Laurie B. Owen-Schaub

Wild-Type Human p53 and a Temperature-Sensitive Mutant Induce Fas/APO-1 Expression

Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.

DNA fragmentation and cell death is selectively triggered in activated human lymphocytes by fas antigen engagement

Fas is a mouse monoclonal antibody-defined cell surface antigen of an unknown physiologic function. Previous studies demonstrated that the anti-Fas antibody mediated apoptosis in those cells sensitive to tumor necrosis factor (TNF) and, further, triggered the co-downregulation of tumor necrosis factor receptors (TNF-Rs). These findings led to speculation that Fas may be associated with TNF-Rs. The present studies were undertaken as an extension of our previous work on the obligate requirement for TNF in development and maintenance of cytotoxic lymphocytes and were designed to analyze the expression and consequences of Fas engagement in these cells. Herein, we demonstrate that, in contrast to TNF-R expression, both resting and IL-2-activated lymphocytes express Fas. In accordance with previous studies using tumor cell lines, lymphocytes rapidly downregulate TNF-Rs after treatment with anti-Fas. The ability of anti-Fas to mediate apoptotic cell death in lymphocytes, however, was dependent upon the status of cellular activation. For example, lymphocytes activated in IL-2 for longer than 4 days underwent rapid DNA fragmentation and cell death after anti-Fas treatment. Despite their expression of Fas, nonactivated lymphocytes and those activated for periods less than 4 days were refractory to antibody-mediated cell killing. Because anti-Fas-mediated lethality is selective for chronically activated lymphocytes, Fas may prove to be an appropriate target for immunosuppressive intervention.

Activation-Dependent Transcriptional Regulation of the Human fas Promoter Requires NF-κB p50-p65 Recruitment

ABSTRACT Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair required for immune homeostasis. Lymphocyte activation results in the upregulation of Fas expression and the acquisition of sensitivity to FasL-mediated apoptosis. Although Fas upregulation is central to the preservation of immunologic tolerance, little is known about the molecular machinery underlying this process. To investigate the events involved in activation-induced Fas upregulation, we have examined mRNA accumulation, faspromoter activity, and protein expression in the Jurkat T-cell line treated with phorbol myristate acetate and ionomycin (P/I), pharmacological mimics of T-cell receptor activation. Although resting Jurkat cells express Fas, Fas mRNA was induced approximately 10-fold in 2 h upon P/I stimulation. Using sequential deletion mutants of the human fas promoter in transient transfection assays, we identified a 47-bp sequence (positions −306 to −260 relative to the ATG) required for activation-driven fas upregulation. Sequence analysis revealed the presence of a previously unrecognized composite binding site for both the Sp1 and NF-κB transcription factors at positions −295 to −286. Electrophoretic mobility shift assay (EMSA) and supershift analyses of this region documented constitutive binding of Sp1 in unactivated nuclear extracts and inducible binding of p50-p65 NF-κB heterodimers after P/I activation. Sp1 and NF-κB transcription factor binding was shown to be mutually exclusive by EMSA displacement studies with purified recombinant Sp1 and recombinant p50. The functional contribution of the κB-Sp1 composite site in P/I-inducible fas promoter activation was verified by using κB-Sp1 concatamers (−295 to −286) in a thymidine kinase promoter-driven reporter construct and native promoter constructs in Jurkat cells overexpressing IκB-α. Site-directed mutagenesis of the critical guanine nucleotides in the κB-Sp1 element documented the essential role of this site in activation-dependentfas promoter induction.

Differential involvement of the CD95 (Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells

The CD95 (Fas/APO-1) system regulates a number of physiological and pathological processes of cell death. The ligand for CD95 induces apoptosis in sensitive target cells by interacting with a transmembrane cell surface CD95 receptor. We previously reported that the recombinant adenovirus-mediated transfer of the wild-type p53 gene caused apoptotic cell death in a variety of human cancer cells. To better understand the mechanism responsible for this cell death signaling, we have investigated the potential involvement of the CD95 receptor/ligand system in p53-mediated apoptosis. The transient expression of the wild-type p53 gene up-regulated the CD95 ligand mRNA as well as protein expression in H1299 human lung cancer cells deficient for p53 and in DLD-1 and SW620 human colon cancer cells with mutated p53, all of which constitutively expressed CD95 receptor as shown by a flow cytometric analysis, and induced rapid apoptotic cell death as early as 24 h after gene transfer. However, the sensitivity to the cytolytic effect of agonistic anti-CD95 antibody (CH11) varied among these cell lines: CH11 induced apoptosis in H1299 cells, but not in DLD-1 and SW620 cells despite their abundant CD95 receptor expression, suggesting that the CD95 receptors on DLD-1 and SW620 cells might be inactivated. In addition, an antagonistic anti-CD95 ligand antibody (4H9) that interfered with the CD95-receptor-ligand interaction partially reduced the apoptosis induced by the wild-type p53 gene transfer in H1299 cells, whereas apoptosis of DLD-1 and SW620 cells occurred in the presence of 4H9. Taken together, these findings led us to conclude that the CD95 receptor/ligand system is differentially involved in p53-mediated apoptosis, suggesting that the restoration of the wild-type p53 function may mediate apoptosis through CD95 receptor/ligand interactions as well as an alternative pathway.

Loss of Fas-Ligand Expression in Mouse Keratinocytes during UV Carcinogenesis

Skin cells containing excessive ultraviolet (UV) radiation-induced DNA damage are eliminated by apoptosis that involves the p53 pathway and Fas/Fas-Ligand (Fas-L) interactions. To determine whether dysregulation of apoptosis plays a role in skin cancer development through disruption of Fas/Fas-L interactions, hairless SKH-hr1 mice were exposed to chronic UV irradiation from Kodacel-filtered FS40 lamps for 30 weeks. Their skin was analyzed for the presence of sunburn cells (apoptotic keratinocytes) and for Fas and Fas-L expression at various time points. A dramatic decrease in the numbers of morphologically identified sunburn cells and TUNEL-positive cells was detected as early as 1 week after chronic UV exposure began. After 4 weeks of chronic UV exposure, these cells were barely detectable. This defect in apoptosis was paralleled by an initial decrease in Fas-L expression during the first week of chronic UV irradiation and a complete loss of expression after 4 weeks. Fas expression, however, increased during the course of chronic UV exposure. p53 mutations were detected in the UV-irradiated epidermis as early as 1 week after irradiation began and continued to accumulate with further UV exposure. Mice exposed to chronic UV began to develop skin tumors after approximately 8 weeks, and all mice had multiple skin tumors by 24 weeks. Most of the tumors expressed Fas but not Fas-L. We conclude that chronic UV exposure may induce a loss of Fas-L expression and a gain in p53 mutations, leading to dysregulation of apoptosis, expansion of mutated keratinocytes, and initiation of skin cancer.

DIFFERENTIAL P53 PHOSPHORYLATION AND ACTIVATION OF APOPTOSIS-PROMOTING GENES BAX AND FAS/APO-1 BY IRRADIATION AND ARA-C TREATMENT

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.

TGF-Beta inhibits the in vitro induction of lymphokine-activated killing activity

SummaryEmploying serum-free media, human peripheral blood mononuclear cells, and purified recombinant interleukin-2 (IL-2), conditions were observed in which the development of IL-2-driven cytotoxic activity was suppressed. The cytotoxic activity of such IL-2-generated lymphokine activated killing (LAK) was tested against natural killer-resistant cultured tumor cells (Daudi, Raji, and a glioma). LAK generation was inhibited by addition of some normal sera, normal platelets, or some tumor cells. Because recent reports have indicated that transforming growth factor-beta (TGF-beta)-like factors are often secreted by tumors and the acidic alpha granules of platelets and can be present in sera, we tested the effect of purified human TGF-beta on the activation of LAK. Our results indicated that TGF-beta is very suppressive for LAK induction, and can completely prevent both the IL-2-driven proliferation and cytotoxicity at concentrations as low as 5 ng/ml. Titrations of IL-2 and of TGF-beta indicated that the suppression is dose-dependent and can be avoided by employing higher levels of IL-2. It was also found that the suppressive effect of TGF-beta can be overcome by washing suppressed cell populations and further culture in low levels of IL-2. Collectively, these data indicate that TGF-beta can be a potent inhibitor of LAK generation under standard activation conditions, but that this effect is regulated by the relative level of IL-2 and may be overcome and/or reversed in vitro.

Apoptosis in Mycobacterium tuberculosis infection in mice exhibiting varied immunopathology

This study examined mechanisms contributing to pulmonary immunopathology following acute Mycobacterium tuberculosis (MTB) infection in vivo in a murine model. A/J and C57BL/6 mice were intravenously infected with MTB (Erdman). Pathological differences were found between strains, unrelated to pulmonary load of bacilli. A/J mice developed progressive interstitial pneumonitis, while C57BL/6 mice maintained granuloma formation. The contribution of FAS and FAS ligand‐mediated apoptosis was assessed via bioluminescent reverse transcription‐polymerase chain reaction (RT‐PCR), immunohistochemical staining, and TUNEL assessment of DNA fragmentation. Cytokine messages for pulmonary tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), as well as for the lytic molecules perforin and granzyme B, were quantified. Immunohistochemical staining for CD3 receptor was performed to monitor lymphocytic lung infiltration. Soon after infection, A/J mice exhibited increased pulmonary IFN‐γ message, concurrent with the appearance of CD3+ lymphocytes distributed throughout the lung. C57BL/6 mice exhibited perivascular cuffing, with no accompanying increase in IFN‐γ message. A/J mice also had elevated levels of FAS and FAS ligand message and protein early after infection, while the C57BL/6 mice had no increased expression of these molecules. Both strains exhibited qualitatively similar numbers of TUNEL‐positive cells throughout infection, with a marked increase on day 7. Apoptotic cells appeared to co‐localize with acid fast bacilli. It is therefore proposed that apoptosis during initial granuloma formation following MTB infection may occur through a FAS/FAS ligand‐independent pathway. Moreover, a failure of completion of the FAS/FAS ligand‐mediated apoptosis pathway in the A/J mice may contribute to inefficient elimination of lymphocytes, thus further aggravating pulmonary pathology. Copyright

Induction of lymphokine-activated killer cytotoxicity with interleukin-2 and tumor necrosis factor-α against primary lung cancer targets

SummaryHuman peripheral blood mononuclear cells (PBM) activated with recombinant interleukin-2 (IL-2) generate potent lytic activity (LAK) against a variety of malignant cells. IL-2 alone is sufficient for LAK generation, but high concentrations are needed to generate optimal cytotoxicity. Our recent studies based on combinations of biological agents indicated that alternative activation pathways may exist. Synergy for LAK induction was investigated using IL-2 and tumor necrosis factor-α (TNF). Single-cell suspensions of primary human lung carcinomas were prepared from seven established cell lines and 32 fresh tumor specimens. Not only were all cell lines sensitive to allogeneic LAK, but also all fresh tumors were sensitive to some degree to both autologous and allogeneic LAK lysis measured by a 4-h 51Cr-release assay. LAK-mediated cytotoxicity, induced with a combination of human recombinant IL-2 (Cetus, 100 U/ml) and TNF (Genentech, 500 U/ml), showed a mean fourfold increase (range 0.7–16.3) over IL-2 alone. No lytic activity was generated from PBM incubated with media or TNF alone. The sequence dependence of adding IL-2 and TNF in enhancing cytolytic activity was also studied. In vitro kinetics data revealed that the addition of TNF 2–6 h before the addition of IL-2 greatly increased LAK activity over that obtained from the simultaneous addition of the two cytokines. These results demonstrated (a) the synergy of IL-2 and TNF for generating LAK; (b) the lysis of fresh primary lung cancer cells by LAK; and (c) the sequence dependence of IL-2 and TNF for the induction of optimal LAK activity.

Development of a Cancer-Targeted Tissue-Specific Promoter System

Present cancer gene therapy using proapoptotic genes has had limited success because the therapy is prone to cause side effects as a result of the lack of tissue and cancer specificity. To target cancer cells without damaging normal cells, we have designed a novel dual promoter system in which a tissue-specific transcription system under the control of a cancer-specific promoter drives expression of a therapeutic gene. The applicability of this system was demonstrated by adapting it to target lung cancer. We termed this lung cancer system TTS (TTF1 gene under the control of human telomerase reverse transcriptase promoter and human surfactant protein A1 promoter). The TTS system showed much higher promoter activity in lung cancer cells compared with other kinds of cancer and normal lung cells, including stem cells. Moreover, insertion of negative glucocorticoid responsive elements in the system allows it to be drug controllable. The approaches that we have used could be adapted to target other types of cancer. We report a novel cancer-targeted tissue-specific dual promoter system designed for gene therapy.