Charles H. Evans

Tumor necrosis factor-? promotes human papillomavirus (HPV) E6/E7 RNA expression and cyclin-dependent kinase activity in HPV-immortalized keratinocytes by aras-dependent pathway

Tumor necrosis factor‐α (TNF‐α) inhibits growth of normal cervical keratinocytes but stimulates proliferation of human papillomavirus (HPV)–immortalized and cervical carcinoma–derived cell lines when mitogens such as epidermal growth factor (EGF) or serum are depleted. Current work identifies the mechanism of growth stimulation. TNF‐α promoted cell cycle progression by increasing expression of HPV‐16 E6/E7 RNAs and enhancing activity of cyclin‐dependent kinase (cdk)2 and cdc2 after 3 d. Increased kinase activity was mediated by upregulation of cyclins A and B and decreases in cdk inhibitors p21waf and p27kip. TNF‐α stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV‐16 E6/E7 and cyclin A RNAs. To define which components of the EGF receptor signaling pathway were important, HPV‐immortalized cells were transfected with activated or dominant negative mutants of Ha‐ras, raf, or MAPKK. Expression of activated Ha‐ras maintained HPV‐16 and cyclin gene expression and promoted rapid growth in the absence of EGF. Furthermore, ras activation was necessary for TNF‐α mitogenesis as transfection with a dominant negative ras mutant (Asn‐17) strongly inhibited growth. Thus, activation of ras promotes expression of HPV‐16 E6/E7 RNAs, induces cyclins A and B, and mediates growth stimulation of immortal keratinocytes by TNF‐α. These studies define a pathway by which ras mutations, which occur in a subset of cervical cancers, may contribute to pathogenesis. Mol. Carcinog. 27:97–109, 2000. Published by Wiley‐Liss, Inc.

Endothelial cell growth supplement: a cell cloning factor that promotes the growth of monoclonal antibody producing hybridoma cells

The efficiency of endothelial cell growth supplement (ECGS), a commercially marketed extract of bovine neural tissue, human endothelial cell supernatant (HECS) derived from freshly isolated endothelial cells, and feeder layers of murine peritoneal cells (PEC), were compared for their ability to support cell fusion, clonal growth, and monoclonal antibody production of murine hybridoma cells. ECGS at 25-100 micrograms/ml was similar to a 1:5 dilution of HECS in supporting the growth of hybridoma colonies; both ECGS and HECS were superior to PEC feeder cells. Furthermore, hybridomas cloned in ECGS produced anti-lymphotoxin antibodies. The commercial availability and stability of ECGS together with its ability anti-lymphotoxin antibodies. The commercial availability and stability of ECGS a superior growth supplement for the fusion and growth of hybridoma cells in monoclonal antibody production.

Immunologic approaches to the treatment of human cancer based on a guinea pig model

SummaryLocal immunotherapy is a form of cancer treatment where exogenous antigen is introduced into the area of the tumor. Under favorable circumstances, the perfused tumor regresses, systemic tumor-specific transplantation immunity is augmented, and distant microscopic metastases regress. Successful local immunotherapy requires an immunologically competent host, small tumor burden, and tumor located usually in the skin. A wide variety of biologic agents are capable of promoting local immunotherapy. BCG has been most widely studied. The antitumor activity of two different preparations of the Tice substrain of BCG were compared. No significant differences in antitumor activity were found. Alternative approaches to intralesional injection were sought. Intradermal injection of BCG adjacent to dermal tumors, prior to surgery, led to eradication of axillary metastases and to the development of tumor-specific transplantation immunity.Successful local BCG immunotherapy is a by-product of the host response to BCG infection. Involved are lymphocytes specifically sensitized to mycobacterial antigens, lymphocyte mediators, and macrophages which develop the capacity to kill tumor cells. Tumor-cell killing may be mediated by exocytosis of macrophage lysosomes into tumor cells. Complete and permanent tumor eradication probably requires the development of tumor-specific transplantation immunity mediated by sensitized lymphocytes. Local infection of the tumor may augment the development of this tumor-specific immunity.

Lymphotoxin — An immunologic hormone with anticarcinogenic and antitumor activity

SummaryLymphotoxin, a glycoprotein lymphokine, possesses direct cytostatic and cytolytic activity for a wide variety of tumor cells. Lymphotoxin has been detected in the plasma from patients with neoplasia as well as individuals with various other diseases. Lymphokine preparations containing lymphotoxin activity can inhibit tumor growth in vivo, and recently lymphotoxin has been shown to irreversibly prevent carcinogen-induced morphological transformation during carcinogenesis. The anticarcinogenic activity of lymphotoxin is more potent than its tumor growth-inhibitory activity; the latter activity, furthermore, is frequently cytostatic rather than cytolytic. Lymphotoxin can also induce an increased susceptibility of tumor cells to cytolytic destruction by natural killer cells. The anticarcinogenic and tumor growth-inhibitory activities of lymphotoxin may be effected through alterations in cellular glycoproteins as the lymphokine stimulates incorporation of glucosamine into larger-molecular-weight glycoproteins in normal cells but inhibits incorporation of glucosamine into the glycoproteins in tumor cells.

Lymphotoxin cytotoxicity, a combination of cytolytic and cytostatic cellular responses

Guinea pig lymphotoxin inhibits the growth of mouse alpha L929 and guinea pig 104C1 tumor cells with lethal doses (LD 50s) of 0.4 and 200 units lymphotoxin/ml, respectively, in a colony inhibition assay. Refeeding with lymphotoxin-free medium is followed by resumption of 104C1 but not alpha L929 cell growth. This suggests that growth inhibition of alpha L929 is primarily due to cytolytic mechanisms, while that of 104C1 cells is due to cytostatic mechanisms. This is confirmed by radionuclide (3H, 51Cr, and 75Se) release assays with LD 50s of 1.0, 1.9, and 2.4 units lymphotoxin/ml, respectively, for alpha L929 cells, whereas as many as 100 units lymphotoxin/ml produce no radionuclide release from 104C1 cells. The L cell variant L929M is 10-fold more resistant to lymphotoxin colony inhibitory activity and 40-300-fold more resistant to cytolytic lymphotoxin activity as measured by the three radionuclide release assays than are alpha L929 cells. L929 and 2071 L cell variants are more resistant as a result of smaller cytolytic and cytostatic responses and some tumor cells, such as one strain of L1210 mouse leukemia cells, exhibit no detectable cytolytic or cytostatic responses to 100 units of guinea pig lymphotoxin. These observations demonstrate that the divergent susceptibilities of different cells to lymphotoxin result in part from constitutive variations in cellular cytolytic and reversible cytostatic responses to lymphotoxin.

Expression of initiated and promoted stages of irradiation carcinogenesis in vitro

Evidence obtained utilizing X-irradiation and ultraviolet light irradiation with 12-O-tetradecanol-phorbol-13-acetate (TPA) for the induction of transformation of Syrian hamster embryo cells (HEC) suggests that there is more than 1 type of initiation of carcinogenesis. Initiated HEC are phenotypically transformed in the presence of TPA (15--20-fold enhancement) but are not necessarily committed to be transformed because some colonies reverted to normal morphology when TPA was removed. Both initiated and promoted HEC are also sensitive to hamster lymphotoxin. The frequency of transformation was reduced by at least 70%. The ability to recognize discrete morphologically altered colonies after TPA addition in vitro indicates that promotion is not merely escape from growth control.

Species specificity of guinea pig and human lymphotoxin colony inhibitory activity

The species specificity of lymphotoxin colony inhibitory activity was measured by in vitro colony growth of neoplastic and nonneoplastic cells in the presence of lymphotoxin from the same or from a xenogeneic species. Growth of guinea pig, rat, and mouse neoplastically transformed cells was inhibited by guinea pig lymphotoxin. Colony formation of benzo(a)pyrene transformed 104C1 guinea pig cells was inhibited 45% with lymphotoxin at 42 units/ml medium; nontransformed 104 and 118 guinea pigs cell were not inhibited. Alpha L929, a spontaneously transformed mouse cell, was completely inhibited. Dimethylbenz(a)anthracene (DMBA) 3T3-D and R4-2 mouse transformed cells were inhibited 40 and 100%, respectively, but parental nontransformed 3T3 cells were not inhibited. DMBA transformed R-2 and R-6 rat cells were inhibited 20 and 40%, respectively, but secondary nontransformed fetal rat cells were unaffected. In contrast, normal human xeroderma pigmentosum, Lesch-Nyhan and 1220 fibroblast cells were inhibited 50-60%. No inhibition of HeLa or RPMI 2650 human tumor cells occurred. HeLa and RPMI 2650 cells, however, were inhibited 30 and 60%, respectively, by human lymphotoxin, but normal human and tumorigenic guinea pig cells were unaffected. Thus, guinea pig and human lymphotoxin exhibit species specific and differential cytotoxicity mechanisms for normal and tumor cells.

Biological and physicochemical characterization of keyhole limpet hemocyanin-lnduced guinea pig lymphotoxin

Abstract Biological and physicochemical properties of keyhole limpet hemocyanin (KLH)-induced guinea pig lymphotoxin (LT) were compared to those of LT induced by another antigen, ovalbumin, or by the mitogen, phytohemagglutinin (PHA). Mitogen- and antigen-induced LT had similar colony inhibitory activities toward guinea pig and rat tumorigenic cells but none toward nontumorigenic cells. Conversely, human nontumorigenic but not tumorigenic cells were inhibited by the three LT preparations. The molecular weight of each LT was 45,000 daltons on gel filtration chromatography but sucrose density gradient ultracentrifugation indicated greater heterogeneity. The KLH-, ovalbumin-, and PHA-induced LTs were resolved into multiple peaks upon preparative column isoelectric focusing (IEF) with IpHs in the range of 4.5 to 5.3. Analytic IEF of the KLH-induced LT revealed the presence of three distinct biologically active peaks with IpH at 4.77, 5.02, and 5.20. LT activity was stabilized during preparative IEF by inclusion of 0.1% polyethylene glycol (4000 molecular weight) in the ampholine gradient. This resulted in 90% recovery of LT activity with a concomitant removal of 96% of the KLH used as the antigenic stimulus for LT production. Fractionation of LT activity during diafiltration and IEF when exogenous proteins were included as protective agents altered the number and the isoelectric characteristics of the peaks obtained suggesting that LT was being bound by the added protein. Colony inhibitory (cytostatic) and 3 H-release (cytolytic) LT activities copurified during fractionation. Thus, this study demonstrated that high yields (approximately 50% of the starting material) of LT, free of antigen or exogenous serum proteins, can be obtained by a combination of diafiltration and IEF performed in the presence of polyethylene glycol.

Cytokine Modulation of Immune Defenses in Cervical Cancer

Human papilloma viruses (HPV) are major factors in the etiology of cervical cancer. Natural killer (NK) lymphocytes, an important defense against viral diseases, are present in most HPV-associated lesions and cervical intraepithelial neoplasia. HPV positive cervical cancer cells and HPV-immortalized human cervical epithelial cells which possess properties similar to cervical dysplasia, however, are resistant to NK but are sensitive to lymphokine-activated killer (LAK) lymphocyte lysis. Sensitivity can be enhanced by treatment of cervical cells with leukoregulin, a cytokine secreted by lymphocytes. Combination treatment with leukoregulin and a chemotherapeutic drug, e.g. cisplatin, further enhances sensitivity of HPV-infected cells to LAK lymphocyte lysis. In contrast, gamma-interferon treatment of cervical cells can result in decreased sensitivity to LAK lysis illustrating the potential balance cytokines can exert in the immunologic control of cervical cancer.

Leukoregulin up-regulation of tumor cell sensitivity to natural killer and lymphokine-activated killer cell cytotoxicity

SummaryThe present investigation demonstrates that leukoregulin, a cytokine secreted by natural killer (NK) lymphocytes up-regulates the sensitivity of tumor cells to lymphokine-activated killer (LAK) cell cytotoxicity. It has been previously established that leukoregulin increases the sensitivity of sarcoma, carcinoma and leukemia cells to natural killer (NK) cell cytotoxicity. Tumor cells were treated with leukoregulin for 1 h at 37°C and tested for sensitivity to NK and LAK cytotoxicity in a 4-h chromiumrelease assay. NK-resistant Daudi, QGU and C4-1 human cervical carcinoma cells became sensitive to NK cytotoxicity after leukoregulin treatment, and their sensitivity to LAK was increased two- to sixfold. Y-79 retinoblastoma cells, which are moderately sensitive to NK and very sensitive to LAK, became increasingly sensitive (two- to fourfold) to both NK and LAK cell cytotoxicity. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant interleukin-1 (α and β), recombinant interferon γ, recombinant tumor necrosis factor or combinations of the latter two failed to up-regulate tumor cell sensitivity to NK and LAK cell cytotoxicity. However, treatment with recombinant interferon γ for 16–18 h, GM-CSF and interleukin-1β for 1 h induced a state of target cell resistance to both NK and LAK cell cytotoxicity. Leukoregulin may have an important physiological function in modulating NK and LAK cell cytotoxicity by increasing the sensitivity of target cells to these natural cellular immunocytotoxicity mechanisms.