Stability of specific immunoglobulin secretion by EBV-transformed lymphoblastoid cells and human-murine heterohybridomas

The purpose of this project was to determine if stability of specific antibody secretion improved after fusion of Epstein-Barr virus (EBV)-transformed lymphoblastoid cells with P3X63Ag8.653 murine myeloma cells. Production of human monoclonal antibodies by Epstein-Barr virus transformation and somatic cell fusion has been used by many laboratories, however the steps involved have not been fully optimized. B lymphocytes isolated from the peripheral blood of normal donors were enriched for Thomsen-Friedenreich (T) antigen-reactive cells by panning on asialoglycophorin. The EBV-transformed lymphoblastoid cell lines generated from asialoglycophorin-adherent B lymphocytes were treated in three different manners: (1) cloned and maintained in culture as monoclonal lymphoblastoid cell lines, (2) cloned and fused with murine myeloma cells or (3) fused shortly after transfomation without prior cloning. Cloned lymphoblastoid cell lines maintained in culture without fusion either died or lost specific antibody secretion within five months. Uncloned lymphoblastoid cells remained viable for up to three months but lost specific antibody secretion within two months probably due to overgrowth by nonspecific clones. In an attempt to increase longevity and to stabilize specific antibody secretion by these cells, the cloned lymphoblastoid cells were fused with murine myeloma cells. In nine of ten fusions no hybrids were recovered. As an alternate approach, uncloned lymphoblastoid cells secreting T antigen-specific antibody were hybridized with murine myeloma cells, hybrids secreting T antigen-specific antibody were recovered in six of seven fusions. Furthermore, T antigen-specific antibodies of high titer were secreted by the heterohybridoma clones for more than five months of continuous culture. These heterohybridoma cells secreted more immunoglobulin, produced greater titers of antibody and maintained specific antibody secretion longer than either monoclonal or polyclonal EBV-transformed lymphoblastoid cells. These studies have conclusively demonstrated that fusion of polyclonal lymphoblastoid cells secreting T antigen-specific antibody with murine myeloma cells results in prolongation of human monoclonal antibody production compared with unfused monoclonal or polyclonal lymphoblastoid cell lines. This procedure should be generally applicable for the production of stable human monoclonal antibody-secreting cells lines from peripheral blood lymphocytes. ^

Mechanism of damage sensing and cell killing following the inhibition of nucleotide excision repair by fludarabine in quiescent cells

Inhibition of DNA repair by the nucleoside of fludarabine (F-ara-A) induces toxicity in quiescent human cells. The sensing and signaling mechanisms following DNA repair inhibition by F-ara-A are unknown. The central hypothesis of this project was that the mechanistic interaction of a DNA repair initiating agent and a nucleoside analog initiates an apoptotic signal in quiescent cells. The purpose of this research was to identify the sensing and signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A. Lymphocytes were treated with F-ara-A, to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Pre-incubation of lymphocytes with 3 μM F-ara-A inhibited DNA repair initiated by 2 J/m2 UV and induced greater than additive apoptosis after 24 h. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 μM aphidicolin considerably lowered the apoptotic response. ^ Wild-type quiescent cells showed a significant loss in viability than did cells lacking functional sensor kinase DNA-PKcs or p53 as measured by colony formation assays. The functional status of ATM did not appear to affect the apoptotic outcome. Immunoprecipitation studies showed an interaction between the catalytic sub-unit of DNA-PK and p53 following DNA repair inhibition. Confocal fluorescence microscopy studies have indicated the localization pattern of p53, DNA-PK and γ-H2AX in the nucleus following DNA damage. Foci formation by γ-H2AX was seen as an early event that is followed by interaction with DNA-PKcs. p53 serine-15 phosphorylation and accumulation were detected 2 h after treatment. Fas/Fas ligand expression increased significantly after repair inhibition and was dependent on the functional status of p53. Blocking the interaction between Fas and Fas ligand by neutralizing antibodies significantly rescued the apoptotic fraction of cells. ^ Collectively, these results suggest that incorporation of the nucleoside analog into repair patches is critical for cytotoxicity and that the DNA damage, while being sensed by DNA-PK, may induce apoptosis by a p53-mediated signaling mechanism. Based on the results, a model is proposed for the sensing of F-ara-A-induced DNA damage that includes γ-H2AX, DNA-PKcs, and p53. Targeting the cellular DNA repair mechanism can be a potential means of producing cytotoxicity in a quiescent population of neoplastic cells. These results also provide mechanistic support for the success of nucleoside analogs with cyclophosphamide or other agents that initiate excision repair processes, in the clinic. ^

THE ROLE OF MUCOSAL EPITHELIAL CELLS IN HIV-1 INFECTION

The predominant route of human immunodeficiency virus type 1 (HIV-1) transmission is infection across the vaginal mucosa. Epithelial cells, which form the primary barrier of protection against pathogens, are the first cell type at these mucosal tissues to encounter the virus but their role in HIV infection has not been clearly elucidated. Although mucosal epithelial cells express only low levels of the receptors required for successful HIV infection, productive infection does occur at these sites. The present work provides evidence to show that HIV exposure, without the need for productive infection, induces human cervical epithelial cells to produce Thymic Stromal Lymphopoietin (TSLP), an IL7-like cytokine, which potently activated human myeloid dendritic cells (mDC) to cause the homeostatic proliferation of autologous CD4+ T cells that serve as targets for HIV infection. Rhesus macaques inoculated with simian immunodeficiency virus (SIV) or with the simian-human immunodeficiency virus (SHIV) by the vaginal, oral or rectal route exhibited dramatic increases in: TSLP expression, DC and CD4+ T cell numbers, and viral replication, in the vaginal, oral, and rectal tissues, respectively within the first 2 weeks after virus exposure. Evidence obtained showed that HIV-mediated TSLP production by cervical cells is dependent upon the expression of the cell surface salivary agglutinin (SAG) protein gp340. Epithelial cells expressing gp340 exhibited HIV endocytosis and TSLP expression and genetic knockdown of gp340 or use of a gp340-blocking antibody inhibited TSLP expression by HIV. On the other hand, gp340-null epithelial cells failed to endocytose HIV and produce TSLP, but transfection of gp340 resulted in HIV-induced TSLP expression. Finally, HIV-induced TSLP expression was found to be mediated by TLR7/8 signaling and NF-kB activity because silencing these pathways or use of specific inhibitors abrogated TSLP expression in gp340-postive but not in gp340-null epithelial cells. Overall these studies identify TSLP as a key player in the acute phase of HIV-1 infection in permitting HIV to successfully maneuver the hostile vaginal mucosal microenvironment by creating a conducive environment for sustaining the small amount of virus that initially crosses the mucosal barrier allowing it to successfully cause infection and spread to distal compartments of the body

Role played by serum, a biological cue, in the adherence of Enterococcus faecalis to extracellular matrix proteins, collagen, fibrinogen, and fibronectin.

BACKGROUND: Most previous studies have found that Enterococcus faecalis isolates do not show significant adherence to fibronectin and fibrinogen. METHODS: The influence of various conditions on E. faecalis adherence to extracellular matrix (ECM) proteins was evaluated using a radiolabeled-cell adherence assay. RESULTS: Among the conditions studied, growth in 40% horse serum (a biological cue with potential clinical relevance) elicited adherence of all 46 E. faecalis strains tested to fibronectin and fibrinogen but not to elastin; adherence levels were independent of strain source, and adherence was eliminated by treating cells with trypsin. As previously reported, serum also elicited adherence to collagen. Although prolonged exposure to serum during growth was needed for enhancement of adherence to fibrinogen, brief exposure (<5 >min) to serum had an immediate, although partial, enhancing effect on adherence to fibronectin and, to a lesser extent, collagen; pretreatment of bacteria with chloramphenicol did not decrease this enhanced adherence to fibronectin and collagen, indicating that protein synthesis is not required for the latter effect. CONCLUSION: Taken together, these data suggest that serum components may serve (1) as host environmental stimuli to induce the production of ECM protein-binding adhesin(s), as previously seen with collagen adherence, and also (2) as activators of adherence, perhaps by forming bridges between ECM proteins and adhesins.

Molecular signals in B lymphocyte activation: The role of intracellular calcium ion concentration, protein kinase C activation, and autocrine interleukin-2

Calcium ionophore, ionomycin, and phorbol myristate acetate (PMA) were used to activate rabbit peripheral blood B cells to study the role of increased intracellular calcium ion concentration ( (Ca$\sp2+\rbrack\sb{\rm i}$), protein kinase C (PKC) activation, and autocrine interleukin (IL-2) in inducing cell cycle entry and maintaining activation to DNA synthesis. When stimulated with a combination of ionomycin and PMA the B cells produced a soluble factor that supported the IL-2 dependent cell line, CTLL-2. The identity of the factor was established as IL-2 and its source was proved to be B cells in further experiments. Absorption studies and limiting dilution analysis indicated that IL-2 produced by B cells can act as an autocrine growth factor. Next, the effect of complete and incomplete signalling on B lymphocyte activation leading to cell cycle entry, IL-2 production, functional IL-2 receptor (IL-2R) expression, and DNA synthesis was examined. It was observed that cell cycle entry could be induced by signals provided by each reagent alone, but IL-2 production, IL-2R expression, and progression to DNA synthesis required activation with both reagents. Incomplete activation with ionomycin or PMA alone altered the responsiveness of B cells to further stimulation only in the case of ionomycin, and the unresponsiveness of these cells was apparently due to a lack of functional IL-2R expression on these cells, even though IL-2 production was maintained. The requirement of IL-2 for maintenance of activation to DNA synthesis was then investigated. The hypothesis that IL-2, acts in late G$\sb1$ and is required for DNA synthesis in B cells was supported by comparing IL-2 production and DNA synthesis in peripheral blood cells and purified B cells, kinetic analysis of these events in B cells, effects of anti-IL-2 antibody and PKC inhibitors, and by the response of G$\sb1$ B cells. Additional signals transduced by the interaction of autocrine IL-2 and functional IL-2 receptor on rabbit B cells were found to be necessary to drive these cells to S phase, after initial activation caused by simultaneous increase in (Ca$\sp2+\rbrack\sb{\rm i}$ and PKC activation had induced cell cycle entry, IL-2 production, and functional IL-2 receptor expression. ^

Tumor cell-associated epitopes recognized by lymphokine-activated killers (LAK)

Interleukin-2 activated lymphocytes, designated lymphokine-activated killers (LAK), acquire the unique capacity to express potent cytologic activity against a broad spectrum of abnormal and/or transformed NK-sensitive and NK-resistant target cells while sparing normal cell types. Investigations into the target spectra exhibited by cloned effector cells indicate that LAK cells express a polyspecific recognition mechanism that identifies an undefined class of cell surface-associated molecules expressed on susceptible targets. This report extends our previous investigations into the biochemical nature of these molecules by characterizing the functional role of two tumor cell-surface-associated epitopes implicated in conferring target cells with susceptibility to LAK-mediated cytotoxicity. The first moiety is implicated in the formation of effector/target cell conjugates. This binding ligand is preferentially expressed on tumor cells relative to LAK-resistant PBL target cells, sensitive to trypsin treatment, resistant to functional inactivation by heat- and detergent-induced conformational changes, and does not require N-linked glycosylation to maintain binding activity. In contrast, a carbohydrate-associated epitope represents the second tumor-associated molecule required for target cell susceptibility to LAK cells. Specifically, N-linked glyoprotein synthesis represents an absolute requirement for post-trypsin recovery of target cell susceptibility. The minimal N-linked oligosaccharide residue capable of restoring this second signal has been identified as a high mannose structure. Although ultimately required for tumor cell susceptibility, as measured in $\sp{51}$Cr-release assays, this N-glycan-associated molecule is not required for the differential tumor cell binding expressed by LAK cells. Furthermore, N-glycan expression is not adequate in itself to confer target cell susceptibility. Additional categories of cell surface components have been investigated, including O-linked oligosaccharides, and glycosaminoglycans, without identifying additional moieties relevant to target cell recognition. Collectively, these data suggest that tumor cell recognition by LAK cells is dependent on cell surface presentation of two epitopes: a trypsin-sensitive molecule that participates in the initial conjugate formation and an N-glycan-associated moiety that is involved in a post-binding event required for target cell killing. ^

Design, synthesis and biological evaluation of 5$\sp\prime$-mononucleotide prodrugs: Strategies to introduce nucleotides into cells

The neutral bis ((pivaloyloxy)methyl) (PIV$\sb2\rbrack$ derivatives of FdUMP, ddUMP, and AZTMP were synthesized as potential membrane-permeable prodrugs of FdUMP, ddUMP, and AZTMP. These compounds were designed to enter cells by passive diffusion and revert to the parent nucleotides after removal of the PIV groups by hydrolytic enzymes. These prodrugs were prepared by condensation of FUdR, ddU, and AZT with PIV$\sb2$ phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV$\sb2$-FdUMP, PIV$\sb2$-ddUMP, and PIV$\sb2$-AZTMP were stable in the pH range 1.0-4.0 (t$\sb{1/2} = {>}$100 h). They were also fairly stable at pH 7.4 (t$\sb{1/2} = {>}$40 h). In 0.05 M NaOH solution, however, they were rapidly degraded (t$\sb{1/2} < 2$ min). In the presence hog liver carboxylate esterase, they were converted quantitatively to the corresponding phosphodiesters, PIV$\sb1$-FdUMP, PIV$\sb1$-ddUMP, and PIV$\sb1$-AZTMP; after 24 h incubation, only trace amounts of FdUMP, ddUMP, and AZTMP (1-5%) were observed indicating that the PIV$\sb1$ compounds were poor substrates for the enzyme. In human plasma, the PIV$\sb2$ compounds were rapidly degraded with half-lives of less than 5 min. The rate of degradation of the PIV$\sb2$ compounds in the presence of phosphodiesterase I was the same as that in buffer controls, indicating that they were not substrates for this enzyme. In the presence of phosphodiesterase I, PIV$\sb1$-FdUMP, PIV$\sb1$-ddUMP, and PIV$\sb1$-AZTMP were converted quantitatively to FdUMP, ddUMP, and AZTMP.^ PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP were effective at controlling HIV type 1 infection in MT-4 and CEM tk$\sp-$ cells in culture. Mechanistic studies demonstrated that PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP were taken up by the cells and converted to ddUTP and AZTTP, both potent inhibitors of HIV reverse transcriptase. However, a potential shortcoming of PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP as clinical therapeutic agents is that they are rapidly degraded (t$\sb{1/2}$ = approx. 4 minutes) in human plasma by carboxylate esterases. To circumvent this limitation, chemically-labile nucleotide prodrugs and liposome-encapsulated nucleotide prodrugs were investigated. In the former approach, the protective groups bis(N, N-(dimethyl)carbamoyloxymethyl) (DM$\sb2$) and bis (N-(piperidino)carbamoyloxymethyl) (DP$\sb2$) were used to synthesize DM$\sb2$-ddUMP and DP$\sb2$-ddUMP, respectively. In aqueous buffers (pH range 1.0-9.0) these compounds were degraded with half-lives of 3 to 4 h. They had similar half-lives in human plasma demonstrating that they were resistant to esterase-mediated cleavage. However, neither compound gave rise to significant concentrations of ddUMP in CEM or CEM tk$\sp-$ cells. In the liposome-encapsulated nucleotide prodrug approach, three different liposomal formulations of PIV$\sb2$-ddUMP (L-PIV$\sb2$-ddUMP) were investigated. The half-lifes of these L-PIV$\sb2$-ddUMP preparations in human plasma were 2 h compared with 4 min for the free drug. The preparations were more effective at controlling HIV-1 infection than free PIV$\sb2$-ddUMP in human T cells in culture. Collectively, these data indicate that PIV$\sb2$-FdUMP, PIV$\sb2$-ddUMP, and PIV$\sb2$-AZTMP are effective membrane-permeable prodrugs of FdUMP, ddUMP, and AZTMP. ^

MONOCLONAL ANTIBODY CHARACTERIZATION OF RAT TRANSFORMATION ASSOCIATED PROTEINS INDUCED BY MOLONEY MURINE SARCOMA VIRUS

By the use of Moloney murine sarcoma virus (Mo-MSV)-induced rat bone tumor (RBT) cells as immunogens, and the hybridoma technique, a mouse hybridoma clone was isolated in Dr. Chan's lab (Chan et al., 1983), which produced a monoclonal antibody, designated MC. MC detected specific antigens in three different Mo-MSV-transformed rat cell lines: 78A1 WRC, RBT and 6M2 (NRK cells infected with the ts110 mutant of Mo-MSV), but not in their untransformed counterparts. These antigens are tentatively termed transformation associated proteins (TAP). In this study, TAP were hypothesized to be the rat specific proteins which are activated by Mo-MSV and play an important role in cellular transformation, and were further investigated. Their properties are summarized as follows: (1) TAP may represent cellular products localized in the cytoplasm of 6M2 cells. (2) The expression of TAP is temperature-sensitive and related to cellular transformation, and probably activated by the v-mos gene products. The optimal temperature for the expression of both P85('gag-mos), the only known viral transforming protein in 6M2 cells, and TAP was 28(DEGREES)C. The expression of both P85('gag-mos) and TAP was proportional to the degree of transformation of 6M2 cells. (3) There were four antigenically-related forms of intracellular TAP (P66, P63, P60 and P58) in 6M2 cells. After synthesis, the 58Kd TAP was probably converted to one of the other three forms. These three polypeptides (P66, P63 and P60) were rapidly converted to two (P68 and P64) and subsequently secreted to the extracellular medium with a 50% secretion rate of 78 min. The conversion of these molecular sizes of TAP is probably related to glycosylation. Inhibition of TAP glycosylation by 0.5 ug/ml of tunicamycin could retard the secretion rate of TAP by 39%. (4) TAP are phosphoproteins, but not associated with any protein kinase activity. (5) TAP have been purified, and found to be mitogenic NRK-2 cells. TAP can bind to the receptors of NRK-2 cells with a K(,d) of 1.4 pM and with about 2 x 10('5) binding sites for TAP per NRK-2 cell. (6) Some weak proteolytic activity was found to associate with purified TAP. ^

ROLE OF MIR-19A RELEASED BY INFLAMMATORY BREAST CANCER CELLS IN THE REGULATION OF DENDRITIC CELL FUNCTIONS: IN VITRO MODEL OF CROSSTALK IN THE TUMOR MICROENVIRONMENT OF INFLAMMATORY BREAST CANCER

Inflammatory breast cancer (IBC) is a rare but very aggressive form of locally advanced breast cancer (1-6% of total breast cancer patients in United States), with a 5-year overall survival rate of only 40.5%, compared with 85% of the non-IBC patients. So far, a unique molecular signature for IBC able to explain the dramatic differences in the tumor biology between IBC and non-IBC has not been identified. As immune cells in the tumor microenvironment plays an important role in regulating tumor progression, we hypothesized that tumor-associated dendritic cells (TADC) may be responsible for regulating the development of the aggressive characteristics of IBC. MiRNAs can be released into the extracellular space and mediate the intercellular communication by regulating target gene expression beyond their cells of origin. We hypothesized that miRNAs released by IBC cells can induce an increased activation status, secretion of pro-inflammatory cytokines and migration ability of TADC. In an in vitro model of IBC tumor microenvironment, we found that the co-cultured of the IBC cell line SUM-149 with immature dendritic cells (iDCSUM-149) induced a higher degree of activation and maturation of iDCSUM-149 upon stimulation with lipopolysaccharide (LPS) compared with iDCs co-cultured with the non-IBC cell line SUM-159 (iDCSUM-159), resulting in: increased expression of the costimulatory and activation markers; higher production of pro-inflammatory cytokines (TNF-a, IL-6); and 3) higher migratory ability. These differences were due to the exosome-mediated transfer of miR-19a and miR-146a from SUM-149 and SUM-159, respectively, to iDCs, causing the downregulation of the miR-19a target genes PTEN, SOCS-1 and the miR-146a target genes IRAK1, TRAF6. PTEN, SOCS-1 and IRAK1, TRAF6 are important negative and positive regulator of cytokine- and TLR-mediated activation/maturation signaling pathway in DCs. Increased levels of IL-6 induced the upregulation of miR-19a synthesis in SUM-149 cells that was associated with the induction of CD44+CD24-ALDH1+ cancer stem cells (CSCs) with epithelial-to-mesenchymal transition (EMT) characteristics. In conclusion, in IBC tumor microenvironment IL-6/miR-19a axis can represent a self-sustaining loop able to maintain a pro-inflammatory status of DCs, leading to the development of tumor cells with high metastatic potential (EMT CSCs) responsible of the poor prognosis in IBC patients.

Mechanisms of multidrug resistance: Differences between natural and acquired adriamycin -resistant human colon carcinoma cells

Mechanisms of multidrug resistance (MDR) were studied in two independent MDR sublines (AdR1.2 and SRA1.2) derived from the established human colon carcinoma cell line LoVo. AdR1.2 was developed by long-term continuous exposure of the cells to adriamycin (AdR) in stepwise increments of concentration, while SRA1.2 was selected by repetitive pulse treatments with AdR at a single concentration. In this dissertation, the hypothesis that the mechanism of drug resistance in SRA1.2 is different than that in AdR1.2 is tested. While SRA1.2 demonstrated similar biological characteristics when compared to the parental LoVo, AdR1.2 exhibited remarkable alterations in biological properties. The resistance phenotype of AdR1.2 was reversible when the cells were grown in the drug-free medium whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions. Km and Vmax of carrier-mediated facilitated diffusion AdR transport were similar among the three lines. However, both resistant sublines exhibited an energy-dependent drug efflux. AdR1.2 appeared to possess an activated efflux pump, and a decreased nucleus-binding of AdR, whereas SRA1.2 showed merely a lower affinity in binding of AdR to the nuclei. Southern blot analysis showed no amplification of the MDR1 gene in either of the two resistant subclones. However, Western blot analysis using the C219 monoclonal antibody against P170 glycoprotein detected a Mr 150-kDa plasma protein (P150) in AdR1.2 but not in SRA1.2 or in the parental LoVo. In vitro phosphorylation studies revealed that P150 was a phosphoprotein; its phosphorylation was Mg$\sp{2+}$-dependent and could be enhanced by verapamil, an agent capable of increasing intracellular AdR accumulation in AdR1.2 cells. The phosphorylation studies also revealed elevated phosphorylation of a Mr 66-kDa plasma membrane protein that was detectable in the AdR1.2 revertant and in AdR1.2 when verapamil was present, suggesting that hyperphosphorylation of the Mr 66-kDa protein may be related to the reversal of MDR. SDS-PAGE of the plasma membrane protein demonstrated overproduction of a Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa protein was not immunoreactive with C219, but its absence in the AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein. In conclusion, the results from this study support the author's hypothesis that the mechanisms responsible for "Acquired" and "Natural" MDR are not identical. ^

IL -12 inhibits the metastatic potential of osteosarcoma cells and induces tumor regression via a mechanism involving Fas /Fas ligand pathway

Despite multiple changes in the adjuvant chemotherapy regimens used to treat osteosarcoma (OS), the 2-year metastasis-free survival has remained at 65–70% for the past 10 years. Characterizing the molecular determinants that permit metastatic spread of tumor cells is a crucial element in developing new approaches for the treatment of osteosarcoma. Since OS metastasizes almost exclusively to the lung, an organ with constitutive Fas ligand (FasL) expression, we hypothesized that the expression of Fas (CD95, APO-1) by OS cells may play a role in the ability of these cells to form lung metastases. Fas expression was quantified in human SAOS-2 OS cells and selected variants (LM2, LM4, LM5, LM6, LM7). Using northern blot, FACS and RT-PCR analysis, low Fas expression was found to correlate with higher metastatic potential in these cell lines. The highly metastatic LM7 cell line was transfected with the full-length human Fas gene and injected into athymic nude mice. The median number of metastatic nodules per mouse fell from over 200 to 1.1 and the size of the nodules decreased from a range of 0.5–9.0 mm to less than 0.5 mm in the Fas-transfected cell line compared to the native LM7 cell line. Additionally, the subsequent incidence of lung metastases was lower in the Fas-expressing cell line. IL-12 was seen to upregulate Fas expression in the highly metastatic LM sublines in vitro. To visualize the effects of IL-12 in vivo, nude mice were injected with LM7 cells and treated biweekly for 4 weeks with Ad.mIL-12, saline control or Ad.βgal. Lung sections were analyzed via immunchistochemistry for Fas expression. A higher expression of Fas was found in tumors from mice receiving IL-12. To study the mechanism by which IL-12 upregulates Fas, LM7 cells were transfected with a luciferase reporter gene construct containing the full-length human fas promoter. Treatment with IL-12 increased luciferase activity. We therefore conclude that IL-12 influences the metastatic potential of OS cells by upregulating the fas promoter, resulting in increased cell surface Fas expression and susceptibility to Fas-induced cell death. ^