Expression of human growth hormone in cultured mouse fibroblasts

The new system for the transfer and expression of foreign genes based on retroviral vectors pPS-neo, conferring neomycin resistance was constructed. The BALB/c mouse cell lines producing highly active human growth hormone (more than 7 micrograms/ml into culture medium) were constructed using these vectors. An antibody column was used to purify the growth hormone from cell culture medium. Possibilities of producers to be applied for gene therapy are discussed.     

Level of protein kinase C activity correlates directly with resistance to adriamycin in murine fibrosarcoma cells

In this report, we demonstrate a direct correlation between protein kinase C (PKC) activity and adriamycin (ADR) resistance in mouse fibrosarcoma cells. PKC activity was measured in four murine UV-2237M fibrosarcoma cell lines that differed in the degrees to which they expressed resistance to ADR, which is an inhibitor of PKC. A comparison of the four cell lines revealed a positive correlation between the level of PKC activity and resistance to ADR. Incubation of the cells with the PKC inhibitor H-7 produced a partial reversal of ADR resistance. Taken together, these results suggest a role for PKC in the mechanism of ADR resistance.     

Destruction of tumor cells by monokines released from activated human blood monocytes: evidence for parallel and additive effects of IL-1 and TNF

Summary The incubation of human peripheral blood monocytes with endotoxins activates the cells to lyse tumorigenic targets directly and also induces the production and release into the culture medium of factors that produce lysis of mouse-transformed fibroblasts L-929 (tumor necrosis factor (TNF)-sensitive) and human A-375 melanoma cells (interleukin-1 (IL-1)- and TNF-sensitive). Immunoblotting analysis revealed that the culture medium of endotoxin-activated but not of control monocytes contained both IL-1 and TNF with a molecular weight of 17,000 daltons each. TNF activity was determined by lysis of L-929 cells, and IL-1 activity was measured by the proliferation of D-10 cells. The production of IL-1 and TNF was concentration-dependent, and the amounts of these monokines were paralleled. The antitumor activity of the culture supernates from endotoxin-treated monocytes was significantly decreased by incubation with heterologous antisera to IL-1, TNF, or both. Recombinant human IL-1 and TNF were used in parallel experiments and as positive controls. Each monokine used produced cytotoxic effects in susceptible targets. The combination of IL-1 and TNF, which more likely resembles culture supernates of activated macrophages, produced an additive antitumor cytotoxicity effect.     

Tumor necrosis factor and IL-1 associated with plasma membranes of activated human monocytes lyse monokine-sensitive but not monokine-resistant tumor cells whereas viable activated monocytes lyse both

The purpose of our study was to determine some of the mechanisms involved in macrophage-mediated lysis of tumorigenic cells. A375 human melanoma cells (A375-R) resistant to lysis mediated by TNF and IL-1 were selected from the TNF- and IL-1-sensitive A375 parental melanoma cells subsequent to continuous (2 mo) exposure to rTNF. Peripheral blood monocytes isolated by centrifugal elutriation from healthy donors were incubated with rIFN-gamma and muramyl dipeptide, with a lipoprotein derived from Escherichia coli (CG-31362) or with LPS for 24 h. These activated monocytes lysed both the A375 (monokine-sensitive) and A375-R (monokine-resistant) melanoma cells. Activated tumoricidal macrophages fixed in 2% paraformaldehyde lysed only the TNF- and IL-1-sensitive A375 cells. These fixed monocytes contained both IL-1 and TNF activities as determined by D10 cell proliferation and L929 cytolysis assays, respectively. Nearly identical results were obtained with preparations of plasma membranes from activated human monocytes. Anti-IL-1 and/or anti-TNF sera neutralized the cytolysis of tumor cells mediated by free monokines, by fixed monocytes, or by plasma membrane preparations. In contrast, anti-TNF and/or anti-IL-1 sera did not inhibit tumor cell lysis by viable activated monocytes. We conclude that IL-1 and TNF molecules associated with the plasma membranes of activated monocytes mediate lysis of susceptible target cells. However, because activated monocytes lysed IL-1-and TNF-resistant target cells, molecules other than these monokines must also be involved in the antitumor activity of monocytes.     

Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice

We examined whether fibroblasts from subcutaneous, colon or lung tissues of nude mice influence the invasive potential of highly metastatic human colon carcinoma KM12SM cells. Primary cultures of nude mouse fibroblasts from skin, lung and colon were established. Invasive and metastatic KM12SM cells were cultured alone or with fibroblasts. Growth and invasive properties of the KM12SM cells were evaluated as well as their production of gelatinase activity. KM12SM cells were able to grow on monolayers of all three fibroblast cultures but did not invade through skin fibroblasts. The conditioned media of KM12SM cells cocultured with skin, colon or lung fibroblasts were examined for the presence of type IV collagenase (gelatinase). KM12SM growing on plastic and on colon or lung fibroblasts produced significant levels of latent and active forms of 64 kDa type IV collagenase, whereas KM12SM cells cocultivated with nude mouse skin fibroblasts did not. In contrast, human squamous cell carcinoma A431 cells produced significant levels of collagenase type IV when cocultured with nude mouse skin fibroblasts, a tissue they invaded and completely penetrated. Incubation of KM12SM cells in serum-free medium containing recombinant human interferon-beta (fibroblast interferon) was associated with significant reduction in gelatinase activity. Since the production of type IV collagenase by human colon cancer cells is specifically inhibited by mouse skin fibroblasts but not by colon or lung fibroblasts the data suggest that organ-specific fibroblasts can influence the invasive and metastatic properties of KM12SM cells.     

In situ activation of mouse macrophages and therapy of spontaneous renal cell cancer metastasis by liposomes containing the lipopeptide CGP 31362

Summary We determined whether the intravenous administration of multilamellar vesicle liposomes (MLV) containing a lipopeptide analogue of a fragment from the cell wall of gram-negative bacteria (CGP 31 362) can render BALB/c mouse alveolar macrophages tumoricidal in situ and reduce the incidence of spontaneous lung metastasis of syngeneic renal carcinoma (RENCA) cells. Alveolar macrophages (a) incubated in vitro with MLV containing CGP 31 362 (MLV-31 362) and (b) harvested from mice injected i.v. with MLV-31 362 were rendered cytotoxic against the RENCA cells. Maximum cytotoxic activity of the macrophages was induced by injecting 5 µmol MLV consisting of 250 mg phospholipids and 0.5 mg CGP 31 362. The single i.v. injection of 5 µmol MLV-31 362 produced activation of macrophages that lasted for up to 4 days. Repeated i. v. injections of MLV-31 362 produced a continuous antitumor activity in alveolar macrophages. To study the lipopeptide's effects on metastasis, we injected the left kidneys of BALB/c mice with RENCA cells. The kidney with growing tumor was resected 10 days later and, after a further 2 days, groups of mice were injected i.v. with MLV-31 362 or with MLV-HBSS (twice weekly for 3 weeks). Treatment with MLV-31 362 significantly decreased the median number of spontaneous lung metastases. These data demonstrate that the systemic administration of MLV-31 362 can activate murine lung macrophages in situ and reduce the incidence of spontaneous RENCA lung metastases.     

Distribution and fate of free and liposome-encapsulated [3H]nor-muramyl dipeptide and [3H]muramyl tripeptide phosphatidylethanolamine in mice

The pharmacokinetics and metabolism of i.v. administered free (unencapsulated) or liposome-encapsulated hydrophilic [3H]-labeled nor-muramyl dipeptide (nor-MDP) and lipophilic [3H]-labeled muramyl tripeptide phosphatidylethanolamine (MTP-PE) were evaluated. In addition we also examined the distribution and fate of these immunomodulators subsequent to intranasal (i.n.) administration. Unique patterns of circulatory clearance, organ distribution, metabolism, and excretion were observed for each of the four preparations. Nor-MDP in saline was rapidly cleared from the circulation and excreted in the urine as intact molecules. MTP-PE dissolved in saline was cleared from the circulation at a slow rate and found within various organs as intact MTP-PE, lyso-MTP-PE, and MDP. Following the i.v. administration of nor-MDP or MTP-PE in liposomes, patterns of clearance and organ distribution corresponded to that of liposome distribution, i.e., the reticuloendothelial system. Extensive dissociation of hydrophilic nor-MDP from the carrier liposomes occurred, and the immunomodulator was recovered in the urine. In contrast, MTP-PE entrapped in liposomes was retained in target organs for the duration of the study. The i.n. instillation of radiolabeled nor-MDP or MTP-PE was associated with the accumulation of these immunomodulators in the brain. Our results demonstrate the feasibility of targeting hydrophilic and lipophilic immunomodulators to cells of the macrophage-histiocyte series.     

A microassay for the rapid and selective binding of cells from solid tumors to mouse macrophages

Summary A microassay was developed to study the rapid binding characteristics of murine macrophages activated by gamma interferon and muramyl dipeptide to adherent neoplastic or nonneoplastic target cells. The binding of tumor cells to both activated and nonactivated macrophages was time- and temperature-dependent, and independent of tumor cell type. Activated macrophages bound more tumor cells than nonactivated macrophages. The initial binding of macrophages to target cells did not necessarily lead to lysis. First, primed macrophages bound tumor cells but did not lyse them, and second, nonactivated macrophages bound nontumorigenic cells without subsequent lysis. The rapid binding assay described here could prove useful in investigating the recognition mechanism(s) between macrophages and tumor cells derived from solid primary and metastatic cancers.     

Potentiation of direct antitumor cytotoxicity and production of tumor cytolytic factors in human blood monocytes by human recombinant interferon-gamma and muramyl dipeptide derivatives

Summary We investigated whether human peripheral blood monocytes isolated by centrifugal elutriation from healthy donors could be acitivated to become tumoricidal and release tumor cytolytic factor (TCF) subsequent to incubation with recombinant human interferon-gamma (r-IFN-) or a derivative of muramyl dipeptide (nor-MDP), or both. Blood monocytes incubated in endotoxin-free medium containing up to 1000 U/ml of r-IFN- or in medium containing less than 1 g/ml of nor MDP were not activated to lyse radiolabeled allogeneic human tumor cells. In contrast, the incubation of monocytes with various dose combinations of r-IFN- and nor-MDP generated significant direct cytotoxic activity as well as production of TCF. Preincubation of the r-IFN- and nor-MDP mixture with polymyxin B did not inhibit the synergism, thus ruling out the possibility that the process was due to endotoxin contamination. TCF harvested from monocyte culture supernatants was cytolytic against five allogeneic tumor targets, but not against a nontumorigenic cell line. Collectively, the data demonstrate that r-IFN- can prime human blood monocytes to allow their activation by synthetic nor-MDP.On leave from the Department of Internal Medicine, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770, Japan