A specific sequence of the noncollagenous domain of the alpha3(IV) chain of type IV collagen inhibits expression and activation of matrix metalloproteinases by tumor cells

The invasive properties of melanoma cells correlate with the expression of matrix metalloproteinases (MMPs) and their physiological modulators (tissue inhibitors of metalloproteinase and membrane-type MMPs) and with that of the alphaVbeta3 integrin. We investigated the effect of anterior lens capsule type IV collagen and of the alpha3(IV) collagen chain on the invasive properties of various tumor cell lines (HT-144 melanoma cells, HT-1080 fibrosarcoma cells). We demonstrated that anterior lens capsule type IV collagen or specifically the synthetic peptide alpha3(IV) 185-203 inhibited both the migration of melanoma or fibrosarcoma cells as well as the activation of membrane-bound MMP-2 by decreasing the expressions of MT1-MMP and the beta3 integrin subunit.     

Distribution of collagen type IV in soft tissue tumors. An immunohistochemical study

The distribution of collagen type IV, one of the major constituents of basement membrane, was studied immunohistologically in a series of 103 soft tissue tumors including those of peripheral nerve origin, smooth muscle origin, striated muscle origin, fibrous tissue origin, fibrohistiocytic origin, adipose tissue origin, synovial tissue origin, and blood vessel origin, paragangliomas, alveolar soft part sarcomas, granular cell tumors, and epithelioid sarcomas. Intensely positive staining for collagen type IV was observed in neurilemomas, neurofibromas, malignant schwannomas, and blood vessel tumors. Weakly to moderately positive staining was seen in leiomyomas, angiomyomas, and leiomyosarcomas. In contrast, synovial, fibroblastic and fibrohistiocytic tumors, benign or malignant, were negative. In paragangliomas, granular cell tumors, and alveolar soft part sarcomas, positive staining was evident surrounding nests or clusters of tumor cells. In all tumors, staining for collagen type IV clearly illustrated the vascular pattern.     

Phytol is a novel tumor promoter on ICR mouse skin.

Phytol is a branched, long-chain aliphatic alcohol which has various biological effects. In this study, we examined phytol as a tumor promoter in a mouse skin initiation-promotion model, and compared its promotion activity with that of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Female ICR mice, 7 weeks of age, were initiated with 100 microg of 7,12-dimethylbenz(a)anthracene, and were then topically promoted twice a week for 16 weeks with 100 mg of phytol or with 2.5 microg of TPA. In this model 95% of animals treated with phytol developed skin tumors within 16 weeks. The average number of lesions per mouse treated with phytol was significantly lower than that in mice treated with TPA, and this significant difference continued up to 16 weeks after the end of promotion treatment. Characterization of hyperplasia 48 h after topical application of agents showed that epidermal thickness and vertical thickness following topical application of phytol were significantly increased compared with vehicle controls, but were significantly smaller than in animals treated with TPA. Ornithine decarboxylase (ODC) activity following topical application of phytol was increased in a dose-dependent manner and showed a weak, delayed induction (which was maximal 11-12 h after treatment) as compared with the case of TPA. The specific binding of [3H]phorbol-12,13-dibutyrate (PDBU) by JB6 cells was not inhibited by phytol at concentrations up to 1 mM. These results indicate that phytol has a weak tumor promoter activity compared to TPA and is a non-TPA-type tumor promoter in this model of mouse skin carcinogenesis.     

一个具有肿瘤转移抑制基因活性的人DNA序列

目的 分离鉴定人类肿瘤转移抑制基因或相应ＤＮＡ序列,探讨转移表型逆转的分子生物学机制。方法 应用Ｉｎｔｅｒ Ａｌｕ ＰＣＲ技术,将导入正常人肺基因组ＤＮＡ片段后转移表型抑制细胞株中的人源性ＤＮＡ片段扩增出来,对其中的７００ｂｐ左右ＤＮＡ片段进行核苷酸序列分析,并在ＧｅｎＢａｎｋ序列数据库进行类似性检索。将该ＤＮＡ再次转染高转移小鼠瘤细胞株,通过体内、体外转移相关性实验,验证其是否具有抑制高转移小鼠     

A novel human DNA sequence with tumor metastasis suppressive activity

　Objective To isolate human tumor metastasis suppressive DNA sequence and to study the molecular mechanisms regulating tumor metastasis. Methods A mouse lung adenocarcinoma cell clone 12 derived from its parent cell line LM2, which had been transduced with normal human genomic DNA, was previously reported. Compared with LM2, the metastatic potential of clone 12 was very much decreased. Clone 12 was used in this study to amplify the human DNA fragments by Inter Alu PCR technique. The human DNA fragments obtained were then transfected into LM2 cells and their malignant phenotype was tested in vitro and in vivo, and compared with that of the untransfected LM2 cells. Results Three human DNA fragments of 700, 500 and 300 bp were isolated. DNA sequencing revealed that the 700bp fragment does not show homology with hitherto reported genes and was accepted by the Genbank (pt712 U67835). In vitro proliferation and colony formation in soft agar of the 700 bp fragment-transfected LM2 cells were significantly inhibited as compared to the untransfected LM2 cells. Upon subcutaneous inoculation to syngeneic T739 mice, the 700bp–transfected LM2 cells grew more slowly and smaller tumors developed compared to the untransfected ones. Moreover, lung metastasis was not found in 6 of 10 mice inoculated with the 700bp-transfected LM2 cells, while it was found in 9 of 10 mice inoculated with the untransfected LM2 cells. The difference was statistically significant (P<0.001). The frequency of lymph node metastasis was also statistically different between the 2 groups of mice. Conclusion The newly isolated 700bp human DNA fragment may be a metastasis suppressor gene of malignant tumor.     

Two-dimensional polyacrylamide gel analysis on nuclear proteins from human tumor cell lines

Two-dimensional polyacrylamide gel electrophoretic analysis was carried out on HeLa, KB, ALL and GW-39 cell "Chromatin Fraction II" proteins. Of the many proteins found, most were visualized in earlier studies on rodent tumors and normal tissues. Of these the greater density of protein CP and the presence of protein CG' differentiates tumors from the nontumor tissues. In samples of normal and mitogen stimulated human lymphocytes, protein CG' was absent and protein CP was present in small amounts. Two-dimensional patterns of 0.4N H2SO4 soluble nuclear proteins showed elevated amounts of proteins C16-18 in the GS-39 cell patterns. Proteins Hu1, G1, G2, G3 and G5 were detected only in human cell nuclei. The increased density of staining for protein CP and the presence of CG' are potential biological markers for neoplastic cells.     

Partial purification and characterization of serum protease from tumor-bearing rats which cleaves type IV collagen

Activity of neutral protease was increased in sera of rats bearing ascites hepatoma AH109A compared to those of normal rats. The protease was isolated from serum protein and partially purified approximately 1,150 times in specific activity after sequential column chromatography of hemoglobin affinity, lysine-Sepharose, Ultrogel AcA34 and TSK-gel G2000SW in that order. The protease fraction still seemed to contain at least two kinds of proteases, serine and cysteine protease. It had a molecular weight of 18-21 kilodaltons with broad optimal pH range of 7.0-9.0, maximum at 8.0. Intradermal injection of the crude preparation of the neutral protease fraction induced extravascular emigration of circulating tumor cells in vivo. Moreover, partially purified protease degraded pepsin-treated chains of bovine glomerular type IV collagen in vitro, but such an in vitro action of the protease was inhibited by an addition of soybean trypsin inhibitor or mercuric chloride. It failed to cleave salt-extracted rat skin type I collagen under the same digestive conditions for bovine type IV collagen. The serum neutral proteases of tumor-bearing host may play some cooperative roles during extravascular emigration of tumor cells by destruction of vascular basement membrane.     

Proteolytic enzymes in tumor metastasis. II. Collagenase type IV activity in subcellular fractions of cloned tumor cell populations

Collagenase type IV degradation activity was examined in metastatic and nonmetastatic clones of the Lewis lung carcinoma (3LL) and of the T10 sarcoma. Conditioned media prepared from cells of both tumors grown in vitro contained low degradation activities, whereas conditioned media from organ cultures of the same clones grown as solid tumors in animals exhibited higher degradation activities. Analysis of subcellular fractions of tumor cells showed that collagenase type IV activity was localized mainly in the cytoplasmic fraction. Crude homogenates or detergent lysates manifested low degradation activities. Little activity was associated with purified plasma membrane preparations and endoplasmic reticular fractions. However, addition of plasma membrane to conditioned media and to cytoplasmic fractions reduced the degradation activities of the cytoplasmic fractions. Possibly a factor that inhibits collagenase type IV exists in the cells in a vesicular form. No correlation between degradation activity and metastatic capacity was demonstrated in the models used in this study. Both metastatic and nonmetastatic clones of the same tumor similarly could degrade basement membrane components.     

The chlorinated pesticide mirex is a novel nonphorbol ester-type tumor promoter in mouse skin.

The hepatocarcinogenic organochlorine pesticide, mirex, was examined as a tumor promoter in the mouse skin initiation-promotion model. Female CD-1 mice were initiated with 200 nmol 7,12-dimethylbenz[a] anthracene and topically promoted three times weekly for 20 weeks with doses of 25, 50, 100, or 200 nmol mirex. Mirex promoted tumors at all dose levels in a dose-dependent manner. At 20 weeks, mice promoted with 25, 50, 100, and 200 nmol mirex developed an average of 0.2, 4, 10, and 16 tumors per mouse with a 10, 60, 93, and 96% incidence of tumor-bearing mice, respectively. With continued treatment to 34 weeks, mice promoted with 25, 50, and 100 nmol mirex developed an average of 0.7, 7, and 12 tumors per mouse with a 27, 85, and 100% incidence of tumor-bearing mice, respectively. These results demonstrate that mirex is a very effective tumor promoter in mouse skin. The effect of mirex on several biochemical and morphological events associated with tumor promotion was then investigated. Mirex did not stimulate epidermal protein kinase C activity in vitro. Unlike the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, a single topical application of mirex (200 nmol) did not increase [3H]thymidine incorporation into epidermal DNA up to 108 h after application. Furthermore, multiple applications of 200 nmol mirex (3 times weekly for 4 weeks) resulted in only a very weak proliferative response; mirex increased the number of nucleated epidermal cell layers from 1 to 2 in acetone-treated controls to 2 to 3 while 2 nmol 12-O-tetradecanoylphorbol-13-acetate produced 6 to 7 nucleated cell layers. Mirex (200 nmol) did not induce ornithine decarboxylase activity up to 56 h after a single topical application. Collectively, these data indicate that mirex is a novel nonphorbol ester-type tumor promoter in mouse skin.     

Prognostic significance of type IV collagen and laminin immunoreactivity in urothelial carcinomas of the bladder

Invasion of a carcinoma involves the degradation and penetration of the subepithelial basement membrane (BM). This phenomenon might be used for histopathologic evaluation of neoplasms of the bladder. The authors studied the clinicopathologic data and tissue specimens of 125 cases of urothelial carcinomas collected prospectively. Penetration of the BM was evaluated by immunohistochemical staining of the BM components laminin and type IV collagen. The use of this parameter as a prognostic indicator in bladder cancer was assessed. The 5-year survival rate of patients having tumors with an interrupted or absent BM was significantly lower than that of patients having tumors with an intact BM. The rate of progression was greater in tumors with an interrupted or absent BM than in tumors with an intact BM. No association was found between BM status and recurrence. However, a significant correlation between tumor stage and BM staining was found. A correlation was also found between ploidy and BM staining as well as between histologic grade and BM staining pattern. When evaluating histologic grade, stage, ploidy, age, and BM score as prognostic parameters, the stage of bladder carcinomas turned out to be the most important factor in predicting the survival rate and the progression-free survival. However, BM staining was found to be of value for early identification of microinvasion and is helpful for correct staging of urothelial carcinomas.     

Antagonistic action between cyclic AMP and estrogen in phosphorylation of mammary tumor nuclear proteins.

A new protein kinase-dependent phosphorylation occurs in the nuclei of hormone-dependent, 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced mammary carcinoma following preincubation of tumor slices with cyclic adenosine 3',5'-monophosphate (cAMP). The presence of 17beta-estradiol in the medium inhibits this effect. Both events have been observed in vivo in the nuclei of DMBA-induced tumors. The phosphorylation pattern of nuclei in hormone-independent mammary tumor, DMBA No. 1, however, is not affected by preincubation with either cAMP or estrogen. These findings suggest that the antagonistic effect of cAMP and estrogen in the growth control of mammary tumors is exerted through a specific action on nuclear protein phosphorylation and that these events correlate with the hormone-dependency of the tumors.     

Detection of nuclear matrix proteins in serum from cancer patients.

Morphological characteristics of the cell nucleus have long been used by pathologists in the clinical diagnosis of cancer. The nuclear matrix of the cell, the structure that serves to organize the chromatin within the nucleus, is known to reflect these morphological characteristics with regard to cell and cancer type. Monoclonal antibodies were developed to extracted nuclear matrix proteins. These antibodies were used in two-site immunometric assays to detect soluble nuclear matrix proteins in the supernatants of two dying cell lines and 15 tumor tissues. Furthermore, nuclear matrix proteins were detected at elevated levels in the sera of cancer patients compared with normal patient sera. In one assay, 63.2% of cancer sera read above 95% of normal sera, and in another assay, 73.7% of cancer sera read above 95% of the normal sera. With the development of monoclonal antibodies with greater cancer specificity, the detection of circulating nuclear matrix proteins may become an important clinical tool in the diagnosis and monitoring of cancer.     

Retinoic acid nuclear receptors and tumor promotion: decreased expression of retinoic acid nuclear receptors by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

During studies to determine the mechanism of tumor promotionby 12-O-tetradecanoylphorbol-13-acetate (TPA), we found thatTPA downregulates mouse epidermal retinoic acid nuclear receptors(RAR), a superfamily of nuclear steroid/thyrold receptors implicatedin mediating effects of retinoic acid (RA). Application of TPAto mouse skin decreased the binding of [³H]RA to RAR from mouseepidermal nuclear extracts. In this experiment, 20 nmol of TPAwas applied to mouse skin and 3.5 h later binding of [³H]RAto RAR was analyzed by chromatography on a size-exclusion column.TPA treatment resulted in an     

Identification of CD47/integrin-associated protein and alpha(v)beta3 as two receptors for the alpha3(IV) chain of type IV collagen on tumor cells.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation independently of its ability to promote cell adhesion; these properties require the presence of the triplet -SNS- at residues 189-191 (J. C. Monboisse et al., J. Biol. Chem., 269: 25475-25482, 1994; J. Han et al., J. Biol. Chem., 272: 20395-20401, 1997). More recently, we demonstrated that native COL IV and -SNS-containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells but also breast, pancreas, and stomach tumor cells up to 82% and prostate tumor cells by 15%. This inhibition was shown to be dependent on a COL IV- or peptide-induced increase in intracellular cAMP (T. A. Shahan et al., Connect. Tissue Res., 40: 221-232, 1999). Attempts to identify the putative receptor(s) on tumor cells led to the isolation of five proteins (Mr 33,000, 52,000, 72,000, 95,000, and 250,000) from melanoma and prostate cells by affinity purification with the alpha3(IV)179-208 peptide. The Mr 52,000, 95,000, and 250,000 proteins were shown to be CD47/integrin-associated protein(IAP), the integrin beta3 subunit, and the alpha(v)beta3 integrin complex, respectively. The Mr 33,000 and 72,000 proteins have not yet been identified. To confirm the specificity of ligand binding to the receptors, cell membranes from either melanoma or prostate tumor cells were pretreated with the unlabeled ligand alpha3(IV)187-191 (-YYSNS-); alternatively, the peptide was pretreated with a peptide-reactive monoclonal antibody (A5D7) before receptor isolation. These treatments inhibited the purification of CD47/IAP, the integrin beta3 subunit, and the alpha(v)beta3 integrin complex from tumor cells. Furthermore, cells treated with CD47/IAP- or the alpha(v)beta3 integrin-reactive antibodies prevented the alpha3(IV)185-203 peptide from inhibiting cell proliferation and the subsequent rise in intracellular cAMP. Pretreating cells with the alpha3(IV)187-191 (-YYSNS-) peptide also inhibited their adhesion to the alpha3(IV)185-203 peptide substrate, whereas the inactive alpha1(IV)185-203 peptide, from the same region of the alpha1 chain as the alpha3(IV)185-203 peptide, had no effect. Incubation of cells with either CD47/IAP and/or alpha(v)beta3 integrin-reactive antibodies inhibited their adhesion to the alpha3(IV)185-203 peptide, whereas antibodies to the beta1 and beta2 integrin subunits were without effect. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation using the receptors CD47/IAP and the alpha(v)beta3 integrin.     

A novel screen using the Reck tumor suppressor gene promoter detects both conventional and metastasis-suppressing anticancer drugs

The membrane-anchored matrix metalloproteinase-regulator RECK is often downregulated in various types of cancers; the levels of residual RECK in resected tumors often correlate with better prognosis. Forced expression of RECK in cancer cells suppresses tumor angiogenesis, invasion, and metastasis in xenograft models. RECK is therefore a promising marker for benignancy and a potential effector in cancer therapy. We established a cell line containing two transgene systems: (1) the secreted alkaline phosphatase (SEAP) gene fused to Reck promoter and (2) the HRAS(12V) oncogene driven by the Tet-off promoter system. This cell line exhibits transformed phenotype in regular medium and flat morphology with increased SEAP activity in the presence of doxycycline, allowing the assessment of RECK-inducing activity of chemicals in the contexts of both transformed and untransformed cells. Our pilot experiments with 880 known bioactive compounds detected 34 compounds that activate RECK promoter; among these, 10 were authentic anticancer drugs. Four selected compounds up-regulated endogenous RECK protein in several human cancer cell lines. The top-ranking compound, disulfiram, strongly suppressed spontaneous lung-metastasis of human fibrosarcoma cells in nude mice. Our data demonstrate the value of this screen in discovering effective cancer therapeutics.