Paul L. Hermonat

Effects of irradiation on the expression of major histocompatibility complex class I antigen and adhesion costimulation molecules ICAM-1 in human cervical cancer

PURPOSE We initiated studies to analyze the effects of high doses of gamma irradiation on the surface antigen expression of MHC Class I, Class II, and ICAM-1 on human cervical carcinoma cell lines. METHODS AND MATERIALS The expression of surface antigens (MHC Class I, Class II, and ICAM-1) was evaluated by FACS analysis on two cervical cell lines at different time points, following their exposure to high doses of gamma irradiation (i.e., 25.00, 50.00, and 100.00 Gy). RESULTS The CaSki and SiHa cervical cancer cells we analyzed in this study expressed variable levels of MHC Class I and ICAM-1 antigens, while Class II surface antigens were not detectable. Whereas irradiation doses of 25.00 Gy were not sufficient to totally block cell replication in both cell lines, exposure to 50.00 or 100.00 Gy was able to completely inhibit cell replication. Range doses from 25.00 to 100.00 Gy significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation but were unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). Importantly, such upregulation was shown to be dose dependent, with higher radiation doses associated with increased antigen expression. Moreover, when the kinetic of this upregulation was studied after 2 and 6 days after irradiation, it was shown to be persistent and lasted until all the cells died. CONCLUSIONS These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy.

The effects of irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical cancer.

PURPOSE Studies were designed to analyse the effects of high doses of gamma-irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical carcinoma cell lines. MATERIALS AND METHODS The expression of heat shock protein gp96 was evaluated at the transcriptional (Northern blot) and post-transcriptional levels (Western blot) in two human cervical carcinoma cell lines following exposure to high doses of gamma-irradiation. RESULTS Doses of gamma-irradiation ranging from 25 to 100 Gy significantly and consistently increased the expression of heat shock protein gp96 on CaSki and HT-3 cervical cancer cells. The increase in the amount of protein was due to transcriptional up-regulation of this gene. Radiation doses unable to inhibit completely cell replication in the totality of tumour cells (i.e. 25 Gy), as well as higher (fully lethal) doses of irradiation (i.e. 50 to 100 Gy), were shown to up-regulate significantly the expression of heat shock protein gp96 mRNA in a dose-dependent manner. CONCLUSIONS Recently, gp96 molecules have been implicated in the presentation of endogenous and viral antigens. A number of key elements in this pathway, including major histocompatibility complex (MHC) class I molecules as well as adhesion/co-stimulation molecules such as ICAM-1, are known to be sensitive to irradiation effects. The results show that radiation can also increase the expression of other immunologically important cell molecules such as a tumour rejection antigen (heat shock protein gp96) in human cervical cancer. Such findings may partially explain the increased immunogenicity of tumour cells following irradiation and further support a role for local radiation therapy as a powerful biologic response modifier.

Secretion of vascular endothelial growth factor in adenocarcinoma and squamous cell carcinoma of the uterine cervix.

OBJECTIVE To determine whether major differences in vascular endothelial growth factor secretion exist between adenocarcinomas of the uterine cervix compared with squamous cell carcinomas. METHODS The secretion of vascular endothelial growth factor by eight fresh cervical cancer cell preparations (four adenocarcinomas and four squamous cell carcinomas) and four established squamous cell lines was evaluated using a sensitive enzyme-linked immunosorbent assay in vitro. RESULTS All cervical tumors secreted significant amounts of vascular endothelial growth factor, and no significant differences between fresh and established squamous cell lines were detectable. In contrast, a highly significant difference in vascular endothelial growth factor secretion was noted between fresh adenocarcinomas (mean = 2712, range between 1700 to 3500 pg/mL/10(5) cells/48 hours) when compared with fresh squamous (mean = 575, range between 200 to 950 pg/mL/10(5) cells/48 hours) or established squamous cervical carcinoma cell lines (mean = 712, range between 400 to 1000 pg/mL/10(5) cells/48 hours) (F-test, P< or =.001). CONCLUSION These data strongly suggest that major differences in the secretion of vascular endothelial growth factor exist between squamous cell carcinoma and adenocarcinomas of the uterine cervix. Therefore, at least some of the differences in the natural biologic behavior of these two histologic types of cervical cancer, including the propensity for earlier lymphatic and hematogenous metastasis as well as the lower response to radiation treatment, could be related to major differences in the secretion of this powerful angiogenic and immunosuppressive cytokine.

The spermicide nonoxynol-9 does not inactivate papillomavirus

Vaginal spermicides are effective contraceptive, and are also capable of inactivating many sexually transmitted pathogens by their detergent effect on bacterial cell membranes and viral envelopes. A 5% concentration of nonoxynol-9, the most frequently used active ingredient of spermicides, was tested for its ability to reduce the transforming activity of bovine papillomavirus type 1 (BPV-1), and the infectivity of BK virus (BKV) and cytomegalovirus (CMV). Nonoxynol-9 markedly reduced the infectivity of CMV, an enveloped virus, but did not significantly affect the activity of the nonenveloped viruses BPV-1 and BKV. Papillomavirus infections are strongly implicated in the etiology of cervical cancer. The reported protective effect of vaginal spermicides against cervical cancer is very likely not mediated by direct inactivation of papillomaviruses by the spermicide.

Development and therapeutic effect of adoptively transferred T cells primed by tumor lysate-pulsed autologous dendritic cells in a patient with metastatic endometrial cancer

We describe a 65-year-old woman with a large surgically unresectable and chemoresistant liver metastasis of endometrial carcinoma who was treated by infusion with peripheral blood T cells stimulated with tumor lysate-pulsed autologous dendritic cells (DC). Extensive in vitro characterization of the DC-activated T cells included phenotypic analysis, cytotoxicity, and intracellular cytokine secretion. High cytotoxicity was observed against autologous tumor cells, but not against NK-sensitive K562 cells, autologous Con-A lymphoblasts, or autologous Epstein-Barr virus-transformed lymphoblastoid cells. Blocking studies demonstrated that lytic activity was HLA class I restricted. Two-color flow cytometric analysis revealed that a significant proportion of CD8+ T cells was also CD56+, and analysis of intracellular IFN-γ and IL-4 expression suggested a type 1 cytokine bias. The patient was treated by three infusions of tumor-specific T cells at 3- to 4-week intervals, and in vivo distribution of the T cells was followed by 111In oxine labeling and serial gamma camera imaging. Tumor localization and accumulation of labeled lymphocytes was consistently detected at serial time points following each injection. However, deep infiltration of the large tumor mass by activated T cells was minimal, as evaluated in 3 dimensions by single photon emission computerized tomography (SPECT) imaging. Transient serum increases of the tumor marker lactate dehydrogenase (LDH), were detectable after each injection. Similar posttreatment elevations were seen for serum uric acid and potassium. Clinically, stabilization of the large liver metastasis was obtained during treatment. Collectively, these results indicate that tumor-specific CD8+ cytotoxic T-cell responses can be generated in patients with endometrial cancer, and suggest that T-cell immunotherapy may be of therapeutic value in patients harboring metastatic disease.

Effects of retinoic acid combined with interferon-gamma on the expression of major-histocompatibility-complex molecules and intercellular adhesion molecule-1 in human cervical cancer

Retinoids and interferons are important regulators of human epithelial cell differentiation and have been successfully used in the clinical treatment of HPV‐involved cervical cancer. In this study, 2 HPV‐positive human cervical‐carcinoma cell lines were analyzed for their surface expression of MHC‐Class‐I, MHC‐Class‐II and ICAM‐1 surface antigens before and after exposure to all‐trans retinoic acid, interferon‐gamma and the combination of the 2 compounds. In addition, the effects on HLA‐Class‐I‐mRNA expression were evaluated after such treatments. Both cell lines expressed MHC‐Class‐I molecules, and their levels were markedly up‐regulated after exposure to IFN‐γ. Similarly, MHC‐Class‐II and ICAM‐1 antigens were either induced or significantly up‐regulated by IFN‐γ. Exposure to all‐trans retinoic acid was also able to significantly increase the expression of MHC‐Class‐I and ICAM‐1 antigens as compared with untreated tumor cells. However, unlike IFN‐γ, retinoids were not able to induce the expression of HLA‐Class‐II surface antigens. Exposure to the combination of IFN‐γ plus retinoic acid significantly up‐regulated (in an additive manner) HLA‐Class‐I and ICAM‐1 molecules as compared with the levels obtainable after exposure to IFN‐γ alone. Finally, Northern‐blot analysis of HLA‐Class‐I‐mRNA expression confirmed that the activity of both of these biologic response modifiers was at transcriptional level. These data indicate that the combination of these modalities could induce an additive effect on the expression of immunologically important surface antigens on human cervical‐cancer cells. These findings, together with the known anti‐proliferative effects mediated by retinoids and IFN‐γ on tumor cells, further support the combination of these agents in the treatment of pre‐invasive and invasive human cervical cancer. Int. J. Cancer 75:254–258, 1998.

Inactivation of Papillomavirus by Low Concentrations of Povidone-Iodine

Background and Objectives A recent report by Hermonat et al showed that nonoxynol 9 is completely inactive against bovine papillomavirus, which is very closely related to human papillomavirus. Finding a vaginal microbicide active against human papillomavirus to reduce the risk of sexual transmission of human papillomavirus would be desirable. Goal of This Study To determine whether povidone-iodine is active in vitro against bovine papillomavirus. Methods A bovine papillomavirus-1 stock prepared by extraction of a fibropapilloma was treated with various concentrations of povidone-iodine. The virus/povidone-iodine samples were incubated at 37°C for 15 minutes and then placed on contact-inhibited cells of mouse fibroblast line C127 in 10-cm tissue culture dishes for the transformation assay. At 2 weeks post-infection, oncogenic foci induced by bovine papillomavirus appeared and were counted after the cells were fixed with 4% formaldehyde and stained with methylene blue. Results Approximately 90% inactivation of papilloma virus was demonstrated with exposure to 0.1% povidone-iodine, and 99.9% inactivation was seen at 0.3%. Conclusions The concentrations of povidone-iodine that were effective in this study are lower than concentrations in available over-the-counter preparations of povidone-iodine. Additional research is needed to verify whether papillomavirus is susceptible to other, more acceptable agents. Clinical trials may be warranted to determine whether povidone-iodine or other agents would reduce the rate of sexual transmission of the human papillomavirus strains associated with cervical cancer.

EXPRESSION OF SURFACE CD40 AND IMMUNOCYTOCHEMICAL ACTIN-BUNDLING PROTEIN FASCIN IN DENDRITIC CELLS FROM MULTIPLE MYELOMA TREATED WITH RETINOIDS DURING THEIR DIFFERENTIATION IN VITRO

Dear Editor: Antigen presenting ceils (APCs) are bone marrow-derived cells with the ability to induce primary immune responses. Dendritic cells (DCs) are considered one of the most important APCs as distinguished by their potency and ability to initiate primary T-cell responses in vitro and in vivo (Romani et al., 1996; Banchereau and Steinman, 1998; Sokoloff et al., 1999; Chiriva-Internati et al., 2001). DCs capture antigens in the peripheral tissues and travel to the T-cell zones of the lymphoid organs where they stimulate naive CD4 and CD8 T-cells. During this migration, DCs differentiate from an immature state, in which they can capture antigens and have low stimulatory capacity, to a mature state, in which they are no longer capable of taking up antigens but have an increased T-cell stimulatory capacity (Rescigno et al., 1999). The high stimulatory capacity of activated DCs is because of increased major histocompactibility restriction (MHC) expression and costimulatory molecules, such as CD40, CDS0, and CD86, the production of different cytokines, and a change in chemokine receptor expression and chemokine production. A wide range of inflammatory stimuli and infection agents such as tumor necrosis factor-c~ (TNF-c~), and regulators of differentiation such as retinoids can induce the maturation of DCs. In the past few yr, some of the molecular mechanisms of the different and complex interactions between a variety of cell types of the immune system, such as DCs have been explained, and several molecules found to be important for these interactions have been identified. Among these molecules, the CD40-CD40 ligand (CD40-CD40L) is well studied (Grewal and Flavell, 1997). CD40 is a 50-kDa transmembrane glycoprotein expressed on APCs, such as B lymphocytes, monocytes, and DCs (Shinagawa et al., 1995). CD40 ligand is a 33-kDa, type II membrane protein that is a member of the TNF gene super-family. The CD40L is of primary importance for the initiation of antigen-specific T-cell responses (Fanslow et al., 1994); it is expressed on CD4 T-cells only when they are activated. The reciprocal communication between APCs and T lymphocytes through CD40-CD40L, therefore, seems to affect not only APC functions but also clonal expression and affector functions of CD4 T-cells (Shinagawa et al., 1995). A number of functional studies have shown that CD40 maturation signals are critical for the direction of cellular immune responses in cancer immunity. In this study we investigated the effect of 13-cis-retinoic acid (RA) and all-trans RA on the kinetics of expression of CD40 antigen and on selected costimulatory molecules on human DCs cells from multiple myeloma (MM) during their differentiation in vitro. Further, we analyzed the effect of RA on the immunocytochemical expression of the actin-bundling protein fascin. Fascin bundles are actin microfilaments within dynamic cellular structures such as microspikes, stress fibers, and membrane ruffles. Fascin overexpression induces membrane protrusions and increased cell motility, and is highly expressed in various transformed cells, and in specialized normal cells including neuronal, endothelial, and DCs (Hu et al., 2000). Briefly, 42 ml of peripheral blood from five MM patients were centrifuged on Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) to obtain peripheral blood mononuclear cells. These cells were then plated (1 • 107/3 ml/well) into 6-well culture plates (Costar, Cambridge, MA) in AIM-V (GIBCO-BRL, Grand Island, NY). After 2 h at 37 ~ C, nonadherent cells were removed, whereas the adherent cells were cultured at 37 ~ C in a humidified 5% COJ95% air incubator, in medium supplemented with recombinant human granulocyte/macrophage-colony stimulating factor (GM-CSF [800 U/ml], Immunex, Seattle, WA and interleukin-4 [IL-4, 1000 U/ml], Genzyme Cambridge, MA). Every 2 d, 1 ml of spent medium was replaced by 1.5 ml of fresh medium containing 1600 U/m1 GM-CSF and 1000 U/ml IL-4, to yield final concentrations of 800 and 500 U/ml, respectively. Some of these adherent cells were treated with 13-c/s RA and all-trans RA at the same concentration (1 IxM, Sigma) for 10 d. Finally the cell culture were treated with TNF-c~ and IL-1 on day 7-10 to stimulate complete DCs differentiation. At various time intervals (i.e., time 0 [cells remaining at 2 h after rinse], days 3, 5, 7, and 10) cells were evaluated for surface marker expression using FACS analysis and a panel of monoclonal antibodies. An average of three experiments was performed for each case and FACS analysis was evaluated using matched control cultures tested at the same time. To visualize their morphology, cells were cytocentrifuged for 4 min at 400 rpm on a microscope slide using a Cytospin-2 centrifuge (Shandon, Southern Products, Astmoor, UK). Slides were then fixed in methanol-acetone, stained with May-Grunwald-Giemsa solution and analyzed by light microscopy on a Zeiss Axiophot microscope (Zeiss, Germany). To investigate the immunocytochemical expression of the actinbundling protein fascin, cells were cytocentrifuged for 4 min at 400 rpm on a microscope slide using a Cytospin-2 centrifuge (Shandon, Southern Products). After dehydration, the cytological specimens were incubated with 3% H202 in distilled water for 30 min to quench endogenous peroxidase activity. Cells were then permeabilized with 0.1% Triton X-100 (0.1% sodium citrate in phosphate