Augmentation of Recombinant CH50 Polypeptide on the Function of Macrophages of Mice during Chemotherapy

The immunosuppressive model of mice was made by intraperitoneal injection of cyclophos- phamide. The effect of CH50, a recombinant polypeptide of human fibronectin, on macrophages of mice was observed. The results showed that continuous intraperitoneal injection of CH50 could prevent the reduction of the number of monocytes in periphery blood and abdominal cavity by chemotherapeutic agents, enhance the metabolic activity and cytotoxicity of macrophages and augment the proliferation of splenocytes. The results suggested that CH50 is a product which could be used to improve the efficacy of chemotherapy of tumors.

Construction of Expressing Plasmids of Recombinant FN Polypeptides with Bifunctional-domain and the Characterization of the Products Expressed in E. Coli

Two expressing plasmids have been constructed and used to express two bifunctional-domain recombinant polypeptides of human fibronectin (FN) in E. coli. One was CH50 (Pro1239-Ser1515 of FN linked with Ala1690-Thr1960 of FN through Met) and the other was CH56 (Pro1239-Thr1960 of FN). Both of two polypeptides were capable of binding heparin and were purified by heparin-a-garose affinity chromatography. The purified products were capable of binding cells. The production of CH50 and CH56 polypeptides provided a fundamental basis for further study of the anti-metastatic function of recombinant fibronectin polypeptides.

Inhibitory Effect of Recombinant FN Polypeptide CH50 onthe Metastasis and Tumor Growth

The inhibition of CH50, a recombinant polypeptide of human fi- bronectin, on the formation of tumor metastases in vivo was studied by inocula-tion of melanoma B16/F1 cells by hypodermic or intraperitoneal injection, and the size and amount of tumor nodes after therapy were measured. In the treatment of hypodermic tumor, local injection of CH50 produced much better efficacy thandistance injection of CH50 did. The inhibitory effect of CH50 on the growth of tumor reached 50 %. In the treatment of peritoneal metastasis, the inhibition ofCH50 on metastases smaller than 1 mm was above 80 %, and above 50 % on metastases larger than 1 mm. A better efficacy was achieved if CH50 was used incombination with hydroxycamptothecine (HCPT ), a chemotherapeutic agent.CH50 displayed a strong inhibitory effect on the formation of small metastasis and growth of larger nodes of tumor, suggesting that CH50 plays a very important role in combined treatment of tumor therapy.

Inhibitory Effect of Recombinant Fibronectin Polypeptide CH50 on Invasion and Metastasis of Melanoma B16 Cells

In order to investigate the inhibitory effect and mechanism of recombinant polypeptide CH50 on invasion and metastasis of melanoma B16 cells, the recombinant polypeptide CH50 was separated and purified by ion exchange chromatographic technique. The melanoma B 16 cells treated with purified CH50 were cultured in vitro, the number was counted at 4, 24, 48 and 72 h and their morphological changes were observed in order to detect their adhesion and spreading abilities. In in vivo study, the melanoma B16 cells were labeled with CFSE and treated with CH50 and then they were injected into mice via mouse-tail veins. After 5 h, the lung tissues were fixed by frozen section. Accumulation and invasion abilities of B 16 cells on lung tissues were observed under the fluorescent microscopy. The results showed that the morphological character of B16 cells treated with CH50 changed greatly and the number of B16 cells treated with CH50 decreased significantly （P〈0.05）. The adhesion and spreading abilities of B16 cells treated with CH50 were weakened obviously and the metastasis foci on lung tissues reduced. It was concluded that the recombinant polypeptide CH50 inhibited invasion and metastasis of melanoma B16 cells on tissues and could be a prospective bio-product in tumor general therapy.

Augmentation of Recombinant Fibronectin Polypeptide CH50 on the Antitumor Function of Macrophages

We prepared an anti-metastatic polypeptide, recombinant fibronectinpolypeptide CH50, and finished the preliminary identification of its functions. In this paper, we studied the effect of this polypeptide on the function of macrophages. CH50 can significantly augment the production of nitric oxide(NO) by macrophages in a dose-dependent manner. The continuous presence of CH50 had a much stronger effect. In the presence of CH50, the cytotoxicity of macrophages to melanoma B16/F1 cells was significantly enhanced, and a stronger effect was obtained if CH50 was present continuously. CH50 polypeptide and IFN-r have a synergistic effect on the production of NO by macrophages and the cytotoxicity of macrophages on tumor cells. In the in vivo experiments, CH50 can inhibit the growth of tumor cells, and have a better effect in the presence of IFN-r. Our results suggest that recombinant fibronectin polypeptide CH50 has two functions: one is to inhibit the metastasis of tumor cells, and the other one is to augment the function of macrophages. And this polypeptide will be potentially useful in tumor therapy.

Investigation on the Purification and Expression of Cell Ⅰ－Hep Ⅱ Recombinant FN Polypeptide in E．Coli

An anti-metastatic polypeptide, bifunctional-domain (Cell Ⅰ -Hep Ⅱrecombinant polypeptide of human fibronectin. was expressed in E. coli and purified. The expression level was found to be about 20% 30 % of the total cell proteins. In BL21 (DE3)/T7, an E. coli expressing system, lactose can be used as an inducer to substitute IPTG thereby reducing the cost by several hundredfold and it is suitable for the large-scale preparation of recombinant FN polypeptide. Cell Ⅰ -Hep Ⅱ fragment is an alkaline polypeptide. In BL21 (DE3)/T7 expressing system' better isolation was achieved if DEAE-52, instead of CM-52,was used for ion-exchange chromatography. The purified product was obtained after heparin-agarose affinity chromatography following ion-exchange chromatography.

Comparative Study on the Inhibitory Effect of RecombinantFN Polypeptide CH50 and CH56 on the Metastasis ofMelanoma Cells

On the basis of preparation of anti-metastatic recombinant FN polypeptides, CH50 and CH56, we further studied the function of these polypeptides.The capacity of CH50 binding with melanoma cells (ED50 30 mM) was higher than that of CH56 (ED50 45 mM). Both of the polypeptides could significantly suppress the binding of melanoma B16 cells to laminin. There was no significant difference in the inhibitory effect between two polypeptides. In the experimental metastasis of melanoma cells, both of CH50 and CH56 could significantly inhibit the metastasis of the tumor cells, and reduce the number of lung metastasis by about 80%. Our results suggest that Ⅲ-11 and ED-A repeats influenced, to some extent, the binding capacity of bifunctional-domain polypeptide to cells, but did not affect the inhibition of the polypeptide on the metastasis of melanoma cells. The presence and connection of cell Ⅰ and Hep Ⅱ domains are the elements which determine the ability of recoinbinant FN polypeptides to inhibit the metastasis of tumor cells.

Construction and Expression of Eukaryotic Expressing Vector pCH510 of Polypeptide CH50 and Its Chemotaxis and Antitumor Function by in vivo Transfection

To construct an eukaryotic expressing vector that expresses CH50, a recombinant CellⅠ HepⅡ bifunctional domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmid in vivo , the plasmid was constructed by DNA recombination. Gene transfection was performed in vitro and in vivo . The expressed product was identified by Western blot. The chemotaxis after gene transfection in vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5′ terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3′ terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3.1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells and in vivo in mouse. The expression of the plasmid in vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor.

Effect of Fragment Asp1961-Glu1978 in Fibronectin on the Expression of Triple-domain Polypeptide in E. coli

Two plasmids were constructed and used to express two triple-domain recombinant polypeptide of human fibronectin (FN). The cDNAs in plasmids code for two polypeptides, CH62 (Pro1239-Ser1515 of FN linked with Ala1690 -Val2049 through Met) and CH63 (CH62 without Ile1850-Glu1978). The expression level of CH62 in E. coli was very low, but that of CH63 was very high.The results suggests that Asp1961-Glu1978 in FN is a key sequence influencing the expression of triple-domain polypeptide in E. Coli. After being dissolved and renatured, CH63 can be purified by heparin-agarose affinity chromatography.Both of the cell-binding domains in the recombinant polypeptide were functional.The production of CH63 provides a fundamental basis for further study of recombinant products with better anti-metastasis function.

Effect of Polypeptide CH50 on Macrophage Activation in vivo and Antitumor Function

The main features of CH50, a recombinant polypeptide of human fibronectin, activating macrophages in vivo and its anti tumor function were investigated. After injection of CH50 and(or) transfection of IFN γ gene in vivo , several kinds of factors produced by macrophages were determined and the growth of tumor in vivo was measured. CH50 could enhance the production of such factors as NO, TNF and IL 1 by macrophages, but the activation of macrophages was relatively slow when CH50 was used in vivo alone. CH50 and IFN γ could synergistically activate macrophages rapidly in vivo no matter whether the injection of CH50 or the transfection of IFN γ gene was performed first. Injection of CH50 alone inhibited the formation of tumor nodes in a dose dependent manner. Low dose of CH50 could strongly inhibit the formation of tumor nodes less than 1 mm, while high dose of CH50 could inhibit those more than 1 mm. A stronger inhibition on the growth of tumor in vivo was obtained by the synergistic effect of CH50 and IFN γ. CH50 and IFN γ, as double signal factors for activation of macrophages, will be potentially useful in tumortherapy.

Construction of Eukaryotic Expressing Vector pCH503 of CH50 and Its Chemotaxis and Anti-Tumor Function by Expression in vivo in Mice

An eukaryotic expressing vector that expresses CH50, a recombinant polypeptide of human fibronectin, in mice was constructed, and its chemotactic and anti-tumor function by in vivo gene transfection was investigated. The plasmid was constructed by recombination techniques. The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5'-terminal noncoding region and coding region of signal peptide of mouse IFN-γ cDNA at 5' side and 3'-terminal noncoden region of human FN cDNA at 3' side. The recombinant cDNA was inserted into plasmid pREP8. The resulted expressing plasmid was designated as pCH503. The macrophages transfected with pCH503 in vivo and cultured in vitro could produce CH50. The expressed product was identified by heparin-affinity chromatography and SDS-PAGE. By counting and Giemsa-staining of coeliac cells and histotomy and staining of muscle tissue, the chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle. The inhibition of gene transfection of pCH503 on melanoma was observed in mice. The number of melanoma 'nodes in mice was reduced by 50 % - 60 % after coeliac transfection with pCH503. The pCH503, an eukaryotic expressingvector of CH50, can express in the in mice. The transfection of pCH503 in vivo has the chemotaxis on immune cells and can inhibit the formation of tumor nodes, suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor.