression and proliferation stimulation in head and neck squamous cell carcinoma in vitro and in situ

BACKGROUND: Stimulation of proliferative activity by urokinase-type plasminogen activator (uPA) has been demonstrated in vitro for cultured primary and carcinoma cells. OBJECTIVE: To examine the effect of uPA stimulation on cultured squamous cell carcinoma cell lines of the head and neck in vitro and to compare the results with the situation in tumor tissue specimens. DESIGN: The uPA-mediated growth stimulation of 2 head and neck squamous cell carcinoma cell lines after suppression of endogenous uPA production was monitored by measuring (3)H-thymidine uptake into cellular DNA. Alternatively, applications of antibodies against the uPA-binding domain of the urokinase receptor were used to suppress autostimulation. To analyze the situation in situ we performed Western blot and zymographic studies on tissue homogenates of 25 squamous cell carcinoma specimens. We tested the expression of proliferating cell nuclear antigen (PCNA), a marker for proliferative activity, and uPA in tissue lysates and correlated uPA and PCNA expression by regression analysis. RESULTS: High-molecular-weight urokinase had a proliferation stimulative effect on both cell lines in vitro. The uPA autostimulation was decreased by blocking the uPA-binding domain of urokinase receptor with antibodies. Regression analysis of zymographic and Western blot data of tumor tissue lysates revealed no significant coherency between PCNA and uPA expression. Immunohistochemical stainings frequently showed different sublocalization of uPA and PCNA within tumors. CONCLUSION: In vitro uPA-mediated growth stimulation is not necessarily transferable to the in situ situation.