Essential role for hematopoietic Fas ligand (FasL) in the suppression of melanoma lung metastasis revealed in bone marrow chimeric mice.

The outcome of cancer metastasis depends on multiple interactions between the malignant cell and the host environment. Such interactions can influence primary cancer growth and metastasis by altering the balance between tumor cell proliferation and death. We have previously reported that the pro-apoptotic protein FasL could potently suppress spontaneous lung metastasis of the Fas-sensitive melanoma, K1735-P. In this report, we have constructed bone marrow chimeric mice using wt and FasL-deficient animals to delineate the source of FasL (hematopoietic or nonhematopoietic) required to control spontaneous metastatic spread from a subcutaneous tumor. Using FasL-deficient animals (gld) reconstituted with wt FasL bone marrow (wt --> gld), and wt animals reconstituted with FasL-derived bone marrow (gld --> wt), we show, for the first time, an essential role for hematopoietic-derived FasL in the suppression of K1735-P metastasis. When FasL was expressed only in the nonhematopoietic compartment (gld --> wt), K1735-P spread was ineffectively controlled with a metastatic phenotype similar to that observed in animals completely lacking FasL (gld --> gld or gld controls). These studies provide evidence for the indispensable role for FasL+ bone marrow-derived cells in the control of melanoma and offer a strategy to target FasL expression in the prevention or therapy of metastatic disease.

Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death.

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.

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.

Adenoviral Bid overexpression induces caspase-dependent cleavage of truncated Bid and p53-independent apoptosis in human non-small cell lung cancers.

Proapoptotic gene transfer to promote death or to augment killing by DNA-damaging agents represents a promising strategy for cancer therapy. We have constructed an adenoviral Tet-Off trade mark vector with tightly controlled expression of Bid (Ad-Bid) (Clontech, Palo Alto, CA). Using the non-small cell lung cancer cell lines H460, H358, and A549, low dose Ad-Bid was shown to induce high levels of full-length Bid as well as caspase-3 and -9 activity. Although only a small fraction of Bid was processed to truncated Bid (a step inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), Ad-Bid gene transfer resulted in mitochondrial changes consistent with apoptosis (mitochondrial depolarization, cytochrome c release), DNA fragmentation, and a dramatic loss of cell viability. The proapoptotic effects of Ad-Bid were independent of p53 status and were augmented markedly by caspase-8 activators such as the DNA-damaging agent cisplatin. When Ad-Bid and cisplatin were used together, chemosensitivity was restored in p53-null H358 cells, increasing death from 35% following treatment with cisplatin and Ad-LacZ to >90% death with Ad-Bid and cisplatin (Ad-Bid alone induced 50% cell death under these conditions). Ad-Bid can induce apoptosis in malignant cells and enhance chemosensitivity in the absence of p53, suggesting this approach as a potential cancer therapy.

Fas function and tumor progression: use it and lose it.

Recent studies have provided evidence that Fas and FasL interactions are important in the control of malignant disease and that changes in the level of Fas expression can determine immune escape and therapeutic responses.