腺样囊性癌两个细胞系体外侵袭能力比较

目的：研究转移潜能不同的两个腺样囊性癌细胞系体外侵袭能力的差异。方法：选用肺低转移细胞系Ａｃｃ－２和从中筛选出肺高转移细胞系Ａｃｃ－Ｍ,利用ＭＴＴ法检测与几种基底膜成分的粘附能力;划痕法检测运动能力;ＰＡＧＥ底物酶谱法检测分泌Ⅳ型胶原酶的能力;改良勃顿小室法观察对重组基底膜的侵袭能力。结果：①１小时后Ａｃｃ－２细胞系对３种细胞外基质的粘附率分别是纤维粘连蛋白（ＦＮ）６１．８％、层粘连蛋白（ＬＮ）４     

全反式维甲酸对CNE—2Z细胞增殖的影响及凋亡诱导

目的：了解全反式维甲酸（ＡＴＲＡ）对ＣＮＥ－２Ｚ细胞增殖生长及凋亡有无影响。方法：采用ＭＴＴ法、细胞核染色、ＤＮＡ裂解率及电泳分析。结果：（１）用ＡＴＲＡ诱导细胞至７２ｈ，细胞增殖抑制率随浓度升高而增加。（２）在终浓度为５×１０－６ｍｏｌ／Ｌ诱导ＣＮＥ－２Ｚ细胞５天，可见部分细胞体积变小，核浓缩及核碎裂；ＤＮＡ裂解率（４４．２０±６．３２）明显高于对照组（１５．４１±５．５０），差别有显著性，Ｐ＜０．０１；ＤＮＡ琼脂糖电泳，出现不连续的典型梯状ＤＮＡ条带。片段大小为２００ｂｐ或其倍数。（３）在浓度为５×１０－７ｍｏｌ／Ｌ诱导５天和５×１０－６ｍｏｌ／Ｌ诱导４天时，均无明显细胞凋亡出现。结论：ＡＴＲＡ对ＣＮＥ－２Ｚ细胞增殖有抑制作用，且随浓度增加而增强；ＡＴＲＡ可诱导ＣＮＥ－２Ｚ细胞发生凋亡，但表现出明显的剂量和时间依赖性。     

IL-4基因及B7-1基因共转染新型瘤苗的制备及其治疗作用的初步研究

本文研究了ＩＬ－２，ＩＬ－４，ＩＬ－６基因转染后Ｂ１６黑色素瘤细胞表面ＭＨＣＩ类抗原及ＩＣＡＭ－１的表达水平，并探讨了基估ＣＴＬ诱导过程中的作用。结果表明，ＩＬ－２，ＩＬ－４，ＩＬ－６基因转染Ｂ１６黑素瘤细胞表面ＭＨＣＩ类抗原及ＩＣＡＭ－１表达均高于野生型Ｂ１６黑色素瘤细胞及转染对照质粒的Ｂ１６黑色素瘤细胞。     

鼻咽癌细胞侵袭不同靶组织的特征观察

以人胚心肌、脐带组织、羊膜组织和脐带动脉为靶组织，观察ＣＮＥ－１、ＣＮＥ－２Ｚ及其单克隆株Ｈ５、Ｆ１、Ｂ４和Ｆ７共６株鼻咽癌细胞的侵袭特征。结果表明，癌组织在侵袭过程中，除具有粘附、溶解破坏及运动功能外，还须有分裂增生能力及以某种未知机制克服靶组织阻力的能力。本文首次使用脐带组织作为肿瘤侵袭实验的靶组织并描述了其优点。此外还初步筛选出侵袭能力较强的Ｈ５和较弱的Ｂ４细胞株。     

人乳腺癌细胞株WAF1/CIP1/p21基因的表达及其与细胞生物学特性的关系

目的研究人乳腺癌细胞株ＷＡＦ１／ＣＩＰ１基因的ＤＮＡ状况、ｍＲＮＡ和蛋白的表达水平及其意义。方法应用细胞培养、分子生物学Ｓｏｕｔｈｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交以及免疫组化染色等技术，检测人乳腺癌表达野生型ｐ５３（ｗｔｐ５３）的ＭＣＦ７细胞和表达突变型ｐ５３（ｍｔｐ５３）的ＭＤＡＭＢ２３１细胞中ＷＡＦ１／ＣＩＰ１基因ＤＮＡ状况、ｍＲＮＡ和蛋白质的表达水平，研究其与ｍｄｍ２、ｐ５３蛋白的表达和细胞生物学特性的关系。结果比较ＭＣＦ７细胞与ＭＤＡＭＢ２３１细胞：（１）两者ＷＡＦ１／ＣＩＰ１基因ＤＮＡ状况无明显差异，前者ｍＲＮＡ和蛋白质的表达水平明显高于后者（Ｐ＜０．０５）；（２）两者ｐ５３蛋白的性质和分布不同，前者ｍｄｍ２蛋白的表达水平明显高于后者（Ｐ＜０．０５）；（３）前者生物学特性好于后者。结论人乳腺癌细胞株ＷＡＦ１／ＣＩＰ１基因ｍＲＮＡ和蛋白质的表达水平与ｐ５３基因表型和细胞生物学特性有关。     

BHRF1表达对~(60)Co照射后CNE2细胞周期分布的影响

目的：了解ＢＨＲＦ１基因表达对６０Ｃｏ照射后ＣＮＥ２细胞周期分布的影响。方法：构建ＢＨＲＦ１高表达载体并转入ＣＮＥ２细胞中，通过检测癌细胞增殖细胞核抗原（Ｐｒｏｌｉｆｅｒａｔｉｎｇｃｅｌｎｕｃｌｅａｒａｎｔｉｇｅｎ，ＰＣＮＡ）表达和在６０Ｃｏ照射后不同时期细胞周期分布的改变。结果：ＢＨＲＦ１可抑制细胞ＰＣＮＡ的表达及降低Ｓ期的百分率；经６０Ｃｏ照射后，ＢＨＲＦ１ＣＮＥ２细胞的凋亡率较低（Ｐ＜００１），Ｓ期细胞数下降不明显（Ｐ＞００５）；在辐射后的２４小时到７２小时，其Ｓ期和Ｇ２期细胞百分率先后升高。结论：ＢＨＲＦ１基因的表达可抑制细胞的增殖并增强其抵抗辐射诱发的细胞凋亡的发生。     

联合应用MLV-rIL-2、TIL、CY抗鼠H22肝癌的实验研究

表达大肠杆菌胞嘧啶脱胺酶（ＣＤ）基因的重组腺病毒ＡｄＣＤ体外转染小鼠黑色素瘤细胞Ｂ１６Ｆ１０，结果显示转梁了ＣＤ基因的Ｂ１６Ｆ１０细胞对５－氟胞嘧啶（５ＦＣ）的敏感性显著提高。将经ＡｄＣＤ／５ＦＣ系统处理的Ｂ１６Ｆ１０细胞止清倍比稀释后，加至野生型了Ｂ１６Ｆ１０细胞中，发现当上清仅占６．２５％时即可对野生型Ｂ１６Ｆ１０细胞发挥明显的杀伤作用，提示ＡｄＣＤ／５ＦＣ介导的旁观者效应可能是通过５ＦＣ经Ｃ     

C－erbB－2癌基因产物在良恶性鼻咽组织及鼻咽癌细胞系和克隆株中的表达

用抗Ｃ－ｅｒｂＢ－２癌基因产物Ｐ１８５多克隆抗体２１Ｎ，应用免疫组化链菌素亲生物素蛋白－过氧化酶（Ｓ－Ｐ）方法，对６９例良恶性鼻咽组织及人鼻咽癌细胞系ＣＮＥ－１、ＣＮＥ－２Ｚ、克隆株ＣＮＥ－２Ｚ－Ｆ１、Ｈ５、Ｂ７中Ｐ１８５的表达进行了检测；此外，还观察了克隆株ＣＮＥ－２Ｚ－Ｂ７在连续传代过程中Ｐ１８５的表达。结果显示：鼻咽癌组织中Ｐ１８５表达率为６６．７％，慢性鼻咽炎中Ｐ１８５阳性表达率为６．３％，人胚鼻咽上皮不表达Ｐ１８５；鼻咽癌细胞系及克隆株中Ｐ１８５表达不一；克隆株ＣＮＥ－２Ｚ－Ｂ７在连续传代过程中Ｐ１８５的表达逐渐升高。结论：Ｃ－ｅｒｂＢ－２癌基因的激活，可能在鼻咽癌发生中起重要的作用；Ｐ１８５的过度表达和肿瘤细胞的分化程度及演进过程有关。     

ＭＭＰ-２特异性ＲＮＡ干扰对人胰腺癌细胞粘附和侵袭的作用

探讨靶向基质金属蛋白酶-２（ＭＭＰ-２）的ＲＮＡ干扰对人胰腺癌细胞粘附和侵袭的作用。方法　设计和构建靶向ＭＭＰ-２的特异性小干扰ＲＮＡ（ｓｉＲＮＡ）表达质粒,将质粒转染入人胰腺癌ＢｘＰＣ-３细胞系中,根据转染质粒靶序列的不同分为５组,即ｐＧＰＵ６-１、ｐＧＰＵ６-２、ｐＧＰＵ６-３、ｐＧＰＵ６-４和阴性对照组ｐＧＰＵ６（－）,并设仅加入转染剂的空白对照组。应用ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔ法检测各组ＢｘＰＣ-３细胞转染后ＭＭＰ-２蛋白及ｍＲＮＡ表达水平,应用ＭＴＴ法及流式细胞术检测各组细胞增殖和凋亡水平,以平板粘附模型和Ｔｒａｎｓｗｅｌｌ小室模型检测各组转染后肿瘤细胞粘附和侵袭能力,比较各组间差异。结果　与空白对照组和阴性对照组相比,ｐＧＰＵ６-１、ｐＧＰＵ６-２、ｐＧＰＵ６-３组ＭＭＰ-２ｍＲＮＡ抑制率分别为７５.３％、６４.５％、５１.６％和７４.７％、６３.７％、５０.５％,差异均有统计学意义;ＭＭＰ-２蛋白表达抑制率分别为７９.１％、６４％、５２.３％和７８％、６２.２％、５０.５％,差异均有统计学意义;粘附抑制率分别为６３.１％、４２.９％、２５.６％和６２.２％、４１.６％、２３.９％,且穿膜细胞数量明显降低,差异均有统计学意义。对照组与ｐＧＰＵ６-４组的ＭＭＰ-２蛋白及ｍＲＮＡ表达水平无明显差异,在肿瘤细胞粘附和侵袭能力方面也未见明显差异。ＭＴＴ法及流式细胞术的结果并没有提示各组间肿瘤细胞增殖和凋亡水平的差异。结论　靶向ＭＭＰ-２的ＲＮＡ干扰能够明显抑制胰腺癌ＢｘＰＣ-３细胞蛋白水平及ｍＲＮＡ水平的表达,从而抑制肿瘤细胞粘附和侵袭的能力,但并没有导致干扰后肿瘤细胞增殖和凋亡能力的改变。     

两株鼻咽癌细胞中TPK,PKC的活性研究

目的：研究ＣＮＥ－１和ＣＮＥ－２Ｚ细胞浆中ＰＫＣ、ＴＰＫ及胞膜ＴＰＫ活性，及槲皮素对它们的影响；方法：常规培养ＣＮＥ－１、ＣＮＥ－２Ｚ和淋巴细胞，用特异底物法、特异激活剂法分别测定其ＴＰＫ、ＰＫＣ活性，两两比较用ｔ检验；结果：ＣＮＥ－１细胞胞浆ＰＫＣ、ＴＰＫ均明显高于脐带血淋巴细胞（Ｐ＜０．０１），而胞膜ＴＰＫ无明显变化；ＣＮＥ－２Ｚ细胞胞浆ＰＫＣ却明显低于淋巴细胞（Ｐ＜０．０１），胞浆、胞膜ＴＰＫ活性均明显增高（Ｐ＜０．０１）。槲皮素（１００μｍｏｌ／Ｌ）使ＣＮＥ－２Ｚ细胞中胞膜ＴＰＫ明显的增高（Ｐ＜０．０１），而对其胞浆ＰＫＣ、ＴＰＫ均无明显影响；该浓度下的槲皮素也能使ＣＮＥ－１细胞中胞膜ＴＰＫ活性明显升高（Ｐ＜０．０１），但强烈抑制其胞浆ＴＰＫ活性（Ｐ＜０．０１），而对其胞浆ＰＫＣ也没有明显影响；结论：胞浆、胞膜ＴＰＫ和胞浆ＰＫＣ可能与鼻咽癌细胞株的分化程度有关     

Blocking of collagenase secretion by estramustine during in vitro tumor cell invasion

The effects of the antitumorigenic drug estramustine on tumor cell membrane penetration (invasion) were investigated in vitro by utilizing a synthetic basement membrane system (a modified Boyden chamber). Tumor cells were plated on a "partition barrier," consisting of a porous filter (8-micron pores) which was coated with a reconstituted basement membrane matrix (Matrigel), and induced to migrate across the barrier with conditioned medium obtained from 9DU 145 human prostatic tumor cells (passage 9). Quantitative radiolabeling studies demonstrated that specially isolated lines (isolated by several passages through the Matrigel) of DU 145 cells, A2058 melanoma, and B16-F10 melanoma cells were highly invasive such that 15 to 20% migrated across a 1-mm-thick Matrigel layer within 5 h at 37 degrees C. NIH-3T3 cells, mouse fibroblasts, and 20DU 145 cells (passage 20) exhibited little or no membrane invasive behavior. Micromolar concentrations of estramustine (30 to 120 microM) inhibited invasion by the invasive cell lines in a dosage-dependent fashion. Quantitative enzymatic assays and radioimmune assays demonstrated that estramustine inhibited membrane invasion by blocking type IV collagenase secretion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots confirmed that 30 to 60 microM estramustine blocked secretion of a Mr 105,000 collagenase protein. Indirect studies showed that a collagenase antibody raised against the Mr 105,000 protein and inhibitors of proteinase activity, including a metalloproteinase inhibitor, and 1,10-phenanthroline, blocked invasion. Because the antibodies inhibited type IV collagenase digestion of 3H-mouse type IV collagen, and invasion simultaneously, it is proposed that collagenolytic activity is involved in invasion. These data demonstrate that estramustine blocks proteinase secretion, and suggest that estramustine may be a useful therapeutic drug for the prevention of metastasis.     

Tumor cell invasion inhibited by TIMP-2.

The 72-kd type IV collagenase is a member of the collagenase enzyme family that has been closely linked with the invasive phenotype of cancer cells. Previous studies have shown that both normal cells and highly invasive tumor cells produce the 72-kd type IV procollagenase enzyme in a complexed form consisting of the proenzyme and a novel tissue inhibitor of metalloproteinases, TIMP-2. The balance between activated enzyme and available inhibitor is thought to be a critical determinant of the matrix proteolysis associated with a variety of pathologic processes, including tumor cell invasion. In the present study, we demonstrate that alteration of the metalloproteinase-metalloproteinase-inhibitor balance in favor of excess inhibitor blocks human fibrosarcoma HT-1080 tumor cell invasion of a reconstituted basement membrane. The HT-1080 cell line produces both the 72-kd and the 92-kd type IV collagenases. Alteration of the type IV collagenase-inhibitor balance was achieved by addition of free TIMP-2 or antibodies to 72-kd type IV collagenase. Native, purified TIMP-2 was inhibitory in the range of 1-25 micrograms/mL. Addition of specific antiserum against the 72-kd type IV collagenase, which did not cross-react with the 92-kd type IV collagenase, inhibited HT-1080 cell invasion to the same extent. These results suggest that metalloproteinases, in particular the 72-kd type IV collagenase, are critical for tumor cell invasion of the reconstituted basement membrane. Our findings demonstrate that addition of the endogenous inhibitor TIMP-2 is able to block invasion. Thus, we recommend initiation of in vivo studies of the therapeutic potential of TIMP-2 to block tumor cell invasion and intravasation into the circulation.     

Inhibition of proteolytic enzymes in the in vitro amnion model for basement membrane invasion

The ability of B16-F10 mouse melanoma cells to cross an amnion basement membrane was determined in the presence of strong inhibitors of both serine and cysteine proteases. The concentrations of inhibitors were at orders of magnitude higher than their Ki values to serine and cysteine proteases implicated in metastasis, thus ensuring a complete inhibition for tumor secreted proteases such as cathepsin B-like proteases, plasminogen activators, and plasmin. Under these conditions of high serine and cysteine protease inhibitor concentrations, no significant decrease in B16-F10 melanoma cell invasion through the amnion was observed. Separate experiments showed that the inhibitors were neither toxic to the cells nor degraded. The results show that neither tumor cell secreted cathepsin B-like proteases nor plasminogen activator have a controlling role in basement membrane crossing in this metastatic model. A possible role for tumor cell membrane proteases in basement membrane invasion, in which the substrates of the protease bind to receptor sites near a membrane associated proteolytic activity, is not eliminated.     

Interaction of metastatic tumor cells with bovine lens capsule basement membrane

The use of bovine lens capsule basement membrane as a model substratum for studies of invasion and extravasation by metastatic tumor cells is described. The abilities of three independently isolated pairs of metastatic variant cell lines to digest the purified substrates, laminin, type IV collagen, and type I collagen, were compared with their abilities to solubilize isotope from 125I-labeled lens capsule basement membrane matrix. The cell lines used were +SA and -SA mouse mammary adenocarcinoma cells, RT7-4bs and RT7-4b-Ls rat hepatocarcinoma cells, and B16-F1 and B16-F10 mouse melanoma cells. In general, imperfect correlations of lytic activity with metastatic ability were found for the purified substrate digestions, but, for each pair of variants, the more metastatic tumor cell line was always able to solubilize more surface-bound isotope from the lens capsule. Visual evidence of tumor cell-associated digestion of lens capsule basement membrane was obtained using transmission electron microscopy. Mouse mammary carcinoma cells attached more rapidly to lens capsule than to endothelial cell monolayers or tissue culture plastic. We next added endothelial cells to the model substrate. Aortic endothelial cells grew well on lens capsules without apparent synthesis of additional basement membrane matrix. In additional studies, the lens capsule was used in a chamber apparatus to demonstrate that cellular invasion of the full thickness of this basement membrane structure could be demonstrated and readily quantitated. Our results indicate that bovine lens capsule is a particularly versatile basement membrane structure useful for studies of tumor cell invasion and extravasation. In addition, the comparison of purified substrate digestions with lens capsule matrix digestion indicates the desirability of also using a matrix digest when correlating lytic abilities of tumor cells with their metastatic abilities.     

小鼠黑色素瘤某些生物学特性与其侵袭潜能的相关性研究

目的 研究具有相同起源而转移能力不同的小鼠黑色素瘤细胞系（B16、B16F10、B16BL6）的某些生物学特性与其侵袭潜能的相关性。方法 用重组基质膜实验考察细胞的侵袭能力;用分光光度法测定黑色素瘤细胞的黑色素含量;利用细胞运动记录分析系统研究细胞在三维胶原基质中的运动状态;用明胶底物酶谱法分析细胞分泌Ⅳ型胶原酶的能力;用端粒重复扩增（TRAP）－PCR法测定细胞的端粒酶活性。结果 B16B6力B16F10具有较高的侵袭能力,其运动能力及分泌Ⅳ型胶原酶的能力亦较强,但B16F10的黑色素含量却较低;端粒酶活性在三者之间无明显差异。结论在所研究的小鼠黑色素瘤不同亚系的声东击西 些生物学特性中,侵袭能力与细胞运动能力、分泌Ⅳ型胶原酶的能力之间有较好的相关性,而与黑色素含量及端粒酶活性无直接相关性。     

Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells

Using both human and murine cell lines, we show that malignant cells are able to invade through basement membrane and also secrete elevated amounts of collagenase IV, an enzyme implicated in the degradation of basement membranes. Using serine proteinase inhibitors and antibodies to plasminogen activators as well as a newly described collagenase inhibitor we demonstrate that a protease cascade leads to the activation of an enzyme(s) that cleaves collagen IV. Inhibition at each step reduces the invasion of the tumor cells through reconstituted basement membrane in vitro. Treatment with a collagenase inhibitor reduced the incidence of lung lesions in mice given i.v. injections of malignant melanoma cells.     

Retinoic acid inhibition of human melanoma cell invasion through a reconstituted basement membrane and its relation to decreases in the expression of proteolytic enzymes and motility factor receptor

Treatment of four A375 human melanoma sublines (A375, A375P, A375P-5, A375M), exhibiting distinct metastatic potentials in vivo, with beta-all-trans-retinoic acid in vitro caused a dose- and time-dependent inhibition of the ability of these cells to penetrate Matrigel-coated filters using a reconstituted basement membrane invasion assay. The possible mechanisms of action responsible for the antiinvasive effect were further investigated, and the data showed that compared with untreated cells the retinoic acid-treated cells: (a) secreted lower levels of collagenolytic enzymes, as demonstrated by a decreased ability of the cells to degrade [3H]proline-labeled type IV collagen substrate and by a reduction in the activity of a secreted Mr 64,000 collagenolytic enzyme detected in type IV collagen-containing polyacrylamide gels; (b) expressed lower levels of the human type IV collagenase mRNA (except in the A375P cells), as detected by Northern blot analysis; (c) exhibited decreased levels of tissue plasminogen activator activity, as demonstrated by a chromogenic assay; (d) were 10-40% less adhesive to a reconstituted basement membrane matrix, as determined by a 60-min Na2(51)CrO4-labeled cell attachment assay; (e) exhibited an increase in the high affinity metastasis-associated cell surface laminin receptor, as determined by flow cytometry after binding of fluorescently labeled laminin receptor antibody; and (f) expressed decreased amounts of gp78, a cell surface receptor for motility factor, demonstrated by immunoblotting and immunofluorescence. Collectively, these data suggest that retinoic acid inhibits tumor cell invasion through a basement membrane-like matrix by suppressing matrix degradation and by altering cell surface receptors.     

Expression of collagenase IV (basement membrane collagenase) activity in murine tumor cell hybrids that differ in metastatic potential

Expression of a basement membrane collagen-degrading metalloprotease activity (collagenase IV) was studied in a series of murine cell hybrids derived from fusions between highly metastatic cells (B16-F10RR) or moderately metastatic cells (UV-2237RR) and tumorigenic cells (K-1735 clone 16) or normal cells [peritoneal macrophages (PEC) or C3H mouse embryo fibroblasts (C3H-F)]. The collagenase IV activity of the parent cells and the hybrids was assayed in vitro and compared to the metastatic propensity of the same cells evaluated in both syngeneic (C57BL/6 X C3H/HeN)F1 mice and BALB/c nude mice. The level of collagenase IV activity secreted by the parent lines correlated with their metastatic capacity. The highly metastatic B16-F10RR line secreted the highest enzyme activity, whereas the tumorigenic but nonmetastatic K-1735 clone 16 and the normal parents PEC and C3H-F secreted the lowest enzyme activity. The enzyme activity was completely inhibited with EDTA. The hybrid derived from fusion of cells from two metastatic cell lines as well as hybrids derived from a metastatic and a nonmetastatic tumor cell line expressed higher levels of collagenase IV activity than either parent, and this expression was associated with a high ability to produce metastases in both nude and syngeneic mice. Fusion of metastatic cells with normal cells produced hybrid cells that exhibited suppression of both collagenase IV activity and metastatic capacity. Collagenase IV activity and metastatic propensity can, therefore, be altered by somatic cell hybridization; in the series of hybrids examined in these experiments the expression of type IV collagen-degrading metalloprotease activity and the metastatic ability were closely correlated, which suggests that collagenase IV activity and other properties required for metastasis are genetically linked.     

Secretion of matrix metalloproteinase-2 (72 kD gelatinase/type IV collagenase = gelatinase A) by malignant human glioma cell lines: implications for the growth and cellular invasion of the extracellular matrix

Human glioma cells (T98G and A172 cell lines) were cultured on various extracellular matrix (ECM) components including type 1, IV and V collagens, fibronectin, laminin, and reconstituted basement membrane (Matrigel), and the role of matrix metalloproteinases (MMPs) in their growth and invasion was examined. T98G glioma cells grew well on these ECM components and invaded the reconstituted basement membrane. In contrast, A172 glioma cells showed growth inhibition on collagen types IV and V and Matrigel without invasion of the Matrigel. Gelatin zymography and enzyme immunoassays demonstrated that T98G glioma cells, but not A172 cells, secrete a large amount of matrix metallproteinase-2 (MMP-2, 72 kD gelatinase/type IV collagenase = gelatinase A), and this was confirmed by immunoblotting and immunohistochemistry. Of the two different tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), T98G cells produced only TIMP-1 during culture on Matrigel, whereas A172 cells secreted both. Although both human recombinant TIMP-1 and TIMP-2 stimulated T98G cell growth slightly on Matrigel, the in vitro invasiveness was significantly reduced by only recombinant TIMP-2. These results suggest that MMP-2 plays an important role in the ECM invasion of T98G human glioma cells in vitro.     

Induction of an invasive phenotype in benign tumor cells with a laminin A-chain synthetic peptide.

K-1735 clones 10 and M2 are cell lines cloned from a UV-induced murine melanoma. While both lines are highly tumorigenic, only the M2 cells are highly invasive in vitro and metastatic in vivo. Here we have exposed the clone 10 cells to the synthetic peptide PA22-2, which contains the IKVAV sequence from the A chain of laminin and which, like laminin, induces collagenase IV production and enhances metastasis formation by B16F10 cells. Zymogram analysis of conditioned media from clone 10 cells cultured on the peptide demonstrated a dose-dependent increase in collagenase IV activity. When clone 10 cells were cultured on a reconstituted basement membrane (Matrigel), this peptide caused an invasive phenotype comparable to the M2 cells. The invasive clone 10 cells were, however, unable to form lung colonies in vivo in the presence of this peptide. We conclude that this peptide represents an active site on laminin which is able to stimulate the invasiveness of this tumor cell line, but that this activity is not sufficient to confer metastatic potential.